New User Special Price Expires in

Let's log you in.

Sign in with Facebook


Don't have a StudySoup account? Create one here!


Create a StudySoup account

Be part of our community, it's free to join!

Sign up with Facebook


Create your account
By creating an account you agree to StudySoup's terms and conditions and privacy policy

Already have a StudySoup account? Login here


by: Murphy Mante

Microbiology BIOL0004

Murphy Mante

GPA 3.68


Almost Ready


These notes were just uploaded, and will be ready to view shortly.

Purchase these notes here, or revisit this page.

Either way, we'll remind you when they're ready :)

Preview These Notes for FREE

Get a free preview of these Notes, just enter your email below.

Unlock Preview
Unlock Preview

Preview these materials now for free

Why put in your email? Get access to more of this material and other relevant free materials for your school

View Preview

About this Document

Class Notes
25 ?




Popular in Course

Popular in Biological Sciences

This 139 page Class Notes was uploaded by Murphy Mante on Tuesday October 20, 2015. The Class Notes belongs to BIOL0004 at Sierra College taught by HarrietWilson in Fall. Since its upload, it has received 32 views. For similar materials see /class/225364/biol0004-sierra-college in Biological Sciences at Sierra College.


Reviews for Microbiology


Report this Material


What is Karma?


Karma is the currency of StudySoup.

You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!

Date Created: 10/20/15
Microbial Nutrition and Growth Some of the information relating to microbial nutrition and growth has been presented in previous lectures or in laboratory sessions This information will be included here as a review Nutritional Categories Microorganisms and macroscopic forms also can be divided into four nutritional categories based on the sources they use for energy and carbon The four categories are l Chemoheterotrophs Organisms requiring preformed organic compounds for both their energy and carbon requirements These organisms may also be referred to as chemoorganotrophs 2 Chemoautotrophs Organisms that use chemicals for energy but are capable of using inorganic compounds e g COZ HCOS etc for carbon If these organisms use only inorganic chemicals to meet their nutritional needs they may be called chemolithotrophs 3 Photoheterotrophs Organisms that use light energy and require preformed organic compounds as their source of carbon 4 Photoautotrophs Organisms that use light energy and are capable of using inorganic compounds eg COZ HCOS etc for carbon Anilnals including hu1nans fungi protozoa and many types of prokaryotic organisms are chemoheterotrophs Of these some are saprotrophs some parasites some hypotrophs and many are freeliving organisms consuming other life forms as food materials Freeliving protozoa may be considered predators carnivores or herbivores depending on the prey they consu1ne but many of these are omnivorous Green plants algae cyanobacteria and some other types of prokaryotic organisms are photoautotrophs These are ecologically considered as producers because they provide much of the organic material consu1ned by various different types of chemoheterotrophs Most photoheterotrophs are prokaryotic but because some singlecelled eukaryotic forms are capable of switching between nutritional categories they might function as photoheterotrophs for short periods of tilne All chemoautotrophs are prokaryotic Culture media Most of the microorganisms grown under laboratory conditions are chemoheterotrophs This is particularly true in clinical laboratories where hu1nan pathogens are of prilnary interest Many chemoheterotrophs can be grown on or in mixtures of materials designed to support their metabolic processes Mixtures of materials providing all the nutrients needed to grow microorganisms in vitro ie in artificial containers are called culture media singular medium When considering the nutrients necessary for growth it is useful to consider the composition of protoplasm and the elements incorporated into various compounds Some conunon nutrients are listed below 1 II V Carbon Carbon is essential for the synthesis of all organic compounds carbohydrates proteins lipids and nucleic acids Though autotrophs can obtain carbon carbon dioxide from the air heterotrophs cannot and must have this element provided to them in the culture mediuln Carbon is often provided in carbohydrate form glucose lactose mannitol etc or as protein or protein breakdown products peptones peptides or proteoses Media without carbon can be used to grow cyanobacteria Nitrogen Nitrogen is essential for the synthesis of proteins nucleic acids and some carbohydrates e g glucosamine Since proteins and protein breakdown products provide both carbon and nitrogen these are common ingredients in culture media Some prokaryotic organisms can obtain nitrogen from the air molecular nitrogen N2 and are called nitrogenfixing organisms Bacteria with this capability can grow readily on nitrogenfree media Minerals Many of the elements incorporated into organic compounds are minerals such as sulfur phosphorous iron calciuln magnesiuln iodine manganese and copper Sulfur is incorporated into certain amino acids so is essential to protein formation Phosphorous is used for the synthesis of phospholipids nucleic acids and a variety of nucleotide derivatives such as coenzymes NAD FAD NADP and high energy compounds ATP GTP etc Iron and copper often serve as prosthetic groups in enzymes with quaternary structure and calciuln magnesiuln and manganese often function as cofactors essential to enzyme activity Electrolytes such as NaCl KCl and CaClz dissociate into ions involved in maintaining membrane potentials activating contractile elements eg microfilaments and other cellular functions lodine is incorporated into various organic compounds Water Though not usually considered as a nutrient water is essential to metabolism and is incorporated into organic compounds as H and OH during hydrolysis reactions The aInount of water present relative to solute materials also in uences tonicity making enviromnents isotonic hypotonic or hypertonic Though bacteria are not daInaged by osmosis when placed in hypotonic environments because they are equipped with walls the nutrient levels in hypotonic media tend to be low and growth may be slow Hypertonic enviromnents tend to inhibit growth because they cause water to exit cells and metabolism to slow or stop For this reason salt and sugar are often used to preserve food materials Since high salt environments tend to occur naturally in various places e g along rocky ocean shores in deserts on skin surfaces and inside nasal passages many microorganisms have adapted to these enviromnents These saltloving organisms are called halophiles Buffers Some of the minerals listed above are also incorporated into buffers ie substances that resist pH change when the acidic or alkaline waste products of various microorganisms are released into media KZHPO4 KHZPO4 and CaCO3 are examples of materials often incorporated into buffer systems Culture media without buffers often contain pH indicators ie substances that change color in response to changes in pH the acidity or alkalinity of the enviromnent Phenol red bromothymol blue and bromocresol purple are pH indicators conunonly used in culture media Other Some organisms will only grow when provided with specific growth factors such as vitamins aInino acids cells tissue fragments or other materials Such media are called enriched media and are used to grow fastidious microorganisms picky eaters Bacteria in the genera Streptococcus and Neisseria tend to be fastidious and are often grown on enriched media such as blood agar or chocolate agar As demonstrated in the laboratory culture media may be categorized in a variety of ways Broth media are liquid in form and when incubated on a moving surface such as an orbital shaker allow all organisms present to maintain contact with the nutrients provided Solid media contain some type of solidifying agent in addition to the nutrients present Though a variety of solidifying agents could be used agar a polysaccharide is the material most commonly used to change broth media into solid media Agar is an ideal solidifying agent for culture media because most microorganisms do not catabolize it break it down Unlike those containing gelatin or starch solidifying agents catabolized by many different types of microorganisms media made with agar will remain solid as microbial cultures grow Another advantage of agar is its response to temperature changes Media made with agar will remain solid at relatively high incubation temperatures considerably above 37 C while media made with gelatin will liquefy Agar is made from algae in the phyluln Rhodophyta Culture media containing agar are prepared as liquids and poured into various containers where they are allowed to solidify Agar deeps slants slopes and plates are commo used in our laboratory Agar plates provide broad surfaces where cultures can be streaked and colonies isolated to check for purity Agar slants and deeps are often used for enzymatic testing but can also be used for longterm storage if the culture tubes used are equipped with screwon caps Factors influencing microbial growth Many factors in uencing microbial growth were described in association with criteria used in microbial classification These same factors can also be associated with microbial control and will be described in that context later Some factors known to in uence microbial growth include 1 Gas requirements Microorganisms can be categorized as obligate aerobes facultative anaerobes obligate anaerobes or microaerophiles based on their gas requirements Obligately aerobic organisms have respiratoryoxidative metabolic processes and use molecular oxygen 02 as a final electron acceptor Aerobic bacteria often grow near the surface in broth media and sometimes form pellicles or skinlike coverings at the mediu1n surface Facultatively anaerobic organisms can often use either respiratory or fermentative metabolic processes and switch back and forth between the two as their environments permit Facultative organisms will grow throughout broth media if they are motile or if agitated during incubation but non motile forms will often form a mass of sediment at the bottom of a stationary tube Obligately anaerobic forms may also form sediments on tube bottoms but typically require special containment or anaerobic media for growth Many of the organisms inhabiting the Winogradsky columns and window are obligately anaerobic 2 Temperature Organisms can be categorized as psychrophiles mesophiles thermophiles or hyperthermophiles based on the temperatures they prefer for growth Bacteria as a group can inhabit environments with extreme temperature differences eg the freezing temperatures of polar ice Vs the near boiling temperatures of hot springs or deepsea thermal vents but individual species typically have relatively narrow temperature ranges Enzyme activity and therefore metabolism is dependent on temperature and enzymes vary considerably with respect to their temperature optimuins Though most of the bacteria grown in our laboratory are mesophiles placing cultures in a 37 C incubator will not necessarily increase their growth rate Many organisms associated with air plates prefer temperatures below this 3 pH requirements The amount of acidic or alkaline material present in culture media can significantly in uence microbial growth Most media used in our laboratory are initially pHadjusted to be near neutral pH 68 72 particularly if they contain pH indicators Many bacteria can tolerate slightly acidic environments shifts toward the low end of the pH scale more readily than they can alkaline environments shifts toward the high end of the pH scale Some organisms eg Ferroplasma grow best in acidic environments and can be considered as acidophiles Although bacteria can form both acidic and alkaline end products through their metabolic processes acidic products tend to stay in the media and have a greater influence on pH alkaline end products such as amines and ammonia are volatile and escape into the air Organic acids eg lactic acid and acetic acid formed by fermentative microorganisms are used extensively in food preservation cheese yogurt pickles sauerkraut and silage because high levels of acid inhibit the growth of organisms likely to cause spoilage 4 Osmotic pressure requirements The effective osmotic pressure or tonicity of an environment can in uence growth by causing water to either enter or exit cells through osmosis Hypotonic environments do not damage organisms equipped with cell walls e g fungi algae and bacteria because walls prevent these cells from taking on excess water and blowing up Hypertonic environments cause water to exit cells and will often inhibit metabolism and growth Since high salt environments occur naturally in various places many microorganisms have adapted to these Some saltloving organisms or halophiles can only grow in hypertonic environments containing high levels of salt 5 Symbiosis In natural environments the growth of microorganisms is significantly in uenced by interactions with other organisms Relationships are often subtle and many have yet to be investigated or are not thoroughly understood A few examples have been described earlier and are included here as reminders Symbiotic relationships between bacteria in the genera Rhizobium and Photobacterium and plants or fish squid respectively are mutually beneficial to all organisms concerned ie are mutualistic Bacteria protozoa and fungi living inside the gastrointestinal tracts of animals including humans gain nutrients provided to them by their hosts but can also provide animals with nutrients they could otherwise not obtain Maintainin balance between the various populations present is essential to good health Parasites such as atworms roundworms and various arthropods take nutrients from animal hosts but generally give nothing in return Fungi form haustoria or mycorrhizae when interacting with plant hosts form antibiotics that inhibit bacterial growth and live symbiotically with algae or cyanobacteria within lichens Ecologically speaking all symbiotic relationships are probably mutualistic because even parasites an pathogens benefit their hosts through population control a concept foreign to many humans When microorganisms are grown in vitro symbiotic relationships are limited but some can still be observed On agar plates exposed to air or soil mixed populations of fungi and bacteria will sometimes interact in ways visible to the naked eye Clear areas or zones of inhibition are often formed by colonies producing chemicals antibiotics inhibiting the growth of others When the effects of antibiotics are synergistic ie greater when combined this is also visible Fungi will often grow on nitrogenfree media if nitrogenfixing bacteria are already present Though fungi cannot fix nitrogen they can and do use byproducts formed by bacteria On blood agar plates hemolysis reactions can be enhanced by the combined action of different bacteria as demonstrated by the CAMPreaction Organisms with this ability can also cause more severe damage when interacting together inside a host Microbial Growth When applied to microorganisms the term growth refers to an increase in cell nulnber rather than to an increase in the size of an individual organism When bacteria are placed on the surface of a solid mediuln such as nutrient agar within a Petri plate each living cell in contact with the nutrients available has the potential to reproduce itself and grow into a mass visible to the naked eye The visible mass is called a colony and though variable in morphology will often appear as a circular form with an entire margin convex elevation smoothshiny surface texture opaque optical character and pigment varying from white to brightlycolored yellow pink purple brown etc Like many singlecelled eukaryotic organisms bacteria typically reproduce themselves by means of an asexual process called binary fission during which one cell divides into two Unlike eukaryotes bacteria do not undergo mitosis Prokaryotic cells do not form microtubules centrioles or a spindle apparatus so the separation of chromosomes requires an alternative mechanism Though fission typically proceeds as a moreorless continuous process it can be broken into a nulnber of steps or phases as described below 1 Replication of DNA and other essential cellular components Before it can divide itself into two parts a cell must replicate its genetic information DNA This insures that each new cell formed will contain the information necessary to function as a separate organism The details of DNA replication will be presented later but replication basically involves the separation of the two nucleotide strands present in each DNA molecule and the formation of new complimentary strands in association with each one Additional cellular components such as ribosomes and enzymes are also made 2 Elongation Though bacteria do not grow extensively as individuals each cell does undergo some degree of elongation before dividing into two parts Since peptidoglycan is not elastic ie does not stretch elongation requires a partial decomposition of this rigid wall material and the deposition of new peptidoglycan The process is somewhat like the growth of long bones in association with epipheseal plates Decomposition typically occurs in several places along the long axes of rod shaped cells bacilli or in one centrally located equatorial region of each spherical cell During elongation the chromosomes are separated Regions of each chromosome are attached to the cell membrane or cytoplasmic membrane recall membranous folds called mesosomes and as the cell elongates the chromosomes are pulled or pushed apart 3 Septum formation Following elongation the cell membrane folds inward across the long axis of the cell until it forms a twolayered membranous septuln separating the cytoplasm into two parts As in eukaryotic cells this inward folding or pinching of the cell membrane involves microfilaments 4 Deposition of new wall within the septum Once the septum has been formed new layers of peptidoglycan are deposited between the membrane layers or within the septum This effectively separates the cell into two parts Many of the diplobacilli observed in bacterial smears are cells at this stage of the fission process 5 Physical separation When sufficient wall material has been deposited the two cells can break away from one another and exist as separate individuals however physical separation does not always occur Cells with a characteristic arrangement eg diplococci streptococci streptobacilli etc are examples of those maintainin connections following fission lf cells do not physically separate their arrangement is in uenced by the orientation of the fission plane Chains of cells streptococci or streptobacilli will be formed if the fission plane always has the same orientation Tetrads sarcinae and staphylococci will form if the orientation of the fission plane changes between fission cycles Population Dynamics in a Batch Culture Bacteria grown in a broth medium within a closed container such as a tube ask or bottle ie grown in vitro form a batch culture Under batch culture conditions the population can receive no additional nutrients and most metabolic wastes will stay within the medium A population of bacteria grown in a batch culture will typically undergo changes in density number of cells per volume that when plotted against time will form a predictable growth curve The phases of this curve can change somewhat with respect to time and cell density but the overall pattern is similar for all populations The growth curve and the phases represented are described below Note that ordinate values are represented on a logarithmic scale 1 Lag phase The lag phase is represented by a straight horizontal line of variable length During this phase of growth the cells present are increasing in DNA content metabolic activity size and dry weight but the population is experiencing no increase in cell number The cells are quotgearing upquot for growth and completing some of the steps involved in fission but physical separation has not yet occurred The number of cells introduced and the batch culture volume will influence cell density line height and the time required to manufacture enzymes needed to complete various metabolic processes will in uence the time involved line and phase length N Exponential growth or Logarithmic growth phase During the exponential or logarithmic growth phase the cells are dividing at a rapid rate and population numbers are represented by a diagonal line extending upward from the lag phase level The population is metabolically very active and its numbers are increasing exponentially 1 cell becomes 2 2 become 4 4 become 8 8 become 16 16 become 32 32 become 64 64 become 128 128 become 256 etc The time required for one cell to divide into two cells is called the generation time or doubling time and for rapidly growing organisms such as E coli Staphylococcus or Salmonella is typically around 20 minutes Since an E coli cell growing under optimum conditions generally requires about 60 minutes to replicate its chromosome this short generation time seems impossible but the bacteria accomplish this amazing feat by overlapping their DNA replication cycles in time ie before the first replication cycle is complete a Ta and 3 cycle have begun It has been estimated that if bacteria growing in a batch culture could be maintained in their exponential growth phase for 45 hours they would form a mass the size of the earth Needless to say this is not possible DJ Stationary phase The stationary phase is represented by a horizontal line again indicating no overall increase in cell number During this phase the number of cells dying is equal to the number of new cells being formed and the population has reached its maximum density referred to as the maximum concentration or m concentration For most bacteria grown in a batch culture the mconcentration is 109 one or more billion cells per ml of culture medium During the stationary phase some of the cells present in the population are dying due to a lack of nutrients and a buildup of toxic metabolic waste products Since conditions within cells and within the environment are not uniform the death of some cells can provide nutrients for others not yet overcome by the toxins present but only for a limited time During this phase of growth population density is influenced by an environmental feature called carrying capacity Carrying capacity refers to the number of organisms an environment can support and is in uenced by limiting factors such as nutrient availability pH redox potential and temperature The numerical value of the carrying capacity is identical to that of the mconcentration because population density cannot exceed the environinent39s capacity to support it All populations are limited by the carrying capacity of their environment 4 Exponential death phase During the death phase represented by a diagonal line extending downward from the stationary level the population is experiencing an exponential decrease in number Many cells are dying due to a lack of nutrients and are being poisoned by their own metabolic waste products The density of live cells decreases rapidly and typically drops below the number of organisms initially introduced into the medium Conditions within the container are generally nasty but a few hardy individuals will often manage to survive especially if metabolism is slowed by a drop in temperature eg if some conscientious individual places the culture into a refrigerator If the growth curve described above were drawn without using a logarithmic scale for the ordinate it would form a Ishaped curve extending steeply upward until the carrying capacity was reached and then dropping in an equally steep downward sweep The growth curve representing earth39s human population shows a similar Ishape Although the carrying capacity of this planet is not uniform and cannot be accurately predicted it is a feature of our environment and cannot be indefinitely ignored Environmental factors such as nutrient availability toxic waste buildup and temperature will eventually limit our population just as they do those of microorganisms As a supplement to this week39s lecture information you may explore the population Web sites listed below Students interested can obtain 5 points extra credit by visiting two or more of these sites and writing a brief report summarizing their content United Nations Population Fund http wwwunfpaorg indexhtm Population Connection http wwwpopulationconnectionorgZ The Population lnstitute http wwwpopulationins1ituteorgZ Though hulnans have considerable potential for intelligent achievement our population growth curve strongly resembles that of Escherichia coli growing in a batch culture Somehow it seems we should know better Growth in a Continuous Culture Microorganisms such as bacteria and yeast can be maintained in continuous cultures if wastes including dead cells are removed and a steady supply of nutrients is maintained Continuous cultivation is often used when microorganisms are being maintained for the synthesis of organic compounds such as enzymes solvents etc or when biochemical processes are being investigated Population density within a continuous culture is much more stable and metabolic processes tend to be more consistent than are those occurring in batch cultures Growth on solid media Bacteria grown on solid media typically form colonies of a characteristic size and shape Although colonies are often surrounded by what appears to be available nutrient colony growth is lilnited by the same factors lilniting growth in a batch culture Bacteria typically release enzymes that break down nutrients beyond the colony border and open agar surfaces are often depleted of nutrients Although the youngest most viable organisms are often found at the colony margin cell reproduction and expansion of the colony is essentially ilnpossible Note Motile organisms and filaInentous forms can temporarily overcome nutrient limitations by swarming swilnming over the agar surface or by extending their filaInents into regions with available nutrients Eventually however the plate edge is reached and all available nutrients are depleted Students seeking to maintain pure cultures of various microorganisms must transfer samples to new media every 23 weeks Nutrient depletion and water loss will eventually cause most of the cells on an agar plate to die even though the colonies may appear unchanged Introduction to Microbiology Microbiology may be defined as the science or study of microscopic organisms ie organisms too small to be observed with the naked eye from the Greek terms micro small Bio life and logos discourse or study of These microorganisms or microbes as they are sometimes called include bacteria archaea protozoa and microscopic forms of fungi and algae Since certain multicellular organisms are microscopic or have microscopic stages during their life cycles and some play a role in disease transmission they are also included in microbiology courses Non cellular forms such as viruses viroids and prions are not true organisms but since they do infect and reproduce within living organisms they fall within the realm of microbiology Eukaryotic cell types Prokaryotic cell types Noncellular types Protozoa Bacteria Viruses Microscopic algae Archaea Viroids Microscopic fungi Prions Microscopic animals When compared to the other natural sciences Microbiology is relatively young ie has not existed for very long Can you think of a reason for this Exactly Although humans have been interacting with microorganisms for thousands of years they are not visible without the aid of a microscope so nobody knew they were there The first living microorganisms were observed a little more than 300 years ago but their significance was not appreciated until nearly 200 years later Today microbiology is recognized as a subject of major importance since microbes play a role in nearly every aspect of our lives Early Uses for Microorganisms Humans began to interact with microorganisms long before they were able to observe them or recognized that they existed Though they sometimes caused disease microbes were also bene cial under various circumstances and people began to put them to work What do you suppose humans first used microorganisms for Records indicate that prior to 6000 BC human societies such as the Sumerians and Babylonians were using microorganisms to ferment grain and make beer This practice probably began as soon as people had excess grain to store for future use Grain stored in depressions in the ground was sometimes wetted by rain would have been fermented by wild yeasts and alcohol was produced As people found the fermented grain and juice palatable they undoubtedly took steps to increase production Thus the first quotbeerquot was made Alcoholic beverages made from rice were produced in China at least as early as 2300 BC and reference to wine is common in association with early cultures Around 4000 BC the Egyptians discovered that bread dough treated in a certain manner would rise into a light airy loaf This was due to yeast cells producing carbon dioxide The practice of saving a small bit of dough as a quotstarterquot probably began long before people recognized yeast as a microbe Cultured foods such as cheese and yogurt were initially produced as the result of storing milk without refrigeration often in containers made of animal skin or stomach When people found that these products would keep without spoiling longer than would fresh milk they were made intentionally So the earliest uses for microorganisms were in food processing and preservation Wine could be stored longer than fresh grape juice and cheese longer than fresh milk The fermentation of materials such as milk grains grapes cabbages cucumbers etc yielded products that remained palatable and could be stored for long periods of time Anton Van Leeuwenhoek 16741676 The discovery of microbiology is usually credited to a Dutch naturalist by the naIne of Anton Van Leeuwenhoek He is sometimes referred to as the quotfather of Microbiologyquot Van Leeuwenhoek ground fine glass lenses which could magnify objects about 266 times and observed living microorganisms which he called quotanimaculesquot from a variety of environments His investigations were apparently made around 1674 but he was rather secretive about his work and did not explain exactly how he made his lenses or his observations Van Leeuwenhoek s observations may not have been the first but they were significant because he made numerous drawings and wrote accurate descriptions of what he saw He documented his ndings For several years starting about 1684 he sent correspondence to the British Royal Society or Royal Society of London and thereby aroused considerable interest in microbiology or I 1 I A l a Van Leeuwenhoek s discoveries did much to revitalize arguments between scientists philosophers and theologians about the origin of life It was at one time generally accepted that living organisms arose spontaneously from nonliving material This belief sometimes called the theory of abiogenesis or spontaneous generation awithout biolife genesisorigins or beginnings was taught by Aristotle around 346 BC He believed that life could and did appear spontaneously from nonliving and or decomposing materials For exaInple he wrote that snakes and frogs caIne from the mud along river banks that insects came from dew that ies arose from decaying meat and that rats sprang from refuse heaps These like many other beliefs of the Greek scholars were maintained until relatively recent times During the 17th century 1600s a Belgian clergyman by the name of Van Helmont wrote a recipe for the generation of mice He suggested that if a dirty garment were placed in a container with wheat grains for 21 days the cloth and grains would give rise to live mice This says little for living conditions at the time and less for powers of observation Although belief in spontaneous generation was based upon inadequate observation and faulty reasoning supporters of this theory were dif cult to refute Around 1665 the Italian naturalist and physician Francesco Redi demonstrated that spontaneous generation did not occur at a macroscopic level using flies Redi placed raw meat into containers and covered some with gauze and some with paper Other containers were left open He found that the meat within the covered containers did not develop ies but that ies did lay eggs on the gauze and on the paper The exposed meat developed maggots but he reasoned that these came from the eggs of ies not from the meat itself Regardless of Redi39s proof people still clung to their belief in abiogenesis and Van Leeuwenhoek39s discoveries seemed to support this theory Van Leeuwenhoek did not conduct experiments to determine the source of his quotanimaculesquot but it is assumed he believed them to have come from the air Those believing in abiogenesis thought the microscopic organisms came from the broths and waters in which they were observed Thus the discovery of microorganisms rekindled an argument that was to wage for years In 1749 John Needham a Catholic priest conducted experiments with mutton broth in asks He boiled the broth and stoppered the flasks with cork but later found the broth to be teaming with microorganisms Needham believed there was a quotvital forcequot present within the broth and that life had arisen spontaneously In 1766 Lazzaro Spallanzani a priest by profession but scientist at heart repeated Needham s experiments Spallanzani boiled his broth longer and sealed his asks with glass After several days the asks were opened and were found to contain no living organisms Needham and others discredited Spallanzani39s work because they said his prolonged boiling had destroyed the quotvital forcequot within the broth and because no air could get in The discovery of oxygen and its importance to life had occurred at about the same time Thus although Spallanzani had actually proven that microorganisms did not arise spontaneously from nonliving materials he was not credited for his work at the time During the 1830s Theodor Schwann and Franz Schultz both German scientists conducted experiments to disprove abiogenesis They allowed boiled broth to come into contact with air that was either heated or passed through solutions of toxic chemicals No microscopic organisms grew in their broth Again the quotvitalistsquot those in favor of spontaneous generation discredited this work because they said the drastic treatment of the air had rendered it inactive About this same time another controversy had developed over the cause of fermentation Biologically inclined investigators including Schwann proposed that the products of fermentation ethanol and carbon dioxide were made by microscopic life forms This idea was opposed by the leading chemists of the time who believed that fermentation was strictly a chemical reaction brought about by chemical entities they called ferments Louis Pasteur 1860s Louis Pasteur a young French chemist and physicist had been hired by French distillers to determine why the contents of their fermentation vats sometimes turned sour vinegar instead of brewing as expected ethanol Pasteur determined that microorganisms including bacteria and yeast fungi were present in the vats Over a period of time he was able to prove that fermentation was indeed the result of microbial activity By taking samples from various vats and transferring them to fresh juice samples he was able to show that each type of fermentation product was mediated by a specific type of microorganism Although they were not pure cultures the collections of organisms in Pasteur39s fermentation vats were predominantly of one type or another and he was able to identify them with a fair degree of accuracy Pasteur also developed a process that could be used to greatly reduce the number of unwanted microorganisms in juice It involved heating the juice brie y to a speci c temperature and thereby killing most of the cells present Can you guess what this process is called Pasteurization Despite mounting evidence to the contrary the proponents of abiogenesis continued to argue their cause and to publish their evidence in support of spontaneous generation Pasteur was irritated by the seemingly endless controversy and set out to settle the question quotonceandforallquot He reported the results of his experiments in 1864 and is usually credited with disproving the abiogenesis of microorganisms By passing air through gun cotton Pasteur was able to show that microorganisms were abundant in air they had been collected and observed on the cotton When placed into flasks of broth these microorganisms grew readily Pasteur also constructed quotgoose neckedquot asks in which he could boil nutrient broths but which by their shape prevented the entrance of microorganisms from air Though these were left open to whatever quotvital forcesquot might be present in air no organisms grew Fortunately Pasteur39s broths contained no endospore forming bacteria since endospores are resistant to boiling and had they been present would have grown Though Pasteur39s work was not universally accepted he had many supporters One of these was an English physicist by the name of John Tyndall Tyndall set up an elaborate box containing only clean filtered air and showed that broths exposed to this clean air did not grow microorganisms Tyndall also discovered that some microorganisms were very resistant to being killed by boiling ie those that produced heat resistant endospores This helped to explain the varied results obtained by other investigators Tyndall found that by alternately boiling and cooling his broths over a period of three days he could eliminate the sporeforming organisms This process is called tyndallization Though many investigators worked to disprove the theory of abiogenesis at the microscopic level it is Pasteur who usually receives credit for finally laying the theory to rest Once this was accomplished the supernatural mysterious or magical aspects of microorganisms were explained away and Microbiology could be recognized as a true science Something to consider Why is the theory of abiogenesis not compatible with science Germ Theory of Disease Even after microorganisms were observed and found to play an important role in fermentation it was a number of years before people recognized their involvement in disease processes Some of the earliest physicians including Hypocrites believed that people could transmit disease from one to another but they did not understand how Around 1546 Girolamo Fracastoro an Italian physician recorded his belief that disease was due to organisms too small to be seen with the naked eye This was referred to as the contagion theory but since Fracastoro had no real proof his writings were largely ignored Prior to Microbiology people generally associated disease with natural phenomena such as earthquakes oods or exposure to bad weather Disease was also attributed to mysterious or supernatural causes Many religious leaders encouraged the belief that disease resulted from disobedience to God People stricken by illness and death were undoubtedly being punished for their evil deeds The threat of such punishment was useful for controlling people Since people were unaware of disease causing microbes and their manner of transmission practices we take for granted today to prevent infection and contamination did not occur to people Around 1840 there was a turning point in surgery due to the advent of anesthesia Prior to that time people undergoing surgery often died of shock unless the surgeon was quick The most successful surgeons therefore were those who were fastest at their work With the advent of anesthesia surgeons could work at a slower pace and their patients did not suffer from shock Unfortunately however longer exposure to the microbes associated with the surgeon39s hands instruments and the surrounding air resulted in more wound infections Physicians did not wash their hands or instruments between patients and most surgery was conducted in open rooms containing large numbers of people Patients no longer died of shock but many died of disease Around 45 of those undergoing surgical procedures died as a result of the associated wound infections Joseph Lister 1867 During the 1860s Joseph Lister an English surgeon reasoned that surgical infection sepsis might be caused by microorganisms Sepsis The condition resulting from the presence of pathogenic microbes or their products in blood or tissues Lister devised methods to prevent microbes from entering the wounds of his patients His procedures came to be known as antiseptic against sepsis surgery and included hand washing sterilizing instruments and dressing wounds with carbolic acid phenol Lister was well aware of microorganisms and is credited with developing techniques to obtain and maintain the first pure bacterial cultures Though he did not publish proof that microbes were responsible for disease he firmly believed they were About this same time 1840s a physician by the name of Ignaz Philip Semmelweis began using antiseptic procedures to prevent quotchildbirthquot or puerperal fever a serious and often fatal disease associated with infection contracted during delivery Semmelweis also strongly discouraged doctors involved in conducting autopsies and teaching anatomy in the basement from practicing their surgical skills on patients upstairs without first washing their hands and instruments The techniques of Lister and Seminelweis were initially scoffed at by some but as they were shown to greatly reduce infection and fatality they were recognized as major improvements to the previously accepted procedures These techniques also provided indirect evidence for the connection between microorganisms and disease The rst microorganisms actually shown to be pathogenic were fungi and protozoa These were found to be infecting silk worms so were impacting an important industry in Europe at the time around 1865 Robert Koch 1876 Direct evidence demonstrating that bacteria were diseasecausing agents etiological agents was provided by Robert Koch a German physician in 1867 Koch was working with a disease of sheep and cattle called anthrax and determined the causative agent to be a bacterium he called Bacillus anthracis Koch established a sequence of experimental steps that could be used to demonstrate beyond a doubt that a specific type of microorganism was responsible for a specific disease These came to be known as Koch39s postulates and are still in use today Koch39s Postulates 1 The suspect causative agent must be found in every case of the disease Koch took samples from hundreds of animals over years of investigation to be certain of his conclusions 2 The specific type of microbe must be isolated from the infected individual and grown in a culture containing no other forms pure culture 3 Upon inoculation into a normal healthy susceptible animal a pure culture of the microbial agent must produce the disease 4 The same type of microbe must be recovered again from the experimentally infected host Fortunately for Koch he was working with a relatively large and easily cultured type of microorganism His postulates are applicable only if the microorganisms associated with a particular disease can be isolated and grown in an artificial environment and for some types of microbes this is much more difficult Because of Koch39s work the etiological agents for many important human diseases were identi ed in rapid succession between the years of 1876 and 1898 By 1900 the microorganisms responsible for major human diseases including cholera diphtheria leprosy plague tetanus tuberculosis and typhoid had been identified The period of years between 1857 and 1914 is sometimes referred to as the Golden Age of Microbiology because rapid advancements and discoveries made during this period led to the establishment of microbiology as a science During their search for disease causing agents Koch and other microbiologists made important contributions to the techniques and materials used in the culture of microorganisms Some of these important developments involved the following people Richard J Petri developed the Petri dish in which microbial cultures could be grown and manipulated Fanny Hesse developed the use of agar as a solidifying agent for microbiological media Hans Christian Gram developed the Gram stain a stain technique that could be used to separate two major groups of disease causing bacteria Immunization Using microorganisms in disease prevention ln science many important discoveries are made accidentally and such was the case with Pasteur39s discovery of imlnunization In 1880 Louis Pasteur had isolated the bacteria responsible for causing chicken cholera organisms similar to the Vibrio choleme causing cholera in hulnans He later arranged for a public demonstration of Koch39s postulates and inoculated a nulnber of animals with the pure culture he had prepared Much to his dismay the animals did not develop disease symptoms but remained perfectly healthy Upon reviewing his records Pasteur found that the experimental animals had been inoculated with a culture several weeks old Pasteur reasoned that this old culture would be weakened attenuated and might therefore be unable to cause disease He arranged to repeat the demonstration and this time inoculated the subject animals with a fresh culture Fortunately he also chose to inoculate a new group of animals with the saIne culture The original animals again did not develop disease symptoms but the newly inoculated animals did As expected they all developed cholera and died Pasteur knew that the experimental animals had all been inoculated with the same type of disease causing bacteria Since they all caIne from a similar source he suspected that exposure to the attenuated culture had somehow made the first ones resistant to the disease He repeated the experiments and eventually concluded that this was indeed the case Bacteria that were killed or attenuated could be used to prevent disease Pasteur called his attenuated cultures vaccines and thus gave credit to an earlier investigator named Edward Jenner In 1796 Edward Jenner a British Physician reported the use of material scraped from the skin of an individual infected with cowpox to immunize a child against smallpox Jenner had noticed that dairymaids young women responsible for milking cows frequently contracted cowpox a relatively mild disease but were resistant to smallpox Since both of these diseases are caused by viruses there was no way for Jenner to see the disease causing agents but his method was successful He called his technique vaccination vacca cow The Magic Bullet By the early 1900s physicians knew that microorganisms could cause disease and under certain circulnstances could be used to prevent disease but they did not know how to cure disease Many strange and sometimes brutal practices had been used in attempts to cure disease but most were useless and some were dangerous for example the ingestion of precious metals gold and silver What was needed was a substance that could be taken into the body and would somehow seek out and kill the pathogenic microorganisms without harming the patient ie a quotmagic bulletquot A German physician by the name of Paul Ehrlich searched for a magic bullet and in around 1910 developed the first effective cure for a bacterial disease The drug he developed was called salvarsan and was an arsenic compound that was effective against syphilis A short time later 1928 Alexander Fleming a Scottish physician discovered penicillin He had noticed that a mold growing on one of his culture plates inhibited the growth of bacteria there and eventually isolated the substance responsible Penicillin was among the first antibiotics to be used in the treatment of disease Although Salvarsan was a synthetic compound and penicillin is produced by mold many compounds now used to treat disease in hulnans and other animals are made by bacteria Thus bacteria play a critical role in health and disease cause prevention and cure During the 20th century microbiology has expanded and increased in importance Immunology virology and molecular genetics recombinant DNA technology have arisen as branches of microbiology New discoveries in microbiology may lead to better methods for food and fuel production as well as environmental remediation that will become more and more critical as the hulnan population continues to expand Or perhaps microbes will eventually force hulnans to live in balance with the natural world Epidemiology quickie version Epidemiology Epidemiology is the quantitative study of the occurrence of disease and factors that in uence disease frequency and distribution It involves data collection and analysis with the goal of disease prevention Within the US data collection analysis and distribution is coordinated through the Centers for Disease Control and prevention CDC the headquarters of which are located in Atlanta Georgia World wide epidemiological efforts involve the World Health Organization WHO centered in Geneva Switzerland Disease categories as they relate to epidemiology include the following 1 Endemic An endemic disease is one that is always present within a population ie one involving relatively few cases but occurring all the time The common cold is endemic to most human populations plague is endemic to rodent populations in California and cholera is endemic to some regions of Southeast Asia 2 Sporadic A disease that occurs in small localized unpredictable outbreaks is categorized as a sporadic disease Legionnaires disease associated with water misters fountains etc and malaria in some places would qualify as sporadic diseases 3 Epidemic When the number of cases of a disease is significantly above the expected background level the disease is considered to be an epidemic 4 Pandemic When disease is spreading to involve people in multiple nations on more than one continent it is considered to be a pandemic Factors influencing the prevalence of disease The primary factors influencing the prevalence of disease are the reservoirs involved and the mode of transmission possible Reservoirs The term reservoir when applied to epidemiology refers to the total of all potential sources for a disease causing agent Reservoirs can be categorized as non living or living as described below Nonliving reservoirs include 1 Water Water contains variable nutrient levels some microbes can reproduce in water others cannot 2 Air Microbes don39t grow but can stay alive in air Air can pick up microbes from soil water or from living organisms droplet nuclei packets of microbes in sneeze or cough expelled material 3 Soil Soil is an excellent reservoir as it is the natural habitat for many types of bacteria Fungi protozoa and other types of microbes live there also 4 Food Food materials contain lots of nutrient microbes reproduce readily if foods are maintained at moderate temperatures 5 Fornites Fomites are small articles such as utensils bedding clothing money etc Bacteria do not typically reproduce on fomites but can be carried about on them Vehicles The term vehicle when applied to epidemiology refers to non living factors involved in disease transmission Reservoirs such as water air food and fomites can also be vehicles because they carry microorganisms sometimes considerable distances and can play a role in disease transmission Living reservoirs include 1 Humans Some diseases such as small pox and measles are restricted to the human population These could be eliminated if the entire population could be immunized Smallpox was declared eradicated in 1979 but is now making a come back as an agent of germ warfare Humans may be reservoirs but show no disease symptoms Typhoid Mary Mary Malone worked in food preparation and carried the Salmonella responsible for causing typhoid She did not show symptoms and refused to believe that she was a carrier so was ultimately incarcerated to avoid disease transmission N Non human animals manunals birds reptiles Many types of diseases occur primarily in non human animals but can be transmitted to humans so anilnals are i1nportant reservoirs Zoonoses Diseases usually associated with non human animals but can be transmitted to humans Plaque Lyme disease Equine encephalitis Rabies etc are examples of zoonoses S Arthropods lnsects mites etc Arthropods play a duel role as reservoirs and vectors Vectors are living organisms usually arthropods involved in disease transmission Fleas lice ticks and mosquitoes are vectors but so are rabid dogs and pigs with trichinosis The 2 1 Factor in uencing prevalence is mode of transmission or how the disease is transmitted from one host to the next Transmission may be either direct or indirect Direct Transmission occurs between a susceptible host and a living reservoir Syphilis is transmitted directly from one person to the next but plague and rabies are also directly transmitted If a person acquires trichinosis from eating raw pork that is also considered to be direct transmission Indirect Transmission when a susceptible host contacts a vehicle previously in contact with a reservoir Stepping on a rusty nail falling into contaminated water etc would be examples of indirect transmission The prilnary factor in uencing mode of transmission for a particular pathogen is how sensitive the pathogen is to exposure eg to heat cold drying UV radiation etc Factors influencing the severity of epidemics How bad will they be Some of the most important factors in uencing the severity of epidemics include 1 N S 4 E quot 0 Acquired immunity Host resistance due to acquired immunity ie the population has been immunized or exposed before can significantly in uence the severity of an epidemic Genetic background Genetic background may give some people an advantage over others For example people with sickle cell anemia a genetic disorder are more resistant to malaria than are most others Different people have different levels of or potential ability to develop immunity General state of the population The age nutritional status pre existing disease load etc can in uence susceptibility to disease Intensity of exposure Intensity of exposure can vary considerably both in time and space People using water from a well contaminated during a ood are very likely to encounter a new pathogen and become ill People using similar water on a regular basis may have already developed resistance to the pathogens present then a person just stopping by for a glass of lemonade is more likely to become infected Cultural practices or habits of the people People using the same river for drinking washing bathing and carrying excrement are more likely to experience epidemics than those using treated water for drinking washing and bathing People randomly tossing disposable diapers are seriously counterproductive to maintaining sanitation in this country Virulence of pathogen Bacteria vary significantly in terms of their ability to cause disease symptoms and this can also influence the severity of epidemics Control of Epidemics Since disease control and prevention is the goal of epidemiology it is important to consider the best methods for controlling disease these include the following 1 Increase host resistance If possible provide immunization if not education can be helpful On the other hand people are surprisingly resistant to education How easy would it be to convince people they should stop using disposable diapers Decrease exposure to reservoirs and or vectors Within the United States many reservoirs have been eliminated or significantly reduced and programs are maintained to control vectors Hear again education is potentially useful Isolate or segregate infected individuals It is common practice to keep sick children home from school until they enter college and to segregate patients in clinical settings Never the less segregation has negative connotations and often meets with opposition In order to determine how well epidemics are being controlled or prevented it is often useful to know the following Morbidity rate The number of cases of a particular disease per unit population and time period Mortality rate The number of deaths attributed to a specific disease per unit population and time period RNA and Protein Synthesis Considerable evidence suggests that RNA molecules evolved prior to DNA molecules and proteins and that many processes now involving DNA and proteins were previously accomplished by RNA alone Many RNA molecules have enzymatic activity and like DNA contain genetic information stored as nucleotide sequences In living cells encountered today RNA molecules are formed from DNA templates through transcription but this was not always so It is no wonder then that RNA molecules play such important roles within living cells Types of RNA molecules Although cellular RNA molecules are typically made via transcription using DNA as a template they are coded for by different genes and have a variety of different functions RNA molecules typically found in both prokaryotic and eukaryotic cells include 1 MessengerRNA mRNA MessengerRNA molecules are coded for by regions of DNA called structural genes and carry the nucleotide sequences determining what types of proteins the cell will make They carry this quotmessagequot from the nucleus or nucleoid to the ribosomes where proteins are made 2 RibosomalRNA rRNA RibosomalRNA molecules bind with proteins to form ribosomes the sites of protein synthesis In prokaryotic cells rRNA molecules occur as 235 165 and 55 segments The 235 and 55 segments occur within the 505 ribosomal subunits while the 165 segments occur within the 305 subunits RibosomalRNA is largely responsible for ribosome function except for the formation of peptide bonds and coordinates the attachment of tRNA anticodons with mRNA codons during translation Most of the RNA within a prokaryotic cell is ribosomal 3 TransferRNA tRNA TransferRNA molecules are relatively small being only 74 to 93 nucleotides in length Their function is to carry individual amino acid molecules to the ribosomes for protein synthesis Each tRNA molecule is folded upon itself to form a shape roughly similar to a cloverleaf The top or quotstemquot forms the amino acid binding site and typically has the base sequence CCA at the 339 end The side loops are called T and D loops and the bottom loop contains the anticodon region as set of three bases AminoacyltRNAsynthetase enzymes catalyze the formation of covalent bonds binding amino acids to tRNA molecules There are typically many different aininoacyltRNAsynthetase enzymes within each cell because each one catalyzes only the reaction binding a specific amino acid to the CCA sequence of each t RNA molecule The resulting complex is called aminoacyltRNA Eulltaryotic cells typically contain additional types of RNA not found in prokaryotic cells Though these are not directly involved in protein synthesis they are of considerable importance Some additional types of RNA include 1 Small or shortRNA sRNA SmallRNA molecules bind with proteins to form structures called spliceosomes and are involved in a process called post transcriptional modification ln eukaryotic cells nearly all RNA molecules undergo some modification following transcription so these smallRNA molecules play a vital role In the case of mRNA molecules posttranscriptional modification involves the removal of multiple sections called introns intervening regions from the original RNA transcript The remaining sections called exons expressed regions are then bond together to form a shorter mRNA molecule in some cases over half the original mRNA transcript is composed of introns Posttranscriptional modification also involves the application of a methylatedguanine quotcapquot which is attached to the 539 end before transcription is completed and a polyadenine quottailquot The splicing of exons following the removal of introns can occur in a variety of ways in some mRNA molecules and this increases the variety of proteins that can be generated N V MicroRNA miRNA MicroRNA molecules are very small usually only 21 bases in length They regulate the expression of genes by binding with mRNA molecules At this time researchers believe there are probably hundreds of genes coding for different miRNA molecules and that the potential for regulating cellular processes by means of miRNA is enormous 3 Small interferingRNA SiRNA Small interferingRNA molecules also called short interfering or silencingRNA are typically 2025 nucleotides in length and double stranded Like miRNA they play a significant role in regulating the expression of eukaryotic genes Translation Protein synthesis The term quottranslatequot means to give an equivalent in another language therefore when not applied to biological activities translation refers to the process of reproducing a written or spoken text in a different language while retaining the original meaning When applied to biological systems it has essentially the same meaning Translation is protein synthesis During translation information contained in mRNA molecules in the form of nucleotide sequences transcribed from small sections of DNA also nucleotide sequences is translated into information contained in polypeptides in the form of amino acid sequences The language change is from nucleotide sequence to amino acid sequence Some polypeptides are fully functional proteins while others undergo modification to become functional units Recall that proteins with quaternary structure contain multiple polypeptide chains Some background information When the structure of DNA was first determined through the efforts of Rosalind Franklin Maurice Wilkins James Watson and Francis Crick see Wikipedia DNA in the early 1950s researchers were uncertain how this relatively simple molecule could contain the information necessary to control the physiological processes within living cells It becaIne obvious that protein structure was dependent on the nucleotide sequences of DNA molecules but it was not i1nmediately apparent how this could be Proteins typically contain 20 different aInino acids and the primary structure of each polypeptide is determined by the sequence or arrangement of these monomers just as the sequence or arrangement of letters determines the informational content of words on a page Each DNA molecule and each mRNA molecule contains only four different nitrogenous bases so researchers knew individual bases translated separately could not determine amino acid sequences When quotreadquot in pairs groups of 2 the bases could be arranged to yield 16 different sets but this was still not enough Then researchers considered translating base sequences arranged in sets of three In this configuration they form 64 possible sets 3letter quotwordsquot which is more than enough to code for 20 different amino acids By synthesizing nucleotide sequences containing only one type of base ie polyadenine polycytosine polyguanine etc researchers were able to determine portions of the quotgenetic codequot the sets of bases coding for different types of amino acid polyadenine sequences yielded polypeptides containing only lysine polycytosine sequences yielded polypeptides containing only proline polyguanine sequences yielded polypeptides containing only glycine etc Eventually the entire genetic code was revealed The Genetic Code The genetic code typically presented in biology textbooks and in the lecture syllabus is made up of nucleotides bases arranged in groups of three These groups are called codons and occur within messengerRNA molecules and DNA molecules Since nucleotides arranged in sets of three form 64 different combinations 64 3letter quotwordsquot and because proteins typically contain only 20 different amino acids there is considerable overlap Most of the amino acids incorporated into proteins are quotcoded forquot by multiple different codons so the genetic code contains redundancy Some references refer to the code as being degenerate but the overlap has benefit because it reduces the potentially lethal effects of certain types of mutation as will be explained later In addition to the codons translated into amino acids the genetic code contains four codons with alternate functions The codon quotAUGquot codes for the amino acid methionine but also serves as a start or initiator codon The codons quotUAAquot quotUAGquot and quotUGAquot do not code for any amino acids and were initially referred to as nonsense codons these code for quotstopquot or termination of the growing polypeptide chain Named quotOcherquot quotAmberquot and quotUmberquot color names these three terminator codons play an essential role in protein synthesis Although similar in all living organisms the genetic code as presented in most texts is not universal Variations are common within mitochondria For example mitochondria found within animals and eukaryotic microbes use quotUGAquot as the code for tryptophan instead of stop most animal mitochondria use quotAUAquot as the code for methionine rather than isoleucine all mitochondria associated with vertebrate animals use quotAGAquot and quotAGGquot as chain terminators and yeast mitochondria use quotCUA CUG CUC and CUUquot to code for threonine instead of leucine Only plant mitochondria use the universal code Beyond that researchers have now developed strains of E coli and Saccharomyces cerevisiae that use specific redundant codons as the codes for amino acids not normally incorporated into proteins The code apparently has considerable versatility Translation The translation process occurs in association with ribosomes and involves the three types of RNA common to all types of cells ie mRNA rRNA and tRNA Since mRNA molecules carry the genetic message they must interact directly with the ribosomes Translation can be broken into a series of stages as follows 1 Initiation Initially the small ribosomal subunit 305 in prokaryotes 405 in eukaryotes binds to mRNA at a site quotupstreamquot of the initiator codon Some bacterial mRNA molecules carry the nucleotide sequence quot539AGGAGGquot the ShineDalgarno sequence which binds to the complimentary sequence quot339UCCUCCAquot the antiShine Dalgarno sequence on the 165 ribosomalRNA portion of the ribosome In bacteria lacking a ShineDalgarno sequence the binding of mRNA to the small ribosomal subunit involves interaction between ribosomal proteins and purine rich regions of RNA upstream of the initiator In eukaryotes a sequence beginning with quotAquot or quotGquot followed by quotCCACCquot the Kozak concensus sequence serves as the ribosome binding site Once the binding of mRNA to the small ribosomal subunit has occurred a nu1nber of translation initiation factors IF bind to the ribosome along with a tRNA carrying the amino acid methionine In prokaryotes this is Nformylmethionine fmet rather than the methionine commonly incorporated into proteins Following the binding of this aminoacyltRNA molecule the second ribosomal subunit SOS in prokaryotes or 605 in eukaryotes binds to the mRNA Elongation The large ribosomal subunit has three tRNA binding sites designated as the Asite the Psite and the Esite The Asite is the amino acid binding site and binds with incoming tRNA molecules aminoacyltRNA ie those carrying individual amino acids to the ribosome The Psite is the polypeptide binding site and once translation has begun will bind with the peptidyltRNA molecule ie the one holding the growing polypeptide chain The Esite binds with the exitingtRNA ie the free t RNA molecule remaining after the amino acid it was carrying has been added to the growing peptide chain The binding of aminoacyltRNA molecules involves interaction between tRNA anti codons and mRNA codons Hydrogen bonds forming between the complimentary bases insure that each amino acid will be incorporated in the correct location A ribosomal enzyme called pepti dyl transferase catalyzes chemical reactions resulting in the formation of peptide bonds between adjacent amino acids Without this enzyme activity proteins cannot be made Peptidyl transferase activity apparently involves RNA molecules not ribosomal proteins Translation requires that the ribosomes travel along mRNA molecules from the 539 ends toward the 339ends As they do the codons associated with the Asite change and new aminoacyltRNA molecules can bind As peptide bonds are formed between adjacent amino acids the growing peptide chain is transferred from the tRNA in the Psite to the one occupying the Asite explaining the name peptidyl transferase The tRNA released by the transfer of the peptide chain occupies the Esite until the ribosome moves and is then released These quotexitingquot tRNA molecules can again interact with aminoacyltRNA synthase enzymes pick up new amino acids and return to the ribosome ie they can be used over and over again 3 Termination Translation is terminated when the ribosome reaches a stop or terminator codon In both prokaryotic and eukaryotic cells the process involves the interaction of two or more proteins called Chain release factors When a stop codon is recognized the finished polypeptide chain is released from the last tRNA ie the covalent bond attaching the last amino acid added to its corresponding tRNA molecule is hydrolyzed Following this reaction the tRNA is released from the ribosome and the two ribosomal subunits separate releasing the mRNA Since messengerRNA molecules have a relatively short life expectancy about 2 minutes in some eukaryotic cells bacteria often increase translation efficiency by binding multiple ribosomes to the same mRNA during translation A group of ribosomes bound to and translating the same mRNA is called a polyribosome or polysome 0 ln eukaryotic cells mRNA molecules typically carry the information copied from individual genes transcription is monocistronic but in prokaryotic cells mRNA molecules often carry the information copied from multiple genes transcription is polycistronic Polycistronic mRNA molecules carry information that is translated into multiple polypeptides In E coli the m RNA copied from the tryptophan biosynthesis operon contains about 7000bp and is translated into five different enzymes Each protein is coded for by a section of mRNA having its own start and stop signals ie initiation and termination sequences For a fun and simple review of the translation process take a look at this site http wwwphschoolcom science biologyplace biocoach translation termhtml Immunization and Hypersensitivity As explained earlier Louis Pasteur39s discovery of immunization was serendipitous ie occurred through chance It involved knowledge provided by other individuals Robert Koch Edward Jenner Joseph Lister etc during a period when immune system physiology and the antigenic properties of microorganisms were not yet recognized Fortunately current information available on immune system function has greatly improved our understanding of how immunization works and why it provides protection Adaptive immunity involves the production of memory cells both B cells and T cells and these allow the body to respond quickly when challenged by a previously encountered pathogen The effectiveness of immunization is dependent on proper immune function and the body39s ability to launch an anamnestic response Immunization Immunization is the process of conferring specific immunity by artificial means As described earlier adaptive immunity acquired through artificial means may be either active or passive depending on if or not the body is induced to produce its own immune cells and substances Therefore immunization may involve one or more of the following I Vaccination Vaccination is the process of inducing active immunity by introducing killed or attenuated microorganisms components of these or their products into the body The term vaccine as initially used by Louis Pasteur applied to a substance containing killed or attenuated microorganism Attenuated forms were weakened in some manner so that although they could initiate an immune response they did not cause disease or at least not in most individuals Currently vaccines may contain genetically modified microorganisms or components of microorganisms Vaccines containing microbial parts rather than whole organisms are called subunit vaccines Since the toxic by products of microorganisms are often responsible for causing disease symptoms vaccination can often be accomplished by inducing immunity against these substances Toxoids are detoxified microbial toxins and are often included in combined vaccines such as the DPT vaccine In this case toxoids induce immunity to the toxins of C orynebacterium diphtheriae and C lostridium tetam39 while a vaccine provides protection against Bordetella pertussis the causative agent of whooping cough In either case vaccination will provide protection only if the individual receiving it has a fully functional immune system capable of producing B cells antibodies T cells and lymphokines N Administration of immune serum Immune serum or antiserum is a substance containing a high antibody titer and capable of providing protection against a specific pathogen Antibody in the isotype IgG gamma globulin is most commonly used but other types could be Historically inunune serum was produced by inoculating various types of anilnals horses goats pigs etc with specific antigens allowing them to produce antibodies against these antigens and then harvesting their serum blood plasma with clotting factors removed Unfortunately anti serum produced by non human anilnals will trigger potentially deadly hypersensitivity reactions if used more than once on the same individual To avoid the potential daInage resulting from this practice anti serum is now made using cells in tissue culture or monoclonal antibody techniques This technique involves fusing B cell myoloma cells cancer cells with indefinite life spans with plasma cells capable of secreting a specific type of antibody Once such a cell line is established it can theoretically continue producing human antibody indefinitely When a patient is receiving i1nmune serum his her body is not required to produce any i1nmune cells or substances so immunity is passively acquired Trends in Immunization Although immunization has become a topic of some controversy of late for a variety of reasons it remains the method of choice for preventing disease and avoiding epidemics Immunization is considered appropriate for the mass of a population ie everyone present only under specific circumstances as indicated below 1 The disease in question is one that will spread rapidly and affect a large number of people most of the population 2 The risk of contracting the disease outweighs the risk of being inununized ie the consequences of becoming infected with the disease causing agent are likely to be life threatening or otherwise damaging Currently recomInendations provided through the Centers for Disease Control and Prevention CDC indicate that all persons living in this country should be immunized against measles mumps rubella German measles diphtheria tetanus whooping cough B ordetella pertussis poliomyelitis hepatitis A and B meningitis and chicken pox Adult individuals are also encouraged to be i1nmunized against in uenza and pneumococcal pneumonia The Hib vaccine providing i1nmunity to Haemophilous in uenzae strain b bacteria causing meningitis and pneumonia is also recommended for children Since one of the factors determining if or not inununization is appropriate is weighing the risk of contracting a disease against the risk of receiving inununization there must be some risks involved or at least the potential for risks Not surprisingly there are potential risks associated with i1nmunization as indicated below a Vaccine failure Vaccine failure may leave individuals unprotected and potentially expose them to virulent pathogens they would otherwise seek to avoid Vaccine failure is uncommon in this country but can occur if vaccines toxoids serum saInples etc are i1nproperly stored e g not maintained at the proper temperature or have been stored too long ie beyond a given expiration date b Infection Vaccines containing attenuated microorganisms can sometimes cause disease when used under certain circumstances Live viral vaccines can sometimes result in fetal infection if the virions involved can cross the placenta Although the live viral vaccine used to i1nmunize adult individuals against rubella does not harm adults it can cross the placenta and cause severe damage to a developing fetus Live vaccines may also cause infection in imInunocompromised individuals living with those receiving vaccines Contarninati on Although contamination is rarely a problem in facilities where needles are used only once and where vaccines toxoids serum samples etc are provided in single use containers contamination is a serious problem in some regions of the world Reusing syringes and containers without means for sterilization can result in serious contamination problems Toxicity Vaccines made with Gram negative bacteria can cause toxic side effects because the lipopolysaccharide LPS associated with the outer membrane of the Gram negative cell wall is highly toxic to humans and other manunals Preservatives added to vaccines e g mercury compounds can also have toxic side effects e Hypersensitivity Hypersensitivity or allergic reactions can be initiated by ilnmunizations however this is not the most common cause f1 9 Various individuals and organizations suggest that ilnmunization is unwarranted in this country and that the risks far outweigh the benefits These individuals argue that the risk of contracting diphtheria poliomyelitis whooping cough measles and other potentially life threatening diseases is low in this country and that imInunization poses a far greater threat This is only true because most of the population is already ilnmunized and the disease causing agents are unlikely to be encountered If the majority of individuals chose to refuse ilnmunization the risk of contracting disease would increase significantly Hypersensitivity Hypersensitivity is listed above as one of the risks associated with imInunization but is more commonly initiated by other factors Like immunization hypersensitivity involves the ilnmune system so familiarity with imInune system physiology is essential to understanding it Hypersensitivity can be defined as an abnormal physiological state during which an ilnmune reaction causes tissue damage or malfunction within the body Commonly referred to as allergic reactions hypersensitivity reactions can involve either humoral or cellmediated imInune responses and can be categorized as ilnmediate or delayed as indicated below 1 Immediate hypersensitivity reactions involve B lymphocytes and antibodies and typically occur within minutes of exposure to a specific antigen or allergen There are at least three different types of immediate hypersentivity reactions sometimes categorized as type I type II and type III involving different types of antibodies and other ilnmune substances N Delayed hypersensitivity reactions involve T lymphocytes and cytokines and occur 24 48 hours or more after exposure to a specific antigen or allergen Sometjlnes categorized as type IV delayed hypersensitivity reactions involve Killer T cells and can result in severe tissue daInage A somewhat simplified explanation for each type of hypersensitivity reaction is presented below Note that allergic reactions involving the same types of antibodies are sometjlnes given different naInes based on whether they are localized ie restricted to specific areas or occur body wide Type 1 hypersensitivity Type 1 hypersensitivity reactions are the most common and involve antibodies in the isotype IgE and mast cells Mast cells mastocytes are conunon throughout the body in association with loose connective tissues and near blood vessels They are similar to basophils granular leukocytes containing granules rich in histamine and heparin They also have receptors with high affinity for lgE so tend to bind irreversibly with these antibodies Type 1 hypersensitivity reactions can be localized atopy or involve most of the body anaphylaxis and vary considerably in their severity a Atopy or atopic allergy Type 1 hypersensitivity reactions involving specific regions of the body eg the eyes nasal passages patches of skin etc are examples of atopy or atopic allergy During such reactions lgE binding with antigens at the surfaces of mast cells cause them to degranulate ie release their granules Heparin is an anticoagulant while histamine causes relaxation of precapillary sphincters increased blood ow into capillaries and increased permeability of blood vessel walls Localized increases in blood ow and the movement of uid into tissue spaces putting pressure on receptors cause symptoms common to type 1 hypersensitivity reactions eg redness swelling itching and sometilnes pain The red watery eyes runny nose and itching sensation conunonly referred to as hay fever is an exaInple of atopic allergy often stimulated by exposure to pollen grains fungus spores dust mites cat hair and other allergens Localized redness swelling and itching of the skin following a mosquito bite or due to hives is another exaInple b Anaphylaxis Type 1 hypersensitivity reactions involving the release of histamine and other in anunatory substances body wide are called anaphylactic reactions or anaphylaxis In this case the binding of lgE with antigen at the surfaces of mast cells occurs throughout the body due to the presence of an allergen within the circulation The human body contains around 70000 miles of blood vessels most of which are capillaries and during an anaphylactic reaction blood ow into capillary beds is increased body wide Since under normal circumstances most capillary beds are closed the pooling of blood in capillaries and venules has seriously detrimental effects Without sufficient blood returning to the heart there is insufficient outflow an body begins to experience circulatory shock due to low venous return Tissues become oxygen deficient and metabolic wastes begin to accumulate causing additional disturbances in blood ow If not corrected the situation can rapidly progress to circulatory failure and death Type 1 hypersensitivity reactions range in severity and are not necessarily restricted to the examples described above Asthma a chronic disease of the respiratory system can involve type 1 hypersensitivity and anaphylaxis typically involves respiratory as well as circulatory symptoms Type 2 hypersensitivity Type 2 hypersensitivity reactions involve antibodies in the isotype IgG sometilnes lgM and the activation of complement factors on the surfaces of cells Hemolytic disease of the newborn HDN sometilnes called a cytotoxic response is one exaInple a U During a cytotoxic response a mother carrying a fetus with Rh positive blood produces anti Rh antibodies Antibodies in the isotype lgG cross the placenta lgM antibodies cannot because they are too large and bind with Rh epitopes on the surfaces of the fetal RBCs The binding of antibody and antigen causes complement factors to be activated and the resulting complement cascade causes destruction of the fetal RBCs Hemolytic disease of the newborn varies in severity but sometimes results in fetal death Since people rarely check blood type before choosing a mate and because Rhesus factors are abundant within the population it is not unconunon for women with Rh negative blood to carry offspring with Rh positive blood Blood exchange is generally minimal during pregnancy but occurs often during delivery so it is the second or subsequent Rh positive fetus that is most likely to be daInaged by a cytotoxic response Women with Rh negative blood and Rh positive mates can avoid the problems associated with type 2 hypersensitivity by receiving RhoGAM or RhoD i1nmune globulin The anti Rh antibodies lgG present in this substance bind with any fetal RBCs entering the mother39s circulation and prevent the activation of B lymphocytes capable of binding with these Thus the mother is prevented from forming anti Rh antibodies of her own Type 2 hypersensitivity is also involved when antibodies bind with transfused RBCs as can occur if transfusion donors and recipients are incorrectly matched A patient with type O positive blood cannot receive RBCs from a donor with type A positive blood because he she will be carrying anti A antibodies potentially both lgG and lgM that can bind with epitopes on the donated RBCs and activate complement factors These will destroy the donated cells Type 3 hypersensitivity Type 3 hypersensitivity reactions sometilnes referred to as immune complex disease involve antibodies in the isotypes lgG and lgM complexing with antigens and the activation of complement factors resulting in cellular daInage Like type 1 reactions these can be either localized or can occur body wide SD U Serum sickness is a body wide reaction involving lgG and lgM binding with soluble antigens and forming complexes throughout the circulation These complexes are eventually deposited in tissues where they activate complement factors and cause tissue damage Serum sickness was a common occurrence when anti serum from animal sources was used to prevent disease People could be given horse antibodies to prevent tetanus but only once because the second time they received such antibodies they could experience potentially deadly serum sickness An Arthus reaction is a localized type 3 hypersensitivity reaction caused by the binding of circulating antibody with antigens introduced into a localized area The complexing of antibodies with antigen causes activation of complement factors and can result in cell damage and in anunation Arthus reactions are sometilnes stimulated by tetanus and diphtheria vaccinations Type 4 hypersensitivity Type 4 hypersensitivity also called delayed hypersensitivity reactions involve the cellmediated portion of the inunune system and often occur 48 hours or more after exposure to an antigen Allergic reactions to poison oak ivy etc and graft rejection fall into this category a Contact dermatitis can result from exposure to allergenic plants such as poison oak and poison ivy and is one example of delayed hypersensitivity In this case oil from the plant becomes associated with skin cells and they are then recognized as antigens by T lymphocytes Killer Tcells can respond to antigen in combination with MHC class I membrane markers which skin cells carry and can begin to attack and kill epithelial cells with perforin and granzymes The extent of tissue damage is variable but in severe cases may involve epithelial surfaces within the mouth and respiratory tract as well as external surfaces U Transplant rejection can also involve T cells and delayed hypersensitivity In the case of acute rejection Tcells recognize the transplanted tissue or organ as foreign and begin to destroy the foreign cells just as they would eukaryotic pathogens Transplant rejection is reduced by matching the MHC proteins of donors and recipients and by treating recipients with iinmunosuppressant drugs Numerous other conditions including rheumatoid arthritis glomerulonephritis endocarditis rheumatic fever scarlet fever and damage associated with tuberculosis and leprosy are due to hypersensitivity reactions Though immune responses are for the most part protective in nature they can sometimes cause considerable damage Fermentation and Cellular Respiration Chemoheterotrophs such as animals fungi protozoa and many bacteria use preformed organic compounds as their source of energy Organic compounds carry potential energy in the covalent bonds holding their atoms together When these bonds are broken the energy released can be used to make ATP through phosphorylation reactions Chemoheterotrophs can use either substrate level or oxidative phosphorylation or sometimes both depending on the type of metabolism they have Since glucose is a compound abundant in the environment most polysaccharides are glucose polymers many types of organisms carry enzymes involved in glucose catabolism Some types of cells use glucose exclusively ie they are not capable of utilizing any other nutrient as a source of energy or carbon It is not surprising then that the enzymes involved in glucose catabolism are constitutive in most types of cells Glycolysis The term glycolysis glyco carbohydrate lysis to split means breakdown of sweets but glycolysis as presented here refers to a specific metabolic pathway or series of chemical reactions catalyzed by enzymes also known as the Embden Meyerhof pathway Glycolysis may be presented diagrammatically as shown below 2 ADP Pi 2 NAD l Glucose molecule 2 pyruvic acid molecules 2 ATP net 2 NADH H Glycolysis is the catabolism of glucose into two molecules of pyruvic acid with the associated production of two molecules of ATP net yield and the reduction of two molecules of NAD to NADH H The ATP made during glycolysis is the result of substratelevel phosphorylation ie the energy required comes directly from the breaking of covalent bonds within glucose molecules As mentioned above the enzymes associated with glycolysis are often constitutive and are found in the cytoplasm of both eukaryotic and prokaryotic cells Although glycolysis is a catabolic pathway and yields energy in the form of ATP it also requires energy for initiation activation energy Each glucose molecule entering the glycolysis pathway must be phosphorylated ie must have a phosphate group bound to it ATP provides both the phosphate group and the energy required to bind it to glucose Enzymes catalyzing chemical reactions resulting in the transfer of phosphate groups between organic compounds are called kinase enzymes recall kinesis movement The enzyme catalyzing the reaction binding a phosphate group to glucose at the beginning of glycolysis is called phosphohexokinase The reaction can be diagrainmed as shown ATP Glucose Glucose 6 phosphate ADP The glucose molecule now has a phosphate group bound to its number 6 carbon atom and one ATP molecule has been converted to ADP adenosine diphosphate The next reaction in the pathway requires an isomerase enzyme An isomerase catalyzes a reaction converting a molecule in this case glucose into its chemical isomer in this case fructose Glucose and fructose are isomers they have the same molecular formula C6H1206 and have the same types and number of bonds The enzyme phosphohexoisomerase catalyzes the following reaction Glucose 6 phosphate gt Fructose 6 phosphate Since no energy is required for or released during this reaction ATP is not involved During the next reaction a second phosphate group is added to the sugar molecule Once again ATP provides the energy and phosphate and a kinase enzyme is involved The reaction catalyzed by phosphofructokinase can be diagramined as shown ATP Fructose 6 phosphate Fructose 16 diphosphate ADP The fructose molecule now has a phosphate group bound to each end ie one on the number 6 carbon and another on the number l carbon Following this step the fructose can be split into two three carbon molecules as shown Fructose 16 diphosphate Glyceraldehyde 3 phosphate 4 Dihydroxyacetone phosphate A new enzyme is required for this reaction but once again energy is not required nor released The double arrow between the two three carbon compounds shown G11 U 3913 r39 r39 andDthJ J r39 r39 indicatesthatthesetwo compounds are in equilibrium ie their concentrations will be maintained as equal by the activity of another isomerase enzyme triose phosphate isomerase Glyceraldehyde 3 phosphate G3P can also be called 3 phosphoglyceraldehyde PGAL or triose phosphate and is a 3 carbon compound with a phosphate group bound to its number 3 carbon Dihydroxyacetone phosphate DHAP has the same molecular formula C3H607P so is a chemical isomer of G3P During the next step in the glycolysis pathway 2 molecules of Glyceraldehyde 3 phosphate are phosphorylated and oxidized to form 2 molecules of bisphosphoglyceric acid The electrons and hydrogen protons removed from each Glyceraldehyde 3 phosphate are passed to NAD reducing it to NADH H The phosphate added during this reaction is provided by inorganic pyrophosphate PPi NAD Glyceraldehyde 3 phosphate PPi r bisphosphoglyceric acid NADH H Next another kinase enzyme catalyzes a reaction transferring one phosphate group from each bisphosphoglyceric acid molecule to ADP forming ATP and 3 phosphoglyceric acid ADP Bisphosphoglyceric acid 3 phosphoglyceric acid ATP The enzyme involved is called phosphoglycerate kinase and the ATP formed is the result of substrate level phosphorylation Since two molecules of bisphosphoglyceric acid were formed from each fructose 16 diphosphate catabolized two molecules of ATP are formed at this step Following this reaction the 3 phosphoglyceric acid is converted to 2 phosphoglyceric acid by a mutase enzyme and then to phosphoenolpyruvic acid by an enolase enzyme Note The glycolysis diagram provided in the lecture syllabus combines some of the reactions occurring between glyceraldehyde 3 phosphate and phosphoenolpyruvic acid Although the information provided here is considerably more accurate students are required to know only kinase and isomerase enzymes The last reaction in the glycolysis pathway involves the transfer of a phosphate group from each molecule of phosphoenolpyruvic acid to ADP yielding ATP and pyruvic acid ADP Phosphoenolpyruvic acid Pyruvic acid ATP The kinase enzyme involved here is called pyruvate kinase and once again the ATP formed is the result of substrate level phosphorylation Since two molecules of phosphoenolpyruvic acid are donating phosphate groups to ADP two molecules of ATP are formed Two molecules of ATP were required toward the beginning of the glycolysis pathway but four molecules of ATP are ultimately formed This means glycolysis has a net yield of two ATP molecules for each glucose molecule catabolized Since oxygen is not required glycolysis can occur under anaerobic conditions however it cannot continue unless it is linked to one or more additional reactions ie glycolysis cannot quotstand alonequot Why not Recall that living organisms can be divided into two categories based on their type of metabolism fermentative organisms and respiratoryoxidative organisms Fermentative organisms typically use some type of organic compound pyruvic acid in the case of eukaryotic cells as their final electron acceptor while respiratory oxidative organisms use some type of inorganic compound e g molecular oxygen In either case a final electron acceptor is required and this explains why glycolysis cannot continue unless linked to one or more additional reactions Glycolysis does not involve a final electron acceptor Without a final electron acceptor the NAD reduced to NADH H during glycolysis cannot be oxidized and without NAD glycolysis will stop Fermentation Fermentation Fermentation can be defined as the anaerobic decomposition of organic compounds usually carbohydrates involving an organic compound usually pyruvic acid as the final electron acceptor The fermentation of glucose to yield lactic acid can be diagramed as shown below 2ADP 2NAD4 2NAD Glucose 2 Pyruvic acids 2 Lactic acids 2ATP 2NADH H gt 2NADHH As explained above glycolysis is used to catabolize glucose into two pyruvic acid molecules but the pathway does not stop there Instead the pyruvic acids serve as final electron acceptors the two molecules of NADHH are oxidized to NAD and the two pyruvic acid molecules are converted into lactic acid molecules Homofermentative Organisms that yield lactic acid as the only end product of their fermentation processes are called homofermentative organisms homo same Bacteria such as Lactococcus lactis Leuconostoc mesenteroides and the Lactobacillus species associated with sauerkraut production Lactobacillus brevis and L plantarum are homofermentative Since lactic acid is the only fermentation product these organisms can make they are often referred to as lactic acid bacteria and are used commonly in food processing ie in the production of sauerkraut cheese sourdough bread and other sour avored foods Heterofermentative Organisms capable of producing a variety of different fermentation products are called heterofermentative organisms hetero different These organisms contain different enzymes and although they can still use pyruvic acid as a final electron acceptor they can convert it into multiple different compounds e g acetic acid butyric acid acetoin acetone acetaldehyde 2 3 butylene glycol ethanol carbon dioxide and hydrogen gas The Saccharomyces cerevisiae used to make wine and rootbeer and the Grain negative fermentative organisms used in Physiological Unknown No l are all heterofermentative Fermentation allows organisms to produce ATP under anaerobic conditions but it is not efficient in terms of capturing the energy potentially available in glucose molecules only about 2 of the energy available is captured During glycolysis only one covalent bond is broken and considerable energy remains in each three carbon pyruvic acid molecule formed This energy is typically lost as pyruvic acid is converted into various fermentation products and released from the cells Another disadvantage of fermentation is the production of potentially toxic waste Acids formed via fermentation can lower the pH of the environment and disrupt enzyme function and alcohols can coagulate cellular proteins Cellular Respiration Organisms capable of cellular respiration can capture more of the energy potentially available in glucose and release only carbon dioxide and water as waste materials Cellular respiration Cellular respiration is a multi step process allowing organisms to completely catabolize glucose into carbon dioxide lf molecular oxygen serves as the final electron acceptor water is also produced Some references represent cellular respiration with the following chemical formula C leo 602 6C02 6HZO Energy This indicates that glucose C6H1206 is reacting with molecular oxygen 02 to form carbon dioxide C02 and water H20 and that energy is being released Though handy and easily memorized this formula is not accurate Within living organisms glucose does not interact with oxygen oxygen is not converted to carbon dioxide and water is not always produced Some respiratory organisms e g Pseudomomts aeruginosa can use inorganic compounds other than oxygen as their final electron acceptors If they do this the formula shown above would not apply Cellular respiration as it occurs in many organisms involves three stages or three separate metabolic pathways these are glycolysis the Krebs cycle and the electron transport chain also called the respiratory chain Glycolysis as presented earlier can be represented diagrammatically as shown below 2 ADP Pi 2 NAD l Glucose molecule 2 pyruvic acid molecules 2 ATP net 2 NADH H Since respiratory organisms do not use pyruvic acid as a final electron acceptor alternative enzymes are employed and the fate of pyruvic acid is quite different Typically an enzyme complex called the pyruvate decarboxylase complex PDC removes a carboxyl group from each pyruvic acid molecule formed through glycolysis then binds the remaining 2 carbon acetyl group to coenzyme A forming acetylCoA a high energy compound During this process the coenzyme NAD is reduced to NADH H and carbon dioxide is released as a waste gas The overall process can be diagramined as shown below C02 NADH H A C Coenzyme A Pyruvic acid gt Acetyl CoA COOH a NAD y Note The pyruvate decarboxylase complex is composed of at least three different quaternary proteins with three different functions Though handy the diagram above is not entirely accurate Details of the process can be found at the following site http wwwbroolltscolecom chemistryd templates studentresources sharedresourc es animations pdc pdchtml The decarboxylation of pyruvic acid and formation of acetylCoA serves as an intermediate step linking glycolysis to the Krebs cycle Following this set of reactions the energy stored in acetyl CoA can be used to bind the 2 carbon acetyl group to oxaloacetic acid 4 carbons forming citric acid 6 carbons and beginning the Krebs cycle Acetyl CoA Oxaloacetic acid gt Citric acid Coenzyme A Citric acid is a ti icarboxylic acid ie a 6 carbon acid with three carboxyl groups in its structure Since the Krebs cycle begins with citric acid it is sometimes called the Citric acid cycle or Tricarboxylic acid cycle TCA cycle Although the chemical reactions associated with glycolysis occur within the cytoplasm of both eukaryotic and prokaryotic cells the decarboxylation of pyruvic acid and chemical reactions of the Krebs cycle do not or at least not usually The pyruvate decarboxylase complex PDC and enzymes associated with the Krebs cycle occur within the cytoplasm of prokaryotic cells but occur within the matrix of mitochondria in most eukaryotic cells with the exception of succinate dehydrogenase which is bound to the inner mitochondrial membrane Krebs Cycle Krebs cycle The Krebs cycle citric acid cycle or tricarboxylic acid cycle is a cyclic metabolic pathway allowing organisms to decarboxylate organic acids and release the potential energy stored within them Most of the energy released is captured in the form of reduced coenzymes NADH H and FADHZ however one molecule of ATP or GTP is also formed during each cycle The carboxyl groups removed from the organic acids are converted into carbon dioxide a waste gas The various reactions occurring during the Krebs cycle are illustrated in the crude diagram shown below Oxaloacetic acid 4C gt Citric acid 6C NADH H4r NAD Malic acid 4C lsocitric acid 6C NAD 02 NADHH H20 Fumaric acid 4C oc Ketoglutaric acid 5C FADH2 NAD FAD co2 Succinic acid 4C Succinyl CoA NADHH GTP GDP Pi In this diagram each arrow represents a chemical reaction being catalyzed by a specific type of enzyme Three of these enzymes require the coenzyme NAD as a helper and in each reaction the NAD is reduced to NADH H We can see here that coenzymes are less specific than are enzymes relative to the chemicals they interact with During one reaction the coenzyme FAD is reduced to FADHZ The reduced forms of these coenzymes have a higher energy potential than do the oxidized forms and this is of considerable significance The acids being decarboxylated during the Krebs cycle include isocitric acid and oc ketoglutaric acid Some of the energy released during decarboxylation is captured as GTP guanidine triphosphate which is equivalent to ATP but contains a different base Each 3 carbon pyruvic acid molecule formed during glycolysis is completely catabolized during the reactions described above The first carbon is removed during the intermediate step prior to the formation of acetyl CoA the second is removed from isocitric acid and the third is removed from oc ketoglutaric acid Each of these carbon atoms is associated with oxygen in the form of a carboxyl group COOH39 When two electrons and one hydrogen proton are removed from each carboxyl group and passed to NAD carbon dioxide is released as a waste gas Respiratory organisms such as humans and other animals release carbon dioxide when they exhale and this is how it is formed Notice that it has nothing to do with molecular oxygen being taken in ie we DO NOT take in OZ slap a carbon atom on it and release it as CO2 and neither does Pseudomonas The Electron Transport ChainSystem The third set of chemical reactions associated with cellular respiration involves enzymes that are bound to membranes In the case of prokaryotic cells these membranes are cell membranes whjle within most eukaryotic cells the membranes involved are the inner folded membranes or cristae of mitochondria The electron transport chain or system involves a series of membrane bound proteins that pick up electrons and hydrogen protons from coenzymes NADH H and FADHZ and pass them from one molecule to the next through a series of oxidation reduction reactions until they are picked up by a final electron acceptor often molecular oxygen The number and types of proteins involved in electron transport systems is variable however all involve cytochromes pigmented enzymes with iron prosthetic groups The diagram provided in the lecture syllabus represents an electron transport system involving a avoprotein a protein bound to avin mononucleotide or FMN a protein bound to coenzyme Q ubiquinone and a series of cytochromes As electrons are passed along the chain initially between different coenzymes and then to a series of prosthetic groups the integral proteins pump hydrogen protons through the membrane generating a concentration and electrical gradient known as the proton motive force The gradient then provides the quotforcequot necessary to move hydrogen protons back across the membrane through an enzyme complex called ATPsynthase Since the hydrogen protons are owing down their concentration and electrical gradients their movement through ATP synthase is passive As the hydrogen protons ow through ATP synthase they provide the energy necessary to convert ADP plus inorganic phosphate into ATP This process is called oxidative phosphorylation and requires that three hydrogen protons pass through the membrane for each molecule of ATP generated For each NADH H oxidized by passing its electrons to the electron transport chain nine hydrogen protons are pumped across the membrane and for each FADH2 oxidized around eight hydrogen protons are pumped across Obviously the protons are not coming from the coenzyme so presumably they are taken from hydronium ions H303 within the matrix of the mitochondrion or within the cytoplasm of a prokaryotic cell The protons moved across the inner membrane of a mitochondrion accumulate within the intermembrane space the potential space between the inner and outer mitochondrial membranes Protons moved across the cell membrane of a prokaryotic cell will accumulate within the periplasmic space In either case a transmembrane electrical potential is generated and this represents potential energy a bit like water behind a dam For each NADH H oxidized by passing its electrons to the electron transport chain nine protons will eventually ow back through ATP synthase and 3 molecules of ATP will be formed For each FADHZ oxidized less than nine protons flow back across the membrane and only 2 molecules of ATP can be formed Because there are ten NAD molecules and two FAD molecules reduced during the catabolism of each glucose molecule as described in the pathways above 34 molecules of ATP can be generated through oxidative phosphorylation Since cellular respiration also generated 2 ATP in association with glycolysis and the equivalent of 2 ATP in association with the Krebs cycle via substrate level phosphorylation the total number of ATP molecules potentially produced is 38 max Though prokaryotic cells often produce close to 38 molecules of ATP for each glucose molecule they catabolize eukaryotic cells are not as efficient and generally produce only 36 ATP per glucose or less This is because in eukaryotic cells glycolysis occurs outside the mitochondria while the reactions of the Krebs cycle and passage of electrons to the electron transport chain occur inside Pyruvic acid must be transported into the mitochondrion and the NADH H formed outside cannot as readily pass its electrons to the proteins bound to the inner mitochondrial membrane as can NADH H formed inside If molecular oxygen serves as the final electron acceptor at the end of the electron transport chain water is formed as a by product of cellular respiration 12 02 2 electrons and 2 hydrogen protons H20 The electrons and hydrogen protons picked up by oxygen are those passed along the electron transport chain not the protons owing through ATP synthase The advantages of cellular respiration over fermentation are multiple and significant and can be sununarized as follows 1 More of the energy potentially available in glucose molecules can be captured in the form of ATP so the process is energetically more efficient about 40 as opposed to 2 The waste products of cellular respiration are considerably less toxic than those of fermentation carbon dioxide and water as opposed to organic acids solvents and alcohols 3 The water formed at the end of the electron transport chain is potentially available for other metabolic processes Organisms such as lichens lizards and kangaroo rats can live in environlnents that are extremely dry because they are able to reduce water loss due to evaporation excretion and because they make metabolic water throug respiration N V 4 Respiration involving oxygen as the final electron acceptor effectively eliminates a potentially toxic substance ie molecular oxygen Oxygen is a powerful oxidizing agent and is potentially damaging to cells and tissues it is lethal to obligate anaerobes Respiratory organisms can eliminate the threat posed by oxygen by converting it into water DNA RNA Replication and Transcription The metabolic processes described earlier glycolysis respiration photophosphorylation etc are dependent upon the enzymes present within cells Most enzymes are proteins a few are RNA and their presence within a cell is determined by the genetic information or hereditary material present This material contained primarily within the nucleus eukaryotic cells or nucleoid prokaryotic cells is deoxyribonucleic acid commonly referred to as DNA Background Information According to a National Geographic article Vol 150 3 1976 the human body contains trillions of cells and each cell contains around 100000 genes segments of DNA This amount of information if written out would fill around 600 1000page books give or take a few as in uenced by font size paper weight etc Within cells the genetic information is tightly coiled but if the DNA from all the cells within the human body were stretched out and laid endto end it would extend to the sun and back over 400 tjines This same amount of DNA would fit in a box about the size of an ice cube DNA is amazing material with respect to its information storage potential Composition of DNA Deoxyribonucleic acid DNA is a polymer ie a long slender molecule composed of many small repeating units called nucleotides Each cellular DNA molecule forms a double helix or duplex ie includes two chains of nucleotides connected to one another by hydrogen bonds and twisted into a helical configuration like a twisted ladder Each nucleotide monomer contains deoxyribose a pentose monosaccharide or 5carbon sugar a phosphate group P04 and one nitrogenous base The bases commonly found in DNA include adenine guanine cytosine and thymine frequently represented by the letters A G C and T Of these adenine and guanine are purine bases purines and have two rings in their structure while cytosine and thymine are pyrimidine bases pyrimidines and have only one ring note that quotyquot words go together The nucleotides within each strand of a DNA molecule are connected together by covalent bonds called phosphodiester bonds formed between the sugar of one nucleotide and the phosphate group of the next Since a water molecule is removed each time one of these bonds is formed DNA synthesis is another example of dehydration synthesis or condensation Each nucleotide chain has a specific orientation determined by the positions of the phosphate P04 groups and hydroxyl OH groups associated with deoxyribose The phosphate is connected to the nuinber5 carbon of the sugar and forms the 539 end of each chain The hydroxyl group is found on the nuinber3 carbon of the sugar and forms the 339 end of each chain The two nucleotide chains within each DNA molecule are antiparallel and complimentary to one another They are antiparallel because their orientation is opposite ie they are upsidedown relative to one another from 539 to 339 They are complimentary because the nitrogenous bases forming the quotrungsquot of the DNA quotladderquot always pair up in a specific manner The purine base adenine is always connected to the pyrimidine base thymine by two hydrogen bonds AT and the purine base guanine is always connected to the pyrimidine base cytosine by three hydrogen bonds GC Note that each base pair contains three rings and that straightsided letters pair together and curvysided letters pair together Although individually hydrogen bonds are weak the two nucleotide side chains in a typical DNA molecule are held together fairly securely because there are so many hydrogen bonds present Most DNA molecules are twisted in a righthanded helix ie when viewed from one end wind away from the viewer in a clockwise direction Each turn contains about 10 base pairs roughly perpendicular to the side chains but each with a slight propellerlike twist between the bases Two grooves form along the surface of each DNA doublehelix a minor groove located between the two nucleotide strands and a major groove formed by the turns of the helix In Prokaryotic chromosomes most plasmids mitochondria and chloroplasts DNA molecules are covalently closed circular structures with no free ends cccDNA In eulltaryotic chromosomes DNA molecules are linear Genes Genes are hereditary units associated with chromosomes As was explained during an earlier lecture chromosomes are made up of chromatin and chromatin is made of DNA and protein recall nucleosomes DNA wrapped around histone octomers Genes are actually small sections of DNA that typically have some specific function within the cell Some genes code for mRNA and polypeptides others code for tRNA rRNA sRNA etc and some serve as regions involved in the regulation of gene expression DNA forms the genetic information within cells because cellular genes are composed of DNA but not all genes are The genes found within some viruses are composed of RNA Composition of RNA Ribonucleic acid RNA is also a polymer and like DNA is composed of nucleotides connected together by phosphodiester bonds Cellular RNA molecules are single stranded ie contain only one chain of nucleotides some viral RNA molecules are doublestranded Each RNA nucleotide contains the pentose sugar ribose a phosphate group P04 and one nitrogenous base Although three of the bases found in DNA are also found in RNA adenine guanine and cytosine the forth base thymine is not Instead some RNA nucleotides contain the pyrimidine base uracil Thymine is 539methyl uracil Although RNA molecules are polymers and often occur as long chains sometimes thousands of bases in length they are much shorter than DNA molecules Prokaryotic cells typically contain three different types of RNA molecules all coded for by different genes some occurring as multiple copies Eulltaryotic cells contain more than three types The functions of the different types of RNA molecules will be explained later DNA Replication Since DNA contains the genetic information within cells it must be reproduced before a cell undergoes fission This insures that each new cell formed contains the information necessary to function Replication sometimes called semiconservative replication is the process involved when DNA molecules reproduce It is a semiconservative process in that each new DNA molecule formed contains half of the original molecule involved in the replication process the original or quotparentalquot strand This is because during replication each strand of the DNA duplex serves as a template or pattern for the new strand being formed Given this feature one might ask just how old is DNA Replication can occur by more than one mechanism and is a complex process involving multiple factors not presented here When considered in simplified form replication always requires three things 1 An existing DNA molecule to serve as a pattern or template 2 Enzymes The heterogeneous proteins found in chromatin 3 Energy Because synthesis reactions are endergonic Within living cells DNA replication typically begins at a specific site called the origin of replication and proceeds in both directions away from that point Prokaryotic cells such as those of E coli generally have only one origin of replication within their circular chromosome but eukaryotic cells have many along their linear chromosomes The origin of replication within an E coli chromosome called oriC is a sequence of nucleotides 245bp in length This region contains specific base sequences recognized by and able to interact with initiation factors and enzymes involved in the process At the origin the two nucleotide strands of the DNA molecule separate hydrogen bonds break and individual bases are exposed between them This separation involves enzymes eg helicases and gyrase topoisomerase II The primary enzyme involved in DNA replication is DNAdependent DNA polymerase often referred to simply as DNA polymerase Prokaryotic cells such as E coli typically have three DNA polymerase enzymes designated as DNA polymerase I H and 111 Of these DNA polymerase III is the primary builder Polymerase enzymes catalyze chemical reactions resulting in the formation of phosphodiester bonds ie they synthesize polymers however DNA polymerase enzymes can only add nucleotides to the free 339 ends of existing nucleotide chains can build from 539 to 339 They cannot initiate the formation of nucleotide strands from individual nucleotides without the presence of pnmers A primer is a short sequence of nucleotides often around 1820 bases in length and when associated with DNA replication is composed of RNA nucleotides primers used in PCR are often made of DNA Enzymes called primase enzymes build the RNA primers associated with replication The first of these is formed near the origin and provides the free 339 end DNA polymerase requires for synthesizing DNA Once replication has been initiated the DNA strands involved appear to form two replication forks ie regions where the double helix separates into two individual strands These will travel in opposite directions away from one another as the original helix unwinds and replication proceeds There is usually only one primer synthesized at the origin of replication and it is associated with the leading strand Once this primer is in place DNA polymerase III can add DNAtype nucleotides to it and build a new complimentary strand the leading strand as a continuous sequence The opposite strand forming the replication fork is called the lagging strand Although nucleotides are also exposed along the lagging strand replication cannot occur there in the same fashion because DNA polymerase cannot build in the 339 to 539 direction Instead a group of proteins including primase form a structure called a primosome and this begins to migrate along the lagging strand traveling in the same direction as DNA polymerase III on the leading strand Periodically as the primosome reaches specific nucleotide sequences along the lagging strand it synthesizes new primers primer synthesis occurs in the 539 to 339 direction so is opposite the direction of primosome migration These primers also made of RNA serve as new start points for DNA synthesis and initiate the formation of a series of DNA fragments called Okazaki fragments Each Okazaki fragment has a short RNA sequence at its 539 end but they are composed primarily of DNA The Okazaki fragment formed nearest the origin serves as the beginning of the leading strand associated with the other replication fork ie the one traveling in the opposite direction away from the origin Since DNA molecules do not contain small segments of RNA all the RNA primers formed during replication must be removed This is accomplished by DNA polymerase I It travels along the newly formed lagging strand degrading the RNA primers and replacing them with DNA it also removes the primer at the beginning of the leading strand However although DNA polymerase can add new nucleotides to a free 339 end of an existing nucleotide strand it cannot form a phosphodiester bond between two existing nucleotide strands This requires a different enzyme called ligase Ligase enzymes form the phosphodiester bonds attaching the multiple Okazaki fragments together and bind the leading strand formed with one replication fork to the lagging strand formed with the other Note There is considerable quotproof readingquot and quotrepairquot associated with the replication process such that most newly formed DNA strands are identical to their quotparentalquot compliments Errors occur at a pace of about 1 per 100 million copies of DNA the spontaneous mutation rate described in a later section In addition to requiring DNA as a template and the enzymes described above replication also requires energy Replication of the E coli chromosome around 46 X 106 bp occurs in about 60 minutes so proceeds at a pace of about 77 thousand nucleotides per minute over 1000 per second The process requires considerable energy because the chemical reaction associated with the formation of each phosphodiester bond is endergonic The energy required is provided by the nucleotides used in the building process ie by dNTPs and rNTPs Nucleoside triphosphates NTPs are highenergy molecules containing pyrophosphate bonds recall the structure of ATP described in an earlier section These bonds are broken as the nucleotides are incorporated into DNA and the energy released is used to form the phosphodiester bonds holding the nucleotides together Transcription RNA Synthesis The term quottranscribequot means to write out an exact copy of something therefore when not applied to biological activities transcription refers to the process of copying written information When applied to biological systems it has essentially the same meaning Transcription is RNA synthesis or the process used to build RNA molecules within living cells Like replication transcription requires DNA as a template or pattern enzymes and energy Transcription occurs in association with DNA so occurs in the nucleus in the nucleoid in association with plasmids and inside mitochondria and chloroplasts The process is si1nilar to replication in that it is initiated by the breaking of hydrogen bonds and the separation of the two strands forming the DNA double helix or duplex This process involves enzymes eg helicases and gyrases Once the strands are separated RNA polymerase enzymes can begin the building process Since transcription does not involve the formation of primers or Okazaki fragments priinase enzymes and ligase enzymes are not required Neither is DNA polymerase DNAdependent RNA polymerase is the enzyme involved in transcription though it is usually referred to simply as RNA polymerase ln prokaryotic cells this is a complex composed of five subunits Four of these designated as CL CL 5 and 539 bind together to form a unit called the core enzyme This unit is primarily responsible for the building of new RNA molecules ie comprises the quotwork forcequot The fifth unit is called sigma factor and has an alternate function When the DNA strands have been separated the sigma factor binds to a specific region on one strand called the promoter site In bacteria such as E coli there are several different sigma factors and each recognizes a different type of promoter but most promoters have some features in common They typically contain highly conserved nucleotide sequences consensus sequences such as TATAAT and a specific start site for transcription often the sequence CAT The position and orientation of the promoter determines where transcription will begin and in which direction it will proceed but only with the help of sigma factor Once sigma factor has attached to the promoter site the core enzyme can bind and transcription can begin Like DNA polymerase the core enzyme builds in the 539 to 339 direction by catalyzing the formation of phosphodiester bonds at free 339 ends Unlike DNA polymerase it can also start the building process The nucleotide sequence of the DNA template determines the sequence of bases incorporated into the newly forming RNA strand just as it would during replication except that the purine base adenine codes for uracil instead of thymine because RNA molecules do not contain thymine The DNA strand serving as the template is often referred to as the sense strand and the opposite strand or complilnent as the nonsense strand Which strand is actually being copied Like replication transcription requires energy and this is provided by rNTPs activated nucleotides containing the sugar ribose ln prokaryotic cells transcription is often polycistronic which means that multiple structural genes are copied as one long mRNA molecule This is because many promoter sites are associated with operons and these typically contain structural genes arranged in a sequence as will be described in greater detail later Polycistronic transcription does not usually occur within eukaryotic cells except within chloroplasts Note many sources divide the processes of replication and transcription into three steps identified as Initiation Elongation and Termination Initiation involves separation of the DNA strands and the binding of DNAdependent RNA polymerase sometimes called primase to the DNA template Elongation involves the binding of nucleotides to form either DNA or RNA polymers and Termination involves the release of polymerase enzymes and in the case of transcription release of the newly formed RNA from the DNA template Both processes are complex and involve numerous details not included here Go to the following sites for more information and illustrations http wwwpbsorgwgbhaso tryit dnaindexhtml Choose quotDNA Workshop Activityquot then quotDNA Replicationquot you will need to have quotShockwavequot installed http wwwvisionlearningcom library science biologyl BlOl lnucleicacidshtm To have even more fun http wwweurekasciencecom lCanDoThat dnaintrohtm http wwwemcmaricopaedu faculty farabee BIOBK BioBookPROTSYnhtml Photosynthesis and Biosynthesis As described earlier chemoheterotrophs obtain the energy they need for growth from the catabolism of preformed organic compounds These organisms make ATP using either substrate level or oxidative phosphorylation or a combination of the two Organisms categorized as phototrophs can make use of a third mechanism for synthesizing ATP called photophosphorylation These organisms use light as their energy source Photophosphorylation Photophosphorylation Photophosphorylation is ATP synthesis involving light as the energy source Organisms capable of photophosphorylation carry one or more types of lighttrapping pigments ie molecules that can respond to light energy in a specific manner In the case of algae cyanobacteria and green plants the pigments involved are green chlorophyll molecules chlorophyll a b or c in the case of anoxygenic phototrophic bacteria they are bacteriochlorophylls bacteriochlorophyll a b c d or e which occur in a variety of colors in the case of Archaea in the genus Halobacterium the pigment is bacteriorhodopsin a red or purplecolored pigment All of these pigment molecules have a feature in common and that is their ability to respond to light energy by releasing electrons Light has both waveform and particleform properties so although light occurs as different wavelengths it also occurs as particles or packets of light energy called photons When photons strike certain atoms associated with lightsensitive pigment molecules the atoms give up electrons ie electrons are activated by light energy and bounce away from atoms within the pigments If the electrons are passed to proteins associated with a membrane integral proteins their ow can trigger the same reactions we saw associated with oxidative phosphorylation ie the transport of hydrogen protons across the membrane to establish a proton motive force and the return ow of electrons through ATPsynthase resulting in the formation of ATP In this case the ATP made would be the result of photophosphorylation Photophosphorylation can be described as cyclic or noncyclic on the basis of whether or not the electrons leaving the pigment molecules return During cyclic photophosphory lation the electrons do return to the pigment molecules while during noncyclic photophosphorylation they do not Photophosphorylation in Anoxygenic Phototrophs Anoxygenic phototrophs include the green and purple sulfur bacteria green and purple nonsulfur bacteria and other phototrophic prokaryotes not included in the phylum Cyanobacteria These organisms are called anoxygenic an without oxy oxygen and genic generation or production of because they do not produce oxygen in association with photophosphorylation Cyclic photophosphorylation as it occurs in these organisms can involve different pigment molecules alone or in combination but in general they are all bacteriochlorophylls These respond to light energy by passing electrons to an electron acceptor e g ubiquinone bacteriopheophytin or others From the acceptor molecules the electrons travel along a series of cytochromes and then back to the pigments The diagraIn shown below is a representation of the pathway taken by activated electrons during cyclic photophosphorylation Bacteriopheophytin In this diagram light is striking or other electron acceptors the pigment molecules and causing the electrons from certain atoms to gain energy and quotbouncequot away sometimes these travel in pairs as indicated here and sometimes they 2e do not The electrons leaving the Cytochrome chain pigment molecules travel to an electron acceptor in this case ADP Pi ATP bacteriopheophytin and from there Light travel along a chain of cytochrome enzymes via oxidation reduction reactions As the electrons exit the OO O 000 cytochrome chain they are picked 000 000 up by proteins associated with the pigments bacteriochlorophylls Bacteriochlorophylls and are ultimately returned to the atoms they caIne from The ow of electrons is associated with ATP synthesis as explained below Since the electrons involved in this process are recycled the synthesis of ATP can continue as long as light energy is available The pigment molecules electron acceptors and cytochromes indicated above are all bound to the bacterial cell membrane so this is where photophosphorylation occurs The electron flow generated by light striking the pigment molecules is linked to the transport of hydrogen protons across the membrane and into the periplasmic space The acculnulation of protons outside the membrane creates a concentration and electrical gradient known as the proton motive force again like water acculnulated behind a dam When the protons flow back across the membrane down their concentration and electrical gradient they pass through ATPsynthase an enzyme complex and supply the energy needed to convert ADP Pi into ATP As long as light is available ATP can be generated Green and purple sulfur bacteria can also capture light energy by means of a noncyclic pathway In this case the electrons leaving the pigment molecules pass to an electron acceptor and then to the coenzyme NADP reducing it to NADPH H The reduced coenzyme has a higher energy potential than it does in its oxidized state so some of the energy available in the light photons is captured but the pigment molecules are now missing electrons Bacteriochlorophylls cannot pull electrons and hydrogen protons away from water molecules so oxygen is not generated but these pigments can pull electrons away from hydrogen sulfide resulting in the formation of elemental sulfur Bacteria samples taken from inside the Winogradsky window at the back of the laboratory often contain sulfur granules within their cytoplasm These appear as tiny refractile particles readily visible when the bacteria are magnified 1000X with a light microscope The diagram shown below represents a noncyclic pathway for capturing light energy as it occurs in green and purple sulfur bacteria Ubiquinone gt NADP In this diagram light is striking or other electron acceptors the pigment molecules and causing the electrons from certain atoms to NADPH H gain energy and quotbouncequot away sometimes in pairs as shown here and sometimes not The electrons 2e travel to an electron acceptor in this case ubiquinone coenzyme Q and from there to the coenzyme NADP reducing it to NADPH H Light Note that NADPH H carries considerable potential energy just as 2e does NADH H In this case the OO O 008 4 HZS pigment molecules replace their 000 00 missing electrons by pulling them away from hydrogen sulfide H25 Bacteriochlorophylls Although this diagram looks similar to the one used to illustrate non cyclic photophosphorylation described below ATP is not being formed so phosphory lation is not occurring The anoxygenic phototrophic bacteria can capture light energy in the form of either ATP molecules or NADPH H molecules but not in both at the same time They can switch back and forth between the two mechanisms as required for maintaining other metabolic processes Photophosphorylation in Algae and Cyanobacteria Algae and cyanobacteria like green plants are oxygenic phototrophs ie organisms capable of generating molecular oxygen in association with their photophosphorylation processes Like the green and purple sulfur bacteria described above algae an cyanobacteria can use both cyclic and noncyclic pathways but since both of these generate ATP both are truly photophosphorylation pathways Algae and cyanobacteria capture light energy with chlorophyll pigments primarily in a manner like that described above however the electron acceptors associated with the photosystems pigment systems of oxygenic organisms are different In addition to this algae and cyanobacteria typically use two different sets accumulations of proteins and pigment molecules capable of responding to different wavelengths of light These two systems are called photosystem I and photosystem II When both photosystems are involved photophosphorylation is noncyclic and energy is captured in the formation of both ATP and NADPH H When only one photosystem is being used photophosphorylation is cyclic and only ATP is generated The diagram below shows a highly simplified representation of noncyclic photophosphorylation as it occurs in algae cyanobacteria and green plants Plastoquinone Ferredoxin gt NADP electron acceptor electron acceptor NADPH H 2e 2e Cytochrome chain Light Light ADP Pi ATP 0 o x 080 o 80 goo 008 O O 4 H20 00 OO Photosystem ll Photosystem I In this diagram light is striking pigment molecules associated with both photosystem I and photosystem II and is causing electrons to leave certain atoms The electrons leaving photosystem II are passed to an electron acceptor e g pheophytin and then to plastoquinone From Plastoquinone the electrons are passed along a chain of cytochrome enzymes and are then transferred to photosystem I At the same time the electrons leaving photosystem I are passed to an electron acceptor usually a quinone and then to ferredoxin an iron sulfur protein From ferredoxin the electrons pass to NADP reducing it to NADPH H The proteins and pigments of photosystem I and II are bound to membranes called thylakoids thylakos sac The passage of electrons along the chain of cytochrome enzymes is linked to the transport of hydrogen protons across these membranes into the region surrounded by them This creates a concentration and electrical gradient ie a proton motive force that drives the passage of hydrogen protons through ATPsynthase resulting in the conversion of ADP Pi into ATP Since the electrons that leave photosystem 11 do not returned the phosphorylation process is noncyclic Photosystem II can replace the lost electrons by pulling them away from water molecules and thus generates molecular oxygen ZHZO 4 electrons and 4 hydrogen protons Oz The photosystems described above are actually very complex each containing multiple proteins lighttrapping pigments and other types of molecules In green algae and higher plants most of the pigments associated with the photosystems are chlorophylls but carotenoid pigments can also be involved In red algae and cyanobacteria chlorophylls are often accompanied by phycobilins Though electrons are depicted as moving in pairs within the diagrams presented here this is not always the case During cyclic photophosphorylation as it occurs in algae and cyanobacteria electrons leave photosystem I are passed to an electron acceptor eg pheophytin and then to ferredoxin however rather than being passed to NADP the electrons are passed to a chain of cytochrome enzymes and then return to photosystem 1 ATP is generated as described above but oxygen is not produced A simplified version of cyclic photophosphorylation is provided in the diagram below Pheophytin gt Ferredoxin In this diagram light is or other electron acceptor striking the pigment molecules causing the electrons from certain atoms to gain energy and quotbouncequot away sometimes 2e inpairs The electrons Cytochrome chain travel to an electron acceptor in this case ADP Pi ATP pheophytin and then Light to ferredoxin From there they travel along a chain amp of cytochrome enzymes and back to photosystem I Since the electrons leaving the photosystem will Photosystem I eventually be returned to it the process is cyclic Note that during cyclic photophosphorylation energy is captured in the form of ATP but not as NADPH H In addition to this there is no splitting of water molecules and oxygen is not produced 00 OO O 0 000 000 Photosynthesis Photosynthesis Photosynthesis photo light synthesis building reactions or light synthesis is often defined as a process allowing organisms to make sugar and oxygen using light energy carbon dioxide and water The chemical equation sometimes used to represent photosynthesis is shown below 6 CO2 6 H20 light energy gt C5H1205 6 02 Although convenient this equation is only partially correct ie is only correct when applied to certain organisms Algae cyanobacteria and green plants produce oxygen in association with their photosynthetic processes but anoxygenic phototrophs do not In addition although photoautotrophs use light energy and inorganic carbon as indicated by this equation photoheterotrophs do not and neither do chemoautotrophs Photosynthesis is actually a complex metabolic process involving two distinctly different phases these are l Photophosphorylation light dependent reactions As explained above photophosphorylation involves making ATP using energy provided by light When the process is noncyclic and occurs within algae cyanobacteria and green plants it also results in the formation of NADPH H 2 The CalvinBenson cycle light independent reactions An anabolic pathway used to convert inorganic carbon carbon dioxide into sugar fructose This pathway requires energy and is driven by the ATP and NADPH H generated in association with photophosphorylation As explained earlier photophosphorylation may or may not result in the formation of oxygen If it does the oxygen is generated when water molecules donate electrons and hydrogen protons to photosystem II The pigment systems of anoxygenic phototrophs do not split water and cannot generate oxygen The CalvinBenson Cycle The CalvinBenson cycle is an anabolic pathway involving enzymes associated with the stroma of chloroplasts in eukaryotic cells algae and green plants and inclusions called carboxysomes in prokaryotes One of the most important enzymes involved in the Calvin Benson cycle is ribulosebisphosphate carboxylase oxygenase RuBisCO This enzyme catalyzes the reaction binding carbon dioxide to ribulose bisphosphate a fivecarbon sugar at the beginning of the cycle As indicated above the light independent reactions do not require light they do however require energy The ATP and NADPH H generated during photophosphorylation can be used to drive the CalvinBenson cycle A simplified version of the CalvinBenson cycle is presented in the diagram below 6 CO2 6 ribulose bisphosphate gt 12 3phosphoglyceric acids 3C ATP ADP Molecular rearrangement l2 13bisphosphoglyceric acids 3C ADP NADPH H ATP NADP 10 GlyceraldehydeBPO4 3C 4 l2 GlyceraldehydeBPO4 3C 1 Fructose molecule 6C Six molecules of carbon dioxide are required to produce one molecule of sugar Although fructose is formed initially this can readily be converted to glucose recall that fructose and glucose are isomers The synthesis of fructose also requires two molecules of ATP and one molecule of NADPH H as shown above The CalvinBenson cycle is not the only pathway autotrophs can use to quotfixquot carbon ie to incorporate inorganic carbon into organic compounds Furthermore highener compounds generated through photophosphorylation processes do not necessarily drive the CalvinBenson cycle Chemoautotrophs can fix carbon but do not have the photosystems necessary to capture light energy Since we have already seen that some phototrophs do not produce oxygen cannot split water and since photoheterotrophs do not fix carbon at all we can see that the chemical equation for photosynthesis is not always applicable Alternate sources of energy and carbon As described earlier chemoheterotrophs use carbohydrates primarily glucose as a major source of energy ie they catabolize carbohydrates and use the energy released to form ATP These organisms also use glucose as a major source of carbon Many of the compounds formed during the catabolism of glucose ie during glycolysis and the Krebs cycle can also be used to form other compounds essential to cell function As we shall see below other compounds can also be catabolized and their byproducts used as sources of both energy and carbon When the catabolism of organic compounds is linked to the anabolism of other compounds which within cells it often is the overall process is called intermediary metabolism Biochemical pathways used both in catabolism and in anabolism are called amphibolic pathways The reactions of glycolysis and the Krebs cycle are of considerable importance because many of the metabolic intermediates formed e g Pyruvic acid oxaloacetic acid and a ketoglutaric acid can have amino groups added to them to form amino acids and these can be used to form proteins The acetylCoA formed just prior to the Krebs cycle can donate twocarbon acetyl groups for use in the synthesis of fatty acids The glyceraldehydes3phosphate formed during glycolysis is in equilibriuln with dihydroxyacetone phosphate and this can be converted into glycerol the 3carbon compound used in the production of triglycerides and phospholipids Most of the nucleotides used to build nucleic acids highenergy compounds coenzymes and regulatory molecules are obtained through the catabolism of nucleic acids ingested by cells ie via salvage pathways however all types of cells can also synthesize nucleotides from components provided by aInino acids and 5 carbon sugars Protein catabolism Chemoheterotrophs cultured in the microbiology laboratory are often provided with proteins or protein breakdown products peptone peptides proteoses as nutrient sources recall that nutrient agar contains beef extract and peptone Like carbohydrates these nutrients can be catabolized to provide cells with both energy and carbon Proteins can be broken into individual amino acids and these can be used to build other different proteins ie via salvage pathways Amino acids can also be deaminated ie can have their amino groups removed and can then be completely catabolized through chemical reactions associated with glycolysis and or the Krebs cycle depending on the type of amino acid initially present Lipid catabolism Triglycerides and phospholipids are abundant within cells and can also be catabolized to yield both energy and carbon The glycerol quotbackbonequot associated with these molecules can be removed and phosphorylated to form dihydroxyacetone phosphate DHAP Since this molecule is in equilibrium with 3phosphoglyceraldehyde PGAL it can readily enter the glycolysis pathway for additional catabolism Fatty acids hydrocarbon chains can be broken into twocarbon units and oxidized through a process called Soxi dation The resulting acetyl groups can then bind with coenzymeA to form acetylCoA and can then enter the Krebs cycle for additional processing Ultimate symbiosis A more complete understanding of metabolic processes often leads to a greater appreciation for the interdependence of the organisms present on this planet In a metabolic sense all living organisms live symbiotically with other organisms human beings included The random devastation of the natural environment perpetuated by human activities is ultimately selfdestructive Without other life forms we cannot survive A brief summary of our obvious interdependence is illustrated in the diagram provided below Photoautotrophs Chemoheterotrophs Green plants algae Fungi protozoa many cyanobacteria and types of bacteria and all other prokaryotes animals including humans Form sugar from CO2 gt Catabolize sugar and other and metabolize sugar to organic compounds releasing form other compounds 4 CO2 and energy to make ATP CalvinBenson cycle Fermentation and respiration Oxygenic forms split gt Respiratory forms use 02 as a water H20 to form 02 final electron acceptor and in molecular oxygen 4 the process generate H20 Oxygenic photophosphorylation Cellular respiration Remember that although photoautotrophs use light as an energy source they can only do this when light is available These organisms use a respiratory type metabolism and consume organic compounds and oxygen just as we do When light is available they produce more oxygen and sugar than they consume but during hours of darkness the reverse is true Recall that eutrophication can lead to fish kills when algae use excessive amounts of oxygen in water during periods of darkness eg at night It is important to remember that the cycling of carbon into and out of organic compounds carbon cycle and the cycling of oxygen with water represents only a fraction of the chemical processes involved in metabolism Bacteria also play crucial roles in the cycling of nitrogen sulfur phosphorous iron and other elements essential to life Global Warming and Other Important Issues A Biological Perspective All life forms require a source of energy and since energy cannot be created produced or destroyed it ows through biological systems Light energy electromagnetic radiation from the sun is the ultimate source of energy for organisms living on this planet Only certain organisms can capture this energy and convert it to highenergy compounds available for metabolism ATP and NADPH H e g green plants algae cyanobacteria certain other bacteria and a few types of archaea These organisms have been capturing light energy for a very long time millions of years at least Some of the energy was used to drive metabolic processes within these organisms and some was used to form organic compounds cellular components for new cells Eventually geologic processes caused some cellular material to become buried under layers of sediments as deposits of coal and oil Cellular respiration and fermentation allow organic compounds to be catabolized often releasing COzinto the atmosphere and these processes have also been occurring for a very long time millions of years Fire the nonbiological conversion of organic compounds and oxygen into carbon dioxide and water has also been around as a natural phenomenon for millions of years volcanoes lightning etc are natural sources of fire Unfortunately within the last few hundred years due to the industrial revolution human activities have significantly increased the rate at which buried organic materials are being burned The increased production of water in association with this activity is hardly significant because 34 of the earth39s surface is covered by water and the amount of liquid water present is influenced primarily by temperature uxuations The release of carbon dioxide is much more important The burning of fossil fuels coal and oil in association with human activities powering automobiles planes ships etc heating and cooling homes offices etc generating electricity driving industrial processes etc has significantly increased carbon dioxide levels within our atmosphere From a biological standpoint increasing CO2 levels can have catastrophic consequences Carbon dioxide is a quotgreenhousequot gas ie a gas that tends to trap heat energy from the sun within the earth39s atmosphere rather than allowing it to radiate into space Although warming the planet39s surface is beneficial to some extent too much heat is likely to have unpleasant consequences Global warming will potentially 1 Melt glacial ice causing sea levels to rise and ooding much of the lowlyin agricultural land currently available imagine California39s central valley filled with sea water 2 Allow parasites and vectors to broaden their ranges and increase the incidence of diseases such as malaria yellow fever sleeping sickness river blindness schistosomiasis etc etc 3 Cause organisms not adapted to warm environments to die Bacteria are not the only organisms in uenced by temperature and many of the plant species currently inhabiting this planet will not survive significant increases in temperature Loss of photoautotrophs means loss of oxygen production 4 Disrupt normal weather patterns and increase the occurrence and severity of hurricanes torrential rains droughts and other natural phenomena recognized as being damaging to human endeavors 5 Cause discomfort and stress within the human population taxing medical facilities and creating unnecessary strife The human population is already greater than can be sustained in the manner we are accustomed to Imagine shrinking the landmasses available for food production increasing damage due to natural phenomena and increasing the ranges of vectors and pathogens known to transmit cause hulnan disease The impact on healthcare facilities and personnel will be enormous In addition to this carbon dioxide reacts with water to form carbonic acid and lowers the pH within water environments Increases in atmospheric carbon dioxide levels are already causing damage in ocean environ1nents where decreases in pH are killing corals and other organisms Recall that pH has a significant impact on enzyme activity Killing organisms in ocean environments can have severe consequences especially if the organisms dying are oxygenic photoautotrophs refer to diagraIn above if you don39t remember why Carbon dioxide is also used as the primary building material for methane within methanogenic prokaryotes Methane released into the atmosphere also acts as a quotgreen housequot gas compounding the potential for global warming What can be done As explained during an earlier lecture hu1nans have tremendous intellectual potential and if properly educated could eliminate many of the problems currently facing mankind Relative to the issues described in this section students can have a positive impact by 1 Voting responsibly Look at the issues consider the consequences and vote in a manner beneficial to all hulnans now and in the future 2 Taking responsibility for your actions Don39t wait for someone else to solve the world39s problems take action yourself Become informed reproduce responsibly recycle eat more vegetables plant trees reduce your dependence on coal and oil carpool turn off the engine when the car is not in use park it and walk take a bus etc etc 3 Promoting education and new energy technology Coal oil and nuclear power are potentially dangerous and alternative energy sources solar wind hydrogen etc are available Promote education and encourage the development use of alternative ecologically quotfriendlyquot energy sources 4 Spreading the word Tell other people the microbiology web site is available to everyone encourage other people to read it We are biological entities and live symbiotically with other biological entities in a complex ecosystem maintained by interactions we do not fully understand Hulnan activity is negatively impacting this ecosystem in ways that will ultimately be damaging to us our future is at stake Fortunately we don39t have to worry about the fate of microorganisms they will be just fine Taxonomy and Identification Classification of Microorganisms Taxonomy Taxonomy may be defined as the science or study of the classification of living organisms It involves separating living organisms and fossil forms of preexisting organisms into groups or categories and developing the criteria to be used for determining which organisms fit into which groups Grouping or categorizing living organisms allows investigators to study and understand them more readily The categories used in the classification of organisms are intended to show the natural relationships between organisms and to re ect phylogeny ie the evolutionary history of organisms Recently new methods for analyzing the biochemical content of organisms have led to the development of new criteria for classification especially in microbiology and although this is exciting for taxonomists it has created inconsistencies in reference sources resulting in considerable confusion for students It is not unusual to find different authors applying different criteria and placing the same organisms into different groups Therefore if you have studied classification schemas in other classes it is likely that the one presented in this class will be different My apologies for the frustration I know this will cause some of you The tendency to categorize vehicles foods clothing etc is common to humans and not restricted to biologists however much of the terminology associated with taxonomy is new to beginning students and can therefore be intimidating For this reason information about specific representative organisms and their taxonomic relationships will be covered in the laboratory where organisms can be observed first hand The information presented here is of a more general nature and includes terminology applicable to taxonomy but also required for understanding other topics introduced later in the semester Binomial Nomenclature Binomial nomenclature refers to the twopart technical name applied to each different type of living organism It is important to biologists because it provides a system for communicating information about specific organisms named in a language universally recognized and accepted The development of the binomial system of nomenclature binomial nomenclature is credited to Carolus Linnaeus a botanist naturalist in association with his S ystema Natura a manuscript containing a classi cation of living organisms first published in 1735 Linnaeus39s text contained lengthy descriptions of multiple living organisms but also included a twopart name for each one based on key characteristics Currently the twopart technical name applied to each different type of living organism includes the genus name which is capitalized and the specific epithet all lower case Both names are Latinized and include either Latin or Greek roots providing descriptive information For example the name Stuphyloco ccus uureus describes a type of organism forming grapelike clusters of sphericalshaped cells and golden or yellowcolored colonies Linnaeus39s text was in Latin because it was the language used in universities at the time however since Latin is no longer a spoken language terms and their meanings remain stable and provide the basis for universally accepted scienti c communication Currently the binomial names of organisms are italicized when in print and underlined when written by hand a convention allowing for easy recognition The twopart name applied to each type of organism indicates where that organism fits into a larger taxonomic schema as indicated below Taxonomic Ranks Taxonomic ranks are the categories used in the classification of living organisms These are nested ranks with each successively lower level being contained within the one above A group of organisms occupying a specific rank is called a taxon pleural taxa or taxonomic group The original taxonomic ranks were as follows Kingdoms singular Kingdom the largest or most encompassing Phyla singular phylum Sometimes called Divisions Classes singular Class Orders singular Order Families singular Family Genera singular Genus Species the most specific category One of several mnemonic forms Kids playing chase on freeways go splat At the time of Linnaeus s work living organisms were grouped into two broad categories the Plantae plants and the Animalia animals These broad categories were c Kingdoms Since then a number of classification categories have been added between the levels of kingdom and genus Similar organisms with the same genus name or genera are grouped within the same family similar families are grouped within the same order similar orders are grouped within the same class similar classes are grouped within the same phylum or division and similar phyla are grouped within the same kingdom The criteria or rules used for the classification of living organisms into taxonomic ranks are quite specific and are determined by groups of biologists from around the world These international groups called congresses meet at varying intervals to determine how plants and animals are to be categorized Although most macroscopic organisms can be readily classified into two kingdoms Plantae and Animalia microscopic organisms cannot Following Van Leeuwenhoek s discovery of microscopic life forms many new organisms were identified that did not meet the criteria for either kingdom One way to solve this problem was to establish a new kingdom In 1866 Ernst Haeckel proposed a third kingdom be established which he called Protista This kingdom would include all singlecelled organisms and those multicellular forms not developing complex tissues A diverse group of organisms including protozoa algae fungi sponges and slime molds were to be classified within this kingdom but their relatedness was minimal In 1957 Roger Stainer and his associates used electron microscopy to demonstrate significant differences between prokaryotic and eukaryotic cells suggesting more than three kingdoms were required In 1969 RH Whittaker proposed a vekingdom system to improve classification This system included three kingdoms of more complex organisms based on three modes of nutrition The Animalia ingest food the Plantae make their own food via photosynthesis and the Fungi Myceteae absorb food in a liquid form The other two kingdoms Protista and Monera include organisms without complex structures that are separated based on their cell types Monera are prokaryotic and Protista are eukaryotic Although the Whittaker fivekingdom system is included in many modern textbooks it is not without problems Recent studies based on biochemical analyses indicate considerable variation among eukaryotic microorganisms and the need for multiple additional kingdoms In 1978 Carl Woese and his associates using biochemical analyses demonstrated signi cant differences within the kingdom Monera and prompted the addition of a new taxonomic rank called Domain above kingdoms in the taxonomic hierarchy This work provided evidence that organisms now recognized as Archaea formerly Archaeobacteria have multiple important characteristics unlike either bacteria or eukaryotic organisms The three domains of life currently accepted by most biologists include the Eukarya all organisms with eukaryotic cells the Bacteria and the Archaea Adding domains to the previously established taxonomic ranks generates a slightly modified hierarchy as shown below Domain pleural Domains the largest or most encompassing Kingdom pleural Kingdoms Phylum pleural Phyla Sometimes called Divisions Class pleural Classes Order pleural Orders Family pleural Families Genus pleural Genera Species the most specific category Mnemonic form Dumb kids playing chase on freeways go splat Since understanding the phylogeny evolutionary history of life on earth is a major goal of taxonomists numerous methods have been employed to determine the evolutionary relationships between organisms Since the 1970s computer technology and a method called cladistics have provided considerable information relative to evolutionary trends In cladistics specific features of organisms are used to determine relatedness A feature that is common to several different types of organisms but shows variation within them is assigned a value or form called a character state Analysis of the character is then conducted to determine which state is primitive ancestral and which is derived evolved from something else Finally the evolutionary relationships determined are portrayed as straightline diagrams evolutionary trees called cladograms Many changes in taxonomy including the addition of the new taxonomic rank domain are due to studies involving cladistics Although the primary features used to determine the relatedness between macroscopic organisms were initially based on morphology the study of external features and mode of reproduction these are not as useful for the classification of microorganisms New features such as types of nutrition and metabolism temperature requirements gas requirements pH and salinity preferences and biochemical properties have proven to be much more useful Taxonomy is an ongoing science and despite multiple new discoveries a complete picture of the relatedness between all living organisms has yet to be developed Because viruses are noncellular entities they are not included in any of the taxonomic schemas described above Viruses are often categorized according to the organisms they infect but new taxonomic schemes are being developed to better demonstrate the natural relationships between viruses Criteria Useful in the Identi cation andor Classi cation of Microorganisms Some of the informationterminology included in this section relates to microbial growth and the culture of microorganisms however since it also relates to identi cation and classi cation it will be presented here 1 Morphology 7 Although highly valuable in the classi cation of multicellular organisms morphology has limited usefulness when applied to prokaryotes Many different types of bacteria form colonies and cells with similar morphology even when subjected to various stain techniques Recall information presented on colony and cell morphology in the laboratory Mode of Reproduction 7 Variation in reproductive structuresmethods is of primary consideration in the classi cation of plants animals and fungi but somewhat less useful in the classi cation of singlecelled organisms Most singlecelled eukaryotes bacteria and archaea reproduce by means of ssion ie one cell divides itself into two daughter cells Nutrition and Metabolism 7 All living organisms can be categorized on the basis of their nutritional requirements and type of metabolism A Nutritional categories are based on energy source and carbon source Organisms can obtain the energy they require either from light or from chemicals Those using light energy are called phototrophs photo light and those using chemical energy are called chemotrophs Chemo chemical In this case the root word troph refers to activity and organisms can be activated either by light or by chemicals Organisms can obtain the carbon they need either from inorganic or organic carbon compounds Organisms using inorganic compounds as carbon sources are called autotrophs auto self while those using preformed organic compounds as their source of carbon are called heterotrophs hetero different The term troph in this case refers to feeding so organisms are either feeding themselves or feeding on a variety of different organic materials By combining energy source and carbon source we obtain four nutritional categories Photoautotrophs Organisms using light energy and inorganic compounds for carbon Photoheterotrophs Organisms using light energy and organic compounds for carbon Chemoautotrophs Organisms using chemical energy and inorganic compounds for carbon Chemoheterotrophs Organisms using organic compounds for both energy and carbon Plants algae and some bacteria are photoautotrophs but only prokaryotic cells function as photoheterotrophs or chemoautotrophs Animals including humans fungi protozoa and many prokaryotes function as chemoheterotrophs so this category is often subdivided Saprotrophs Chemoheterotrophs using dead or decaying organic materials for nutrients These are sometimes called saprophytes or decomposers Parasites Chemoheterotrophs using living organisms as their source of nutrients some living inside their host and others living outside Hypotrophs Obligate intracellular parasites ie organisms able to grow and reproduce only when inside a living cell Viruses some protozoa and some bacteria are hypotrophs Carnivores Chemoheterotrophs obtaining nutrients from meat Herbivores Chemoheterotrophs obtaining nutrients from plant material Omnivores Chemoheterotrophs able to obtain nutrients from both meat and plant material B Metabolism 7 Metabolism includes all the chemical reactions occurring within living organisms anabolism and catabolism and can be categorized as either fermentative or respiratory oxidative Fermentative organisms use organic compounds usually pyruvic acid as the nal electron acceptors in their metabolic processes Respiratory oxidative organisms use inorganic compounds usually molecular oxygen as the nal electron acceptors in their metabolic processes Gas Requirements 7 The gas requirements of organisms based on oxygen utilization can be useful in their classi cation as indicated below Obligately aerobic organisms obligate aerobes Organisms requiring molecular oxygen for growth and reproduction metabolic processes Obligater anaerobic organisms obligate anaerobes Organisms unable to tolerate exposure to molecular oxygen oxygen is often toxic to these and they cannot grow in its presence Facultatively anaerobicaerobic organisms facultative anaerobesaerobes Organisms able to grow and reproduce with or without oxygen available to them Microaerophiles Organisms able to grow best in environments with limited oxygen as might occur in the mud at the bottom of a pond lake sea etc or within the gastrointestinal tract Although obligately aerobic organisms typically have a respiratory or oxidative metabolism and require oxygen as a nal electron acceptor not all obligately anaerobic organisms are fermentative Many types of bacteria can use inorganic compounds other than molecular oxygen as nal electron acceptors for their respiratory metabolic processes Temperature Requirements 7 The temperatures required for optimum growth are variable and can be used to categorize microorganisms as follows Psychrophiles Psychrophiles are coldloving organisms psychro cold phil love These organisms grow best at cold temperatures between 75 and 20 degrees C Mesophiles Mesophiles are moderateloving organisms meso medium or intermediate and grow best at moderate temperatures between 20 and 45 degrees C Thermophiles Thermophiles are warmloving organisms thermo warm and grow best at warm temperatures between 45 and 60 degrees C Hyperthermophiles Hyperthermophiles are hotloving organisms and grow best at hot temperatures e g above 60 degrees C Hyperthermophiles living in hot springs grow at temperatures above 90 degrees C Organisms can also be described relative to their temperature tolerance ie ability to survive or tolerate exposure to temperature extremes Organisms that can tolerate exposure to extreme cold are said to be psychroduric They cannot grow at these temperatures but do not die either Most bacteria are psychroduric and can be maintained in a viable state at 770 degrees C Organisms that can tolerate exposure to heat are said to be thermoduric They cannot necessarily grow in hot environments but are not killed by exposure to them Endospores are thermoduric Acidity Vs Alkalinity or pH Requirements 7 Although most organisms grow best in neutral environments pH between 65 and 75 some prefer acidic environments and others prefer alkaline Many types of culture media contain buffers substances that resist pH change to help stabilize the pH or pH indicators substances that change color in response to changes in pH to indicate the presence of acidic or alkaline metabolic end products Organisms that grow best in acidic environments are called acidophiles but are relatively rare Highly acidic or alkaline environments tend to inhibit microbial growth because cellular enzymes fail to function under these conditions Osmotic Pressure Requirements 7 The effective osmotic pressure tonicity of an environment is in uenced by the solute concentration present and can significantly impact microbial growth Isotonic environments iso same contain solute levels similar to protoplasm so cells placed into them will experience neither a net gain nor net loss of water Hypotonic environments hypo under beneath less than or too little contain lower levels of solute than protoplasm and will cause cells placed into them to gain water Microorganisms equipped with cell walls e g algae fungi bacteria and archaea or contractile vacuoles many types of fresh water protozoa can live comfortably in hypotonic environments Organisms lacking these protective structures will tend to take on water via osmosis until they explode Hypertonic environments hyper over above too much or excessive contain higher levels of solute than protoplasm and will cause cells placed into them to lose water Hypertonic environments containing high levels of salt or sugar are often used to preserve foods ie inhibit microbial growth within those foods Organisms capable of growing and reproducing in environments containing high levels of salt are called halophiles These may be categorized as extreme halophiles obligate halophiles those requiring high levels of salt for growth or facultative halophiles those capable of growing with or without salt Environmental Relationships 7 The types of environmental relationships microorganisms form with other organisms can be useful as criteria for classification however these relationships are often not quot 39 39J J J nor Symbiosis 7 Symbiosis is a condition or circumstance existing when two or more different types of organisms are living together in a close association Although once thought to be unusual symbiosis is now recognized as a common occurrence essential to ecosystem function Pathogen Vs Host 7 Pathogens growing within a host benefit from host resources but the host is harmed and sometimes killed Microorganisms capable of causing infection and disease in humans domestic animals and plants used in agriculture have been extensively studied but represent an extremely small percentage of the total Parasite Vs Host 7 Parasites also benefit from their hosts without giving in return Organisms capable of parasitizing humans and other animals have been studied extensively because some cause disease and others serve as vectors involved in the transmission of diseasecausing agents Mutualistic relationships mutualism ie those involving organisms in mutually beneficial arrangements are the most common form of symbiotic relationships Even pathogens and parasites can be considered beneficial in the sense that they help prevent population overgrowth and maintain balance within ecosystems a concept foreignrepugnant to most humans Biochemical Analysis 7 Biochemical analysis allows for a more technical evaluation of the relationships existing between organisms and has become the method of choice for the classi cation of bacteria and archaea Various subcategories exist as follows A Enzymatic Testing 7 The types of enzymes organisms produce can be determined by testing their ability to catabolize various materials andor to form speci c end products Enzymatic testing will be used extensively during the identi cation of Physiological Unknown No l B Chromatography 7 Various applications of chromatography can be used to identify speci c chemical constituents of cells e g cell wall lipid or amino acid content membrane protein content or the presence of speci c pigments C Serology 7 Serology is the science or study of antibody and antigen interactions in vitro and has multiple applications in the detection identi cation and classi cation of microorganisms Microorganisms are antigenic ie are perceived by the body as foreign agents antigens and typically stimulate the production of immune proteins called antibodies Because the interactions between antigens and antibodies are quite speci c and because antibodies can bind with antigens it is possible to use known types of antibodies to detect or identify speci c types of antigens Several different types of serological reactions will be explained and demonstrated in the laboratory D Phage Typing 7 Phage typing bacteriophage typing involves the use of viruses called bacteriophages Like antibodies these will recognize and bind with speci c types of bacteria however unlike antibodies they cause infection typically resulting in cell death Because these viruses are hostspeci c known types of virus particles can be used to identify unknown types of bacteria Phage typing will be explained and demonstrated in the laboratory E Nucleic Acid Analysis 7 The analysis of nucleic acids DNA and RNA can provide considerable information useful in the identi cation and classi cation of microorganisms Techniques commonly used in nucleic acid analysis include Percent base composition G C or A T 7 Organisms with identical percentages in base composition may or may not be closely related but organisms with very different percentages in base composition are not related 2 Nucleic Acid Hybridization 7 Hybridization the ability of two nucleic acid strands to form hydrogen bonds with one another has multiple applications including PCR and DNA chip technology 3 Polymerase Chain Reaction PCR 7 The polymerase chain reaction involves hybridization and can be used to amplify DNA or RNA in vitro Gel Electrophoresis 7 Gel electrophoresis can be used to separate DNA or RNA fragments on the basis of size by exposing them to an electric field 5 DNA Fingerprinting or RFLP analysis 7 Fragments of DNA generated by restriction endonuclease digestion will form patterns when subjected to electrophoresis These patterns are called DNA ngerprints or RFLP patterns 6 Nucleotide sequencing 7 Determining the sequence of nucleotides in a strand of DNA or RNA can yield information highly signi cant to identification and classification 4 Note 7 Methods 2 3 4 5 and 6 will be covered more extensively in the laboratory where they will be used in the identi cation of Physiological Unknown No 2 F Protein analysis 7 The analysis of proteins other than antibodies can also be useful in the identi cation and classi cation of microorganisms Some methods involved include Gel electrophoresis 7 Similar to methods used with nucleic acids 2 Amino acid sequencing 7 Determining the sequence of amino acids present in a protein can be useful in determining protein function and sometimes protein origin For example the origin of prions infectious protein particles was determined using amino acid sequencing in conjunction with nucleic acid analysis According to the taxonomic system currently used by biologists living organisms are grouped relative to similar characteristics ie they are categorized according to speci c criteria as described above Under the binomial system of nomenclature binomial nomenclature each different type of organism is identi ed with a twopart technical name scienti c name indicating its genus and species In the case of multicellular eukaryotic organisms a species is de ned as a group of closely related organisms that will breed among themselves The classi cation of such organisms is therefore based largely on morphology and mode of reproduction In the case of prokaryotes a species can be de ned simply as a population of cells with similar characteristics a bacterial culture containing only one population of organisms is considered a pure culture and contains only one species Because most prokaryotes have similar morphology and mode of reproduction prokaryotic taxonomy is based primarily on other criteria with biochemical analysis being most important Once the criteria for classi cation have been determined it is necessary to devise methods for comparing the characteristics of speci c groups with those of newly discovered organisms Two methods commonly used for making such comparisons involve the use of dichotomous keys and cladograms Dichotomous keys allow investigators to identify organisms by answering a series of questions each with two possible answers dichotomous cut in two After answering one question the investigator is directed to answer a second a third and so on until the identi cation is made Although useful for identification dichotomous keys provide little information about the evolutionary relationships between organisms Cladograms clado branch are branching tree like patterns developed through cladistic analysis and typically indicate degrees of relatedness between organisms based on specific criteria Cladistics is a method for hypothesizing the evolutionary relationships among organisms and classifying organisms based on these relationships Most cladograms currently being made for bacteria are based on rRNA gene sequence analysis and provide signi cant information about prokaryotic phylogeny evolutionary history Complex cladograms hypothesizing the evolutionary relationships between multiple different types of currently existing organisms and their ancestral forms are called phylogenetic trees Armed with new methods of biochemical analysis and computer technology modern biologists are attempting to use the information gained through cladistic analysis to reconstruct the pattern of events leading to the distribution of life on our planet They are attempting to understand the evolutionary relationships between all living organisms and to determine the mechanisms of evolution involved in their origins This branch of science is called phylogenetic systematics and has been applied extensively to the classi cation of prokaryotic organisms ie archaea and bacteria Harriet Big Introduction to Algae According to the Whittaker fivekingdom system of classification singlecelled algae and protozoa belong to the Kingdom Protista Although sometimes divided into other kingdoms including Chromista Alveolata Parabasala etc the microscopic algae and protozoa are frequently referred to as protists and for the sack of simplicity will be left as protista here The lecture information for algae and protozoa will be presented in two parts with algae described first Microscopic algae singular alga are plantlike organisms occurring as single cells threadlike filaments or colonies of various shapes and composition They are abundant in water both fresh and marine in damp soil and on moist surfaces Some types of algae live inside other organisms and some form symbiotic relationships with fungi in structures called lichens Phycology The science or study of algae is called phycology phykos sea weed and initially involved the investigation of macroscopic organisms common in marine habitats Many types of marine algae are macroscopic sometimes reaching nearly 100 feet in length but since this is a microbiology course those organisms are not included here Algae are oxygenic photoautotrophs and contain greencolored pigments called chlorophylls within folded membranous thylakoids of organelles called chloroplasts recall eukaryotic cell structure and function The green chlorophyll pigments are light sensitive and allow algae to convert light energy into chemical energy ATP through a process called photophosphorylation Algae produce oxygen are oxygenic by splitting water molecules and are sometimes credited with producing up to 70 of the oxygen present in the earth39s atmosphere though reaching this percentage probably requires the inclusion of oxygen produced by cyanobacteria as well As autotrophs algae take in inorganic carbon carbon dioxide from the atmosphere and use it to form organic compounds sugars that can be metabolized in a variety of ways They are selffeeding but also provide food for other organisms Algae are ecologically categorized as producers and play an essential role at the bottom of multiple food chains webs Algae cells like those of fungi are surrounded by rigid nonliving layers called cell walls Most algae have walls made of polysaccharide cellulose or agar but some contain quantities of glass silica dioxide Walls give algae cells their characteristic shape and allow them to live in hyportonic environments without being damaged Though plantlike in some ways algae do not have stems leaves or roots The body of a multicellular alga is called a thallus and may have structures resembling stems leaves and roots Singlecelled filamentous and colonial forms of algae are much simpler in composition Algae Reproduction Algae reproduce both sexually and asexually with asexual reproduction occurring more commonly Some variation in asexual reproduction are described below a Fission Fission in algae like fission in other cells involves mitosis the separation of chromosomes and cytokinesis the division of the protoplasm into two parts Some algae such as Spirogym and Oedogonium undergo binary fission ie divide in half across their long axis Other types such as Chlamydomonas undergo longitudinal fission ie divide in half lengthwise b Shrinking division Asexual reproduction in diatoms involves a specialized type of fission called quotshrinking divisionquot which results in the formation of two cells of unequal size Diatoms have glass walls composed of two parts called frustules or valves depending on sources that fit together like the two sections lid and bottom of a Petri plate When a diatom undergoes fission the two wall sections separate and a new wall is formed on the inside of each The diatom receiving the quotlidquot section of wall will be the same sized as the original cell but the one receiving the quotbottomquot will be smaller Repeating fission cycles will produce smaller and smaller diatoms until a minimum size is reached and then stops This explains the size variation typical of diatoms in the same species Sometimes produce asexual spores Fragmentation Filamentous forms of algae can undergo fragmentation like fungus hyphae and each fragment can grow into a new filament d Spore formation Algae like fungi produce asexual spores of various types V C Sexual Reproduction Sexual reproduction as it occurs in algae is similar to that occurring in fungi in that it involves three stages or steps called plasmogamy karyogamy and meiosis This similarity is largely due to historical events ie algae and fungi were both considered members of the kingdom Plantae and the reproductive processes of both groups were described by botanists Sexual reproduction requires the participation of two genetically dissimilar algae of the same species and typically occurs in three stages or steps as outlined below H Plasmogamy Plasmogamy involves the joining of the protoplasm plasma protoplasm gainous union or marriage and requires decomposition of the cell walls separating the cells involved Karyogamy Karyogamy involves the joining of two haploid nuclei karyon nucleus and results in the formation of a diploid cell called a zygote Ha loid cells nuclei have only one set of chromosomes while diploid cells nuclei have two Meiosis Meiosis reduction division is a process involving separation of chromosomes and the division of the diploid nucleus into two haploid parts An important feature of meiosis is the formation of new genetic combinations not possible through asexual reproduction N DJ These stages may be separated in time and result in the formation of two separate generations of algae known as sporophytes and gametophytes The gametophyte generation is composed of haploid cells that can undergo plasmogainy and karyogamy to form diploid zygotes The diploid cells form the sporophyte generation and these can undergo meiosis to form gametophytes Since cells of the gametophyte and sporophyte generations are often morphologically similar or identical it is difficult impossible to determine if any given sample includes haploid or diploid cells by simple observation Algae Pigments The classification of algae sea weeds was initially based on the types of pigments present and three major groups included the green algae red algae and brown algae Microscopic algae can also contain different types of pigments including those listed below a Chlorophylls Chlorophst a b and c are the green pigments primarily involved in capturing light energy for photophosphorylation making ATP using light energy b Phycobilins Phycobilins are redcolored pigments found in red algae cyanobacteria and certain other groups These pigments absorb light energy and then pass it on to chlorophylls c Carotenoids Carotenoids are yellow and orangecolored pigments associated with several types of algae and green plants eg in carrots yellow and orangecolored owers and orange and yellowcolored fall leaves Betacarotene and lutein a type of xanthophil are two examples Some Interesting features of Algae Eutrophication Eutrophication is an increase in algae populations in a body of water fresh or salt and occurs when nutrients are abundant and light intensity is high When microscopic algae are involved they are referred to as phytoplankton phyton plant and planktos wanderer or drifter Although algae potentially provide a food source for other organisms and are oxygenic eutrophication is usually considered to be detrimental rather than beneficial ln swimming pools fish tanks and clear mountain lakes like Tahoe the growth of algae is unwanted Eutrophication sometjines referred to as an algae bloom or red tide when involving dino agellates in marine habitats causes water to become green or red cloudy and in some cases to have a foul odor Some consequences of eutrophication are described below 1 Algae can kill fish and other organisms living in water Algae are respiratory organisms require an inorganic compound as their final electron acceptor and although they can make oxygen they also use it Algae like other phototrophs can make oxygen only during daylight hours at night they use up oxygen and can cause fish and other organisms living in the water to die from suffocation Fish kills resulting from eutrophication are usually observed during summer months when light intensity is high and can occur in both freshwater and marine habitats for example in Clear Lake and San Francisco Bay 2 Algae can cause paralytic shellfish poisoning When eutrophication occurs in ocean waters and involves dino agellates organisms containing red pigments it can create quotred tidesquot Dinoflagellates like other types of algae can use up oxygen and kill fish but they can also kill large animals bears sea otters humans etc by producing potent neurotoxins gonyautoxins that cause paralytic shellfish poisoning PSP Shellfish such as mussels clams scallops and oysters feed on dino agellates filtered from the water and accumulate neurotoxins in their tissues When animals eat the shellfish they experience PSP and potentially deadly paralysis of skeletal muscles Dinoflagellates in the genera Alexandrium formerly Gonyaulax and Gymnodinium are commonly associated with PSP Though multiple factors may in uence eutrophication quotred tidesquot are most common along California39s northern coast during months without the letter quotrquot in them 3 Pfiesteriu threatens both fish and fishermen Dino agellates identified as Pfiesteria piscicida cause considerable damage along the eastern coast of the US by attacking and killing fish in large numbers Though the fish being killed by these dino agellates are small in size they are important to the fishing industry because they feed other larger fish of commercial interest P esteria also produce neurotoxins that are harmful to humans causing neurological symptoms such as headache dizziness and memory loss When present in high concentrations these toxins may become airborne and cause damage to people not contacting contaminated fish or water 4 Some dinoflagellates are bioluminescent Dino agellates in the genus N octiluca carry lux genes and produce luciferase enzymes involved in converting chemical energy into light energy Like bioluminescent bacteria these dino agellates produce their own light or glow in the dark During red tides N octiluca can cause waves to light up as they break on the shore sand to sparkle under foot and ships to leave light trails as they travel through the water II V Diatom cell walls form diatomaceous earth Diatoms microscopic algae with glass cell walls are often abundant in both fresh and salt water During earlier periods in the earth39s history diatoms inhabited inland seas that are no longer present Their cell walls accumulated at the sea bottom forming a sedimentary rock type called diatomite This material now occurs as deposits on dry land and can be quotminedquot and ground into diatomaceous earth Diatomaceous earth is used extensively in filters for water honey apple juice and other liquids It is also used as an abrasive in car polish toothpaste and cleansers Diatoms have clinical significance because diatomaceous earth can be used to determine blood clotting time Some diatoms produce a neurotoxin called domoic acid that can cause damage to humans eating seafood that was formerly feeding on diatoms 6 Red algae make agar Agar the polysaccharide commonly used as a solidifying agent in microbiological media is made by red algae Phylum Rhodophyta Algae and symbiotic relationships Endophytic The term endophytic applies to algae living inside other organisms Though green chlorophyll pigments occur in nearly all types of algae greencolored protista are not necessarily algae Sometimes algae live inside other organisms causing them to appear green Since algae are photoautotrophs they cannot be considered as parasites so when they live inside another organism they are said to be endophytic Endophytic algae can be found living inside organisms such as sea anemones H ydm atworms sponges and several types of protozoa Lichens Lichens are organisms made up of algae or cyanobacteria and fungi living together symbiotically Though classified as individual organisms lichens are actually composed of two different types of organisms living together The relationship between fungus and alga cells is mutualistic with the algae providing food through photosynthesis by collecting and processing carbon dioxide and the fungi providing protection and metabolic water Lichens are extremely hardy often colonizing environments where few other organisms can grow eg on rock surfaces Sometjlnes they are the first organisms to return after a fire or volcano removes all vegetation Introduction to Protozoa Protozoa are mostly singlecelled animallike organisms Although some are colonial or form loose aggregations most live and function as separate cellular individuals Most protozoa are chemoheterotrophs ie organisms using preformed organic compounds for both energy and carbon Most do not contain green pigments and are not capable of using light as an energy source however there are exceptions They live in a variety of habitats including fresh and salt water and inside multicellular organisms including hu1nans and other animals Most protozoa are freeliving organisms that obtain nutrients from decaying organic materials or feed on bacteria and smaller eukaryotic cells Some are parasites and some are pathogens capable of causing disease in hu1nans and other animals Since protozoa live in diverse habitats and function as individuals subject to a multitude of environmental challenges it is not surprising that they as a group have evolved a variety of specializations Some of these are described below 1 Locomotor structures Locomotor structures are specializations allowing cells to travel through their environment as a means of dispersing locating food sources or escaping potential predators a Cilia cilia singular ciliu1n are short hairlike structures found on the surfaces F V of protozoa called Ciliates phylu1n Ciliophora As described earlier Eukaryotic cell structure and function each ciliu1n is surrounded by the cell membrane and is supported by a cytoskeleton of microtubules arranged in a characteristic 9 plus 2 pattern They are capable of whiplike motion involving MAPs such as dynein and are coordinated by microtubules arranged just inside the cell membrane Cilia can be distributed more or less uniformly all over the cell surface can occur in rows or patches or be grouped together in tufts Most cilia are used for swimming and allow ciliates to move smoothly through their watery habitats some provide a ju1nping motility and cilia arranged in tufts called cirri allow cells to walk or ju1np along solid surfaces Flagella agella singular agellu1n are long whiplike structures found on the surfaces of many eukaryotic cells Like cilia they are surrounded by the cell membrane and contain microtubules arranged in a characteristic 9 plus 2 pattern Some cells such as those in the genus Trypanosoma have a single flagellu1n enclosed in a double layer of membrane running the length of the organism in a finlike manner Flagella are usually ness numerous than cilia and are often used to pull cells through their environments Pseudopodia Pseudopodia or false feet singular pseudopodiu1n are extensions of the protoplasm associated with amoebalike organisms These vary considerably in size and shape but are like cilia and flagella are surrounded by the cell membrane and often supported by microtubules not arranged in a 9 plus 2 pattern Pseudopodia typically form in a flowing fashion and allow amoebalike organisms to creep slowly along solid surfaces Some extend through holes in glass skeletons like a multitude of spokes radiating from spherical wheels 2 Food gathering structures Structures involved in food gathering activities are often those used for locomotion including cilia agella and pseudopodia In addition to these are structures involved in ingestion and digestion of foods Many protozoa are holozoic ie organisms that take in whole organisms for food They have no means of biting off small portions of their prey as would large multicellular animals a Cytostome The cytostome or cell mouth cyto cell stoma mouth is a region on the surface of a cell where endocytosis can occur Ciliated protozoa such as Paramecium can take in food only through their cytostome because the rest of the cell is covered by a tough pellicle b Lysosomes Lysosomes contain the digestive enzymes needed to break down food materials taken in through endocytosis Cilia and flagella can sweep food along the cell surface toward the cytostome and sometimes line a region of the cell called an oral funnel or oral groove Pseudopodia Amoebalike protozoa use their pseudopodia to capture food by extending them out and around the food and fusing them to form food vacuoles ea 9 Osmoregulatory structures The contractile vacuoles present in many types of freshwater protozoa are used primarily to pump excess water out of cells They are connected to the endoplasmic reticulum so also have circulatory function and may be used to eliminate liquid wastes ie also have excretory function a Protective Structures Protozoa live in potentially dangerous environments and have evolved a variety of protective structures that help them survive a Pellicle The pellicle is a tough exible layer found outside the cell membrane on all types of ciliated protozoa It helps provide the cell with a characteristic shape and protects it against physical damage b Skeletons The skeletons of protozoa are usually made of glass silica dioxide or calcium carbonate Radiolaria have glass skeletons perforated with numerous holes allowing pseudopodia to extend out through them Foraminifera have skeletons of calcium carbonate that resemble the shells of mollusks eg the chambered nautilus Skeletons provide protection against predation and also support the protoplasm Trichocysts Trichocysts are dartlike structures that can be shot out from certain cells They are made of protein are often barbed and attached to the cell surface by microscopic threads Trichocysts are released in response to chemical and or physical stimuli and may be used for defense or attachment F V U1 Life cycle stages Protozoa often live in habitats subject to change due to climate and seasons while some live parts of their lives inside different types of hosts To survive variations in living conditions protozoa can switch between two different stages a Trophozoites Trophozoites troph activity are active protozoa sometjines called vegetative cells While in their trophozoite form protozoa are engaged in feeding reproducing and moving about actively Vernal pools ie those filled with water during the spring contain many trophozoites As the weather warms up and pools dry out in the summer sun the protozoa go into a resting state b Cysts Cysts are dormant structures produced by many types of protozoa under certain circumstances They are metabolically inactive and much more resistant to heat drying radiation and chemicals than are trophozoites active vegetative cells Cysts allow protozoa to survive when their watery habitats dry out during summer months or freeze solid during the winter They also allow gastrointestinal parasites to survive passage through the stomach without being damaged by stomach acids Protozoan Reproduction Protozoa like fungi and algae can reproduce themselves both asexually and sexually There are many variations on these basic themes but some of the most commonly encountered forms of reproduction are introduced below H N Asexual reproduction Asexual reproductive processes allow individuals to reproduce without interacting with other cells In eukaryotic organisms asexual reproduction requires mitosis the separation of the chromosome and cytokinesis the separation of the cytoplasm forming new daughter cells Some specific examples include a Binary fission Binary fission is a process involving the separation of the cytoplasm across the long axis of the cell Most protozoa reproduce by binary fission b Schizogony Schizogony or multiplefission is a process involving the splitting of one cell into many daughter cells Sporozoans in the genus Plasmodium reproduce by means of schizogony while inside human RBCs Budding Budding involves an unequal division of the protoplasm and results in the formation of a bud at the margin of a cell If conditions are favorable the bud will grow and eventually separate from the cell but if conditions are poor the bud may die with little consequence to the cell F V Sexual reproduction In order to reproduce sexually protozoa must interact with other genetically different cells Sometjines this involves plasmogamy karyogamy and meiosis but not always and these terms are rarely used in zoology texts Two examples of sexual reproduction include a Syngamy Syngamy involves the fusion of two haploid cells to form a diploid zygote Protozoa in the genus Plasmodium undergo syngamy while inside mosquitoes b Conjugation Conjugation requires that two cells with different genetic content meet and position themselves sidebyside Portions of the cell membranes fuse allowing the formation of a cytoplasmic bridge and then segments of genetic material DNA are exchanged between the two cells Following conjugation the cells separate again but each one is now carrying a new combination of genetic material Medical Protozoology Introducing Some Medically Significant Protozoa Though most protozoa are freeliving organisms feeding on decaying organic materials bacteria and other cells some protozoa are parasites and some are pathogens Protozoa of medical significance are responsible for killing millions of humans and remain a threat to human health throughout the world Some examples of medically significant protozoa include 1 N DJ Giurdiu lumbliu Protozoa identified as Giardia lamblia or G intestinalis are agellated organisms that infect the small intestine and occasionally the bile ducts of humans and other animals causing giardiasis They enter the body in cyst form along with contaminated food or water and each cyst contains two trophozoites The trophozoites released within the intestine attach to the mucosa where they apparently feed on mucous and other epithelial secretions If they are numerous covering muc of the intestinal mucosa they interfere with digestion and absorption of nutrients especially fats resulting in diarrhea often accompanied by large amounts of yellowish fatty mucous Giardia are more commonly pathogenic in children than in adults Trichomonus vuginulis Various species of Trichomonas may live within the human host but only T vaginalis is pathogenic and causes trichomoniasis These organisms live primarily in the vagina but may travel to the cervix or vulva and can infect the urethra and prostate in males Infection causes in ammation accompanied by a creamywhite discharge with severe itching and chaffing Transmission may be direct via sexual intercourse or may involve transfer from mother to infant during childbirth Some infections have been acquired in poorly maintained pools and hot tubs According to some texts around 2040 of women in some areas have Trichomonas infections Entumoebu histo lyticu Although most amoebalike protozoa are freeliving in fresh and salt water many are parasites and some are pathogenic Entamoeba histolytica are amoebae that live in the large intestines of humans and other animals They enter their human hosts in the cyst form usually with water or contaminated vegetable material The cyst walls are digested away in the stomach and duodenum allowing the trophozoites four per cyst to be released The trophozoites live in the caecurn and reproduce by binary fission In most cases they cause no or little damage living on food material that is passing through the host However the name histolytica which means tissue lysing or splitting indicates that these organisms can invade the tissues causing damage In about 10 of infections the amoebae invade the intestinal mucosa causing tissue lysis and ulceration resulting in dysentery In severe infections they may penetrate the submucosa muscularis and serous membrane to enter the peritoneal cavity often resulting in secondary bacterial infection Symptoms vary depending upon severity and location of the infection but typically include nausea cramps and diarrhea More severe infections result in abdominal tenderness dysentery dehydration and general incapacitation Symptoms may develop within days of exposure or as much as a year later depending on host condition Rarely amoebae travel via the portal system to the liver causing amoebic hepatitis 4 Acun thumoebu and other genera There are a nu1nber of other intestinal amoebae and E quot F gt1 there are also cases of a1noebae infecting other areas of the body Amoebae in the genera Acanthamoeba Naegleria and Balamuthia are normally freeliving in soil and water but have been found to cause eye infections keratitis skin infections and primary amoebic meningoencephalitis a rapidly fatal infection of the brain and meninges These a1noebae can enter the nasal cavities and travel via the ethlnoid bone into the brain where they cause extensive hemorrhage and tissue damage Death can occur in less than one week Contaminated outdoor pools and hot baths have been found to harbor aInoebae Plusmodium vivux mu luriue ovule and fulcipurum Protozoa in genus Plasmodium are sporozoans recognized as the causative agents of malaria The vector involved in the transmission of malaria is a mosquito in the genus Anopheles The parasites enter their mammalian host along with saliva from the mosquito bite and in this stage are called sporozoites These enter tissue cells exoerythrocytic stage such as the liver where they may persist indefinitely Eventually some parasites enter the bloodstreaIn erythrocytic stage where they infest RBCs and reproduce asexually via schizogony The red blood cells lyse and the parasites now called merozoites are released in great nu1nbers The maturation of parasites within the RBCs occurs at intervals of 48 hours in Plasmodium vivax ovale and falciparum and of 72 hours in malariae which accounts for the cyclic symptoms of the disease The rupture of infected RBCs releases hemoglobin and causes fever Symptoms begin with severe chills shivering teeth chattering etc followed by fever headache and nausea fever may reach as high as 106 degrees F Fever is followed by sweating and a drop in temperature sometimes to below normal that lasts until the next cycle begins Within the bloodstreaIn some of the merozoites undergo meiosis to form gametocytes A mosquito can pick up gametocytes from the blood of a mammalian host and the parasites undergo sexual reproduction syngamy within the mosquito gut The naIne Malaria means quotbad airquot and was applied due to the association of disease with the air of swamps before the mosquito connection was recognized It was also called black water fever due to the production of dark urine from hemoglobin breakdown Toxoplusmu gondii Sporozoans identified as Toxoplasma gondii are recognized as the causative agents of toxoplasmosis and are known to infect all kinds of mamlnals and birds In the 197039s it was estilnated that between 17 and 35 of all Americans carred the organisms Dogs and cats also serve as reservoirs and recent findings suggest the protozoa change the behavior of rodents to increase their susceptibility to predation by cats Transmission can occur from contact with feces or esh of infected anilnals eating raw meat and can also occur prenatally Symptoms in adults include fever rash enlarged lymph nodes and eye disturbances The parasites may be found free in the blood or within various tissue cells In fetuses or infants born infected nervous system daInage is common and fatal encephalitis is not unusual Pregnant women and infants can be tested for the presence of antibodies against Toxoplasma Cryp tosporidium paryum Protozoa identified as Cryptosporidium parvum are the most common cause of cryptosporidiosis in imlnunocompromised patients The organisms develop within the microvilli of the intestinal mucosa and can be transmitted through animal feces and contaminated water Some noscomial hospital acquired cases have 00 0 also occurred These parasites can also infect the lungs and the gal bladder causing potentially deadly disease symptoms Bu luntidium ca 1139 Although most ciliated protozoa are freeliving some are parasites and some are pathogens Balantidium coli are ciliates that are parasitic in the gut of man and other animals They enter their human hosts in cyst form along with contaminated food Digestive enzymes in the gut dissolve the cyst walls releasing the trophozoites within the colon In most cases these feed on bacteria and fecal debris without causing disease symptoms but rarely they invade the mucosa and submucosa of the gut causing abscesses and ulcerations Symptoms typically include chronic diarrhea alternating with constipation but can result in severe dysentery Fatal cases occur occasionally Hemflagellates Flagellated protozoa inhabiting the bloodstream are known as hemo agellates and include organisms in two genera Trypanosoma and Leishmania Species of Trypanosoma cause African sleeping sickness and Chaga39s disease while Leishmania organisms cause a variety of diseases including Kalaazar and oriental sore All hemo agellates live within the circulatory system of their host and are transmitted by insect vectors a T 39 Hemo agellates identified as I J r o 39 are recognized as the causative agents of African sleeping sickness and are transmitted by insect vectors Tse tse ies in the genus Glossina These trypanosomes may be passed to many alternate hosts antelope pigs monkeys dogs etc that form a huge reservoir in addition to humans The Trypanosomes live in the salivary glands of the flies and so enter their host when the flies bite to feed The bites itch and form lesions and the following infection is accompanied by fever and headache at irregular intervals In this form it may last for weeks or months making the victim susceptible to other diseases Eventually if not treated the diseasecausing organisms may enter the cerebrospinal uid CSF and the symptoms of true quotsleeping sicknessquot occur During this stage the victim quotsleepsquot a lot and is unable to engage in physical activity Muscular convulsions and trembling are common If not treated coma and death are inevitable If the diseasecausing organisms do not enter the CSF the symptoms disappear spontaneously and the hosts recover Prevention requires control of the Tsetse ies or elimination of the alternate hosts which is highly unlikely o b Trypanosomu cruzi Hemo agellates identified as Trypanosomu cruzi are recognized as the causative agents of Chaga s disease and are transmitted by insect vectors quottrue bugsquot in the genus Triatoma called quotkissing bugsquot that feed on human blood The trypanosomes enter the body when infected excrement deposited by feeding bugs is scratched into the wound They then enter the bloodstream or lymphatic system and migrate to tissue cells The trypanosomes reproduce within the tissue cells and are later released when the cells lyse Symptoms include edema at the site of infection at bite or conjunctiva with later development of acute headache fever and sometimes severe skin lesions especially in children Chronic infections may involve enlargement of the liver spleen and lymph glands with anemia and nervous system disorders Treatment is not overly successful and chronic infections may result in death Prevention involves elimination of the vectors Currently the protozoa responsible for causing malaria sleeping sickness Chaga39s disease dysentery and other diseases are more commonly encountered in tropical regions than here in the United States Microbiologists are concerned that changes in world climate will result in more widespread disease as vectors expand their ranges in response to global warming Cell Membrane Structure and Function The cell 39 c r 39 39 39 or plasma membrane a structural component of all living cells is a living dynamic layer that surrounds and limits the cell It is sometimes referred to as an invisible surface layer because it is too thin to be visible with a light microscope A typical cell membrane is about 8nm thick which is considerably thinner than the skin on an apple or the colorful film forming the wall of a large soap bubble The cell membrane is said to form a selectively and differentially permeable barrier because it allows only certain types of substances to pass through into or out of the cell and because it changes over time It is alive and not static Membrane Structure Eukaryotic cell membranes are typically composed of lipids and proteins in a near 5050 ratio ie they contain about 50 lipids and 50 proteins of various types Prokaryotic membranes contain 40 lipid and 60 protein The most commonly accepted model for the structure of the cell membrane the Fluid Mosaic Model was developed by Singer and Nicholson in 1972 and shows proteins quot oating like ice bergs in a sea of lipidquot Most membrane illustrations are based on this model and show lipids arranged in a bilayer with proteins oating in and sometimes penetrating through this layer The lipid bilayer portion of a bacterial cell membrane is composed primarily of phospholipids These are amphipathicamphiphilic molecules ie have both polar hydrophilic and nonpolar hydrophobic portions and are arranged with their hydrophobic tails toward the inside of the membrane and their hydrophilic heads toward the watery environment either inside or outside the cell The orientation of these molecules is in uenced by their solubility in either lipid or water and is a major factor maintaining the structural integrity of the membrane itself In eukaryotic cells the lipid bilayer is about 65 phospholipids 25 cholesterol and 10 other types of lipids Since prokaryotic cells generally lack cholesterol though some bacteria have sterollike lipids the cell membranes represented in most illustrations are prokaryotic bacteria specifically The cell membranes of Archaea do not contain phospholipids at all The proteins associated with cell membranes are usually globular in form and also amphipathicamphiphilic They can be divided into two categories based on their location and degree of interaction with the lipid bilayer Proteins categorized as integral proteins intrinsic proteins extend into and sometimes all the way through the membrane and cannot readily be removed without causing structural damage Proteins categorized as peripheral proteins extrinsic proteins sit on the membrane surface either inside or outside the cell and can be easily removed Many of the proteins associated with cell membranes occur as complexes and some are glycoproteins ie proteins bound to polysaccharide chains Glycoproteins found on external cell surfaces often serve as active or reactive sites involved in the regulation of cellular responses Membrane Functions The cell membrane is a living functioning portion of the cell and is associated with a number of cellular processes including 1 Separating the cell from the environment surrounding it Recall that the cell 2 membrane contains and limits the cell Controlling what types of materials enter and exit the cell Since the movements of materials into and out of cells are essential to cellular function membrane transport mechanisms have been extensively studied Some of the bestunderstood mechanisms are described below A Diffusion Diffusion can be defined as the movement of particles to fill an available space and occurs due to the random activity of molecules It may involve liquids solids or gasses Since it is a passive transport process ie does not require the cell to expend energy the net movement of particles will always be down a gradient either a concentration gradient chemical gradient or an electrical gradient A concentration gradient exists when there is a difference in the concentration of particles between one area and another Although particles move at random the net direction of movement will be from an area of hig concentration to an area of lower concentration from high to low so the particles are said to travel down the gradient Charged particles ions are influenced by concentration but also by the net charge existing between opposite sides of a membrane Particles with different charges cations and anions are attracted to one another while those with the same charge repel one another Charged particles can be induced to move against their concentration gradient by a strong electrical gradient Diffusion can be categorized as simple or facilitated depending upon the types of particles moving and how they pass through the membrane Simple diffusion does not require a membrane but can occur across one while facilitated diffusion requires a membrane because integral proteins are necessary to facilitate the transport process Gasses such as oxygen and carbon dioxide are among those particles able to move into and out of cells via simple diffusion Both can pass through the lipid portion of the membrane but the direction they travel varies depending on the type of cell being considered In the case of animal type cells oxygen 02 tends to move inward while carbon dioxide C02 moves outward This is because much of the oxygen entering an animal type cell is quickly converted into water keeping the oxygen concentration low while carbon dioxide is constantly being formed through the catabolism of carbohydrates In the case of plant type cells actively engaged in photosynthesis just the opposite is true Carbon dioxide will move inward because it is being converted into carbohydrate and oxygen will diffuse outward because it is being produced within the cell as a waste gas from the splitting of water molecules lons charged particles can also move passively across cell membranes but require a protein channel to do so The proteins facilitate the diffusion process make it easier so the process is called facilitated diffusion Channel proteins may be complexes and are sometimes equipped with gates that if closed will restrict ion ow Small organic compounds such as monosaccharides amino acids nucleotides etc can also move across membranes by facilitated diffusion but only down their concentration gradients The proteins involved undergo configurational changes during the process recall revolving door analogy In some cases the proteins facilitating particle movement are specific ie allowing only one type of molecule to pass and in other cases they are more general allowing a variety of similar molecules to pass The term permease is sometimes applied to proteins involved in facilitated diffusion but not all permeases allow passive transport Osmosis Osmosis is sometimes de ned as the diffusion of water though a membrane but has a more technical de nition It is the movement of solvent water in biological systems from an area of low solute concentration high water to an area of higher solute concentration low water through a selectively permeable membrane ie one permeable to the solvent but not to solute particles This can be thought of as the movement of water from an area of high water concentration to an area of lower water concentration or down a concentration gradient however chemically speaking there is no such thing as water concentration Osmosis provides the mechanism by which water in uences cell size and shape because water entering a cell will cause it to swell up while water leaving a cell will cause it to shrink In either case excess water movement can cause cell damage and sometimes death Because the movement of water through cell membranes is in uenced by the concentration of solute particles we can think of these particles as exerting pressure In reference to uid environments surrounding cells the term tonicity is equivalent to effective osmotic pressure ie the pressure causing water to move by osmosis Though technically osmotic pressure is measured by preventing the movement of water An enviromnent is isotonic iso the salne if the concentration of solute particles is equal to that found within the cell The osmotic pressure is equal on either side of the membrane and there is no net movement of water An enviromnent is hypotonic hypo under or beneath if it has a solute concentration lower than that found within the cell as would occur in a water enviromnent Cells placed into hypotonic enviromnents will take on water through osmosis unless they are protected by cell walls Water will readily enter hulnan RBCs placed in hypotonic enviromnents and will cause them to rupture but this doesn39t happen to bacteria because they are protected by cell walls An enviromnent is hypertonic hyper over above or excessive if the solute concentration is greater than that found within the cell In this case water will leave the cell and the cell will shrink sometimes collapsing 0 Exactly how water passes through cell membranes appears to vary depending on the cell type Sometimes it moves through channel proteins but evidence indicates this is not always the case Some cell membranes can prevent osmosis from occurring but others cannot Active Transport Active transport is a process allowing cells to move particles through membranes against their concentration and or electrical gradients Like facilitated diffusion it involves integral proteins but unlike diffusion it requires energy ie it is not passive The protein complexes involved in active transport are often referred to as pumps because they use energy to move particles through or across membranes When a protein pump moves two different types of particles in opposite directions at the same time the particles are moving in antiport the protein may be called an antiporter or antiport The sodiurnpotassiurn pumps associated with cell membranes move particles in this manner If the pump moves two different types of particles in the same direction at the same time the particles are moving in symport the protein may be called a symporter or symport Glucose can be moved in symport with either hydrogen or sodium ions as explained below Some protein pumps are very speci c and will only move one type of particle ie move particles only in uniport Adenosine triphosphate ATP is the highenergy compound most commonly used to drive active transport processes but it may be used either directly or indirectly Some cells use ATP driven protein pumps to move hydrogen ions across membranes in uniport and then use the concentration gradient established to move glucose in symport with hydrogen as the hydrogen ions move back across the membrane down their concentration gradient The glucose is actively transported even though the hydrogen is moving by facilitated diffusion because ATP was required to create the hydrogen gradient in the first place Yeast cells placed in a 5 glucose solution can initially take in glucose passively through facilitated diffusion however when the glucose concentration outside the cells becomes lower than that inside the yeast cells will switch to active transport Diffusion both simple and facilitated osmosis and active transport are used by both prokaryotic and eukaryotic cells but some prokaryotes can also use another process called group translocation or group transport Group translocation Group translocation sometimes called group transport is a mechanism allowing bacteria to move molecules through membranes down their concentration gradients by changing them into different types of molecules during the transport process For example bacteria taking in glucose molecules through group translocation bind a phosphate group to each one as it passes through the membrane The phosphorylated glucose glucose6phosphate cannot exit the cell membrane is not permeable to it so accumulates inside the cell Because this mechanism requires energy it is considered active but it does not involve ATP directly E Endocytosis Endocytosis is a transport mechanism associated with eukaryotic cells only It involves the invagination inward folding of a small section of the cell membrane and the pinching off of that portion to form a vacuole or vesicle inside the cell Typically the materials being taken in are bound to receptors on the outer cell surface and this triggers the invagination process Endocytosis can be divided into two categories based on the size of the particles being taken in If the particles are large cellular in size the process is called phagocytosis or cell eating If the particles being taken in are small molecular in size the process is called pinocytosis or cell drinking Singlecelled anjlnallike organisms known as protozoa typically consulne food materials by means of phagocytosis Phagocytosis is also the process used by human WBCs when they consulne bacteria F Exocytosis Exocytosis is a process allowing cells to release various materials to the outside by joining vacuoles or vesicles with the cell membrane from the inside Although this process is sometimes also called emiocytosis a term meaning cell vomiting the materials being released are not necessarily waste Many cells release enzymes into their environ1nents by this means and enzymes have multiple different functions Both endocytosis and exocytosis are considered to be active transport mechanisms because both require energy ATP 3 Influencing taxis Taxis is the directed movement of cells within their environment and often involves receptors usually glycoproteins associated with the cell membrane Cell membranes are equipped with various receptors telling the cell what is going on in the environment Stimuli perceived by these receptors trigger internal responses resulting in cell movement In the case of prokaryotic cells movement also involves the cell membrane because prokaryotic agella are driven by membrane quotmotorsquot lf movement is directed toward a stimulus it is called positive taxis and if it is directed away from a stimulus it is called negative taxis Different types of stimuli trigger different types of taxis as indicated below Phototaxis movement directed by light Organisms capable of using light as an energy source will frequently display positive phototaxis ie will move toward light Organisms preferring dark habitats will move away from light so will display negative phototaxis Chemotaxis movement directed by chemicals Animallike organisms such as protozoa display positive chemotaxis as they move toward a food source and negative chemotaxis as they move away from toxic chemicals or potential predators Magnetotaxis movement directed by magnetic fields Magnetotaxis involves structures called magnetosomes made up of magnetic crystals surrounded by cell membrane Cells displaying positive magnetotaxis often travel toward iron deposits Geotaxis movement directed by gravity Organisms display positive geotaxis when they move downward or into a sediment layer and negative geotaxis when they move upward or away from sediments 4 Synthesizing ATP and facilitating other metabolic processes Although ATP synthesis occurs in association with internal organelles mitochondria and chloroplasts of eukaryotic cells in prokaryotic cells much of it occurs in association with the cell membrane The processes involved oxidative phosphorylation and photo phosphorylation will be described in detail later Prokaryotic cell membranes contain a variety of enzymes involved in metabolic processes including the synthesis cell wall constituents and portions of the cell membrane the replication of DNA and formation of septa required for cell division light production and some carbon dioxide fixation Quorum sensing Quoruln sensing involves receptors in bacteria cell membranes sensitive to signaling molecules released by other bacteria of the same type When the signaling molecules called autoinducers or pheromones bind with membrane receptors they trigger the activation of genes in uencing a variety of cellular functions including the formation of more membrane receptors Quoruln sensing is involved in such bacterial activities as biofilm formation light production cell aggregation release of toxins and production of enzymes required for digestion of nutrients Harriet Big Introduction to Fungi Fungi singular fungus Fungi can be defined as nucleated achlorophyllous osmotrophic sporebearing organisms that typically reproduce both sexually and asexually and whose usually branching filamentous bodies are surrounded by cell walls composed of cellulose chitin or both The science or study of fungi is called mycology mykes mushroom and originated as the study of macroscopic organisms such as mushrooms puffballs and bracket fungi These organisms were often collected as food items but the ingestion of toxic fungi sometjines resulted in fatality and sometimes caused peculiar neurological reactions quotthey did funny things to the headquot It was important that people collecting fungi knew something about them Fungi are classified within the domain Eukarya and according to the Whittaker five kingdom system are categorized within the kingdom Fungi Myceteae although other references divide them between the kingdoms Chromista and Eumycotina Fungi are eukaryotic organisms with true nuclei surrounded by nuclear envelopes They are equipped with a variety of organelles but unlike plants do not have chloroplasts The term achlorophyllous means without chlorophyll a without chlorophyll green light sensitive pigment Green pigments are commonly associated with lighttrapping ability in green plants algae cyanobacteria and other phototrophic organisms but fungi do not have this ability Fungi are nutritionally categorized as Chemoheterotrophs like us Chemoheterotrophs chemo chemicals hetero different troph feeding or activity are organisms capable of using preformed organic compounds for both energy and carbon Fungi are osmotrophic chemoheterotrophs which means they take in nutrients in liquid form Fungi digest their food or nutrient materials outside their bodies by du1nping digestive enzymes into their environments Once the food has been broken into smaller components molecules it can readily be taken into the fungus cells Most fungi are saprotrophs sapros rotten and use dead or decaying organic material for food these forms are also called decomposers Some fungi are parasites living on other live organisms and some are pathogens causing disease symptoms Fungi can be divided into three general categories yeasts molds and eshy fungi based on their morphology however it should be noted that many types of fungi can change form and do so on a regular basis Yeasts are singlecelled fungi with growth habits similar to bacteria ie when grown on nutrient media form circular convex smooth shiny colonies in various colors Molds are filamentous fungi with microscopic thread like morphology When grown on nutrient media they form fuzzy hairlike masses that often extend considerably above the agar sometjines filling the entire container Fleshy fungi are those forming macroscopic fruiting bodies commonly encountered in gardens fields and forests during periods of wet weather Though these appear solid they are also composed of filaments so are structurally similar to molds The body of a moldtype or eshy fungus body soma or thallus is made up of microscopic threadlike filaments called hyphae singular hypha Hyphae grow and extend outward from each germinating fungus spore reproductive body and penetrate into whatever nutrient material is available to them As the hyphae multiply they eventually form a filamentous mass or mat visible to the naked eye and this is called a mycelium pleural mycelia As the myceliu1n matures it can be divided into two regions with separate and specific functions That portion of the myceliuln extending down into the nutrient material is called the vegetative mycelium and is involved in food getting That portion extending upward into the air is called the aerial mycelium and is reproductive The bluecolored mass on the surface of a rotting orange or the gray fuzzy looking mass on the surface of a rotting strawberry is aerial myceliu1n If the bluecolored mass is on the surface of your favorite cheese remember it is being supported by a vegetative myceliuln extending below the surface Whether to eat or not to eat is a personal decision Hyphae are composed of multiple nucleated cells surrounded by cell walls but these may or may not be separated ie hyphae may be either septate or aseptate Septate hyphae have crosswalls or septa singular septuln between the individual cells while aseptate hyphae do not Sometimes the cells of septate hyphae contain two separate haploid nuclei each and are called dikaryons dikaryon two nuclei The cells of aseptate hyphae are not separated by crosswalls so obviously form a true syncytium ie a multjnucleated mass of protoplasm The crosswalls of septate hyphae are not complete because they have holes in them like lifesavers so the cells of septate hyphae form a true syncytiuln also This is significant because materials can readily move from cell to cell within a fungus and the hyphae of the vegetative myceliuln can share nutrients with those of the aerial myceliuln The wall materials formed by fungus cells are polysaccharide usually cellulose chitin glucan or a combination of these Walls provide support and protection for the protoplasm and give the fungi their characteristic shape singlecelled Vs filaInentous Though all moldtype and eshy fungi have hyphae the size of these structures vary considerably Some hyphae are truly microscopic and only visible with the aid of microscopy while others are thick hairlike and may grow to be several inches in length Specialized types of hyphae The hyphae associated with vegetative mycelia differ from those associated with aerial mycelia in terms of function but both types can be divided into still more specific categories depending on where they are found Some specific examples of specialized hyphae are listed below 1 Hyphae supporting different types of asexual spores can be named in association with the spores they support for example sporangiophores support the saclike sporangia containing r g39 I an 39 39 support the beadlike conidiospores developing at the ends of phialids r 2 Haustoria Haustoria haust to suck are specialized hyphae produced by parasitic fungi These penetrate into the cytoplasm of host cells and absorb nutrients Many fungi considered as plant pathogens form haustoria 3 Mycorrhizae Mycorrhizae are specialized hyphae allowing fungi to form mutualistic relationships with the roots of forest trees and other plants Some mycorrhizae endomycorrhizae penetrate root cells and others ectomycorrhizae wrap around the root surface but both types share materials with their plant hosts Mycorrhizae help plants absorb minerals primarily phosphorous and water from soil while the fungi obtain nutrients primarily sugar from the plant The fungi also produce chemicals antibiotics that help protect the plant from infection by bacteria This is an important symbiotic relationship and without their fungal symbionts many plants cannot develop properly cannot reproduce and sometjnies die Fungus Reproduction Most fungi reproduce both sexually and asexually although asexual reproduction is a silnpler process and much more common Fungus cultures maintained under laboratory conditions will reproduce asexually over and over again and only form sexual structures under specific conditions Would you believe soft music and candlelight Asexual reproduction does not require the reorganization of genetic materials so can be accomplished silnply by one cell dividing itself into two parts This can occur in various ways as indicated below Asexual reproductive Processes 1 Binary fission During binary fission one cell divides itself in half across the lon axis giving rise to two new daughter cells Since most fungi form hyphae fission does not necessarily involve physical separation of the cells present but does involve mitosis separation of the chromosomes 2 Budding Budding involves the uneven division of cytoplasm during the fission process such that one daughter cell receives most of it and the other the bud receives only a small alnount though both cells gain a full compliment of genetic material If conditions are good the bud grows until it reaches full size and separates if not the bud can be eljlninated with little loss to the original cell Asexual spores called blastospores are essentially buds as are microconidia 3 Fragmentation Fragmentation can occur after fission has allowed the formation of numerous cells in multiple hypha During fragmentation the hyphae break into little pieces or fragments and each one has the potential of forming a new fungus Asexual spores known as mquot r and 39 39 J J r are e PnHallv thickwalled fragments formed by the fragmentation of modified hyphae 4 Spore formation sporulation Asexual spores known as sporangiospores and conidiospores are formed at the ends of specialized hyphae usually and typically occur in large nulnbers as described below Characteristics of asexual spores such as arrangement and color are often useful in fungus classification a Sporangiospores are contained within saclike structures called sporangia singular sporangium supported by hyphae called sporangiophores These may be rounded or linear in shape as demonstrated by the Rhizopus and Saprolegm39a observed in the laboratory Some sporangia occur as saclike pustules on the undersides of plant leaves and are essentially formed by the plant epidermis as demonstrated by Albugo b Conidiospores are arranged lillte beads on a string at the ends of hyphae called conidiophores Conidiospores are formed by specialized hyphae called phialids and arise through fission at the phialid tip Older conidiospores are moved farther away from the phialid as new conidiospores form The arrangement of phialids on conidiophores is variable and often useful in fungus classification Sexual Reproductive Processes Sexual reproduction requires the participation of two genetically dissi1nilar fungi of the same species and typically occurs in three stages or steps as outlined below 1 Plasmogamy Plasmogamy involves the joining of the protoplasm plasma protoplasm gaInous union or marriage and requires decomposition of the cell walls separating the hyphae involved 2 Karyogamy Karyogamy involves the joining of two haploid nuclei karyon nucleus and results in the formation of a diploid cell called a zygote Haploid cells nuclei have only one set of chromosomes while diploid cells nuclei have two 3 Meiosis Meiosis reduction division is a process involving separation of chromosomes and the division of the diploid nucleus into two haploid parts An i1nportant feature of meiosis is the formation of new genetic combinations not possible through asexual reproduction These stages may be separated in time and organisms categorized as dikaryotic fungi typically form masses of hyphae carrying pairs of haploid nuclei after undergoing plasmogamy These eventually undergo karyogaIny and meiosis but take their time about it Though meiosis as it occurs in plants and animals has been thoroughly studied and appears to be fairly consistent meiosis occurring in microorganisms is quite variable Fungi typically show no sexual dimorphism ie lack structures recognized as male Vs female so fungi engaging in sexual reproduction are often designated as plus and minus strains During karyogamy plustype and minustype haploid nuclei join and form a structure called a diploid zygote however because the DNA forming the chromosomes typically replicates prior to meiosis the zygote is actually tetraploid each Xshaped chromosome is made up of two chromatids or two copies of the DNA present During meiosis chromosomes line up along the middle of the cell and are pulled apart by microtubules the spindle apparatus Typically there are two sets of cell divisions chromosome separations involved with chromosomes lining up in pairs during the first set and chromosomes lining up in single file during the second The recombination of genetic material resulting in genetic variation occurs during the first division cycle as chromosomes pair up and then separate The chromosomes aligned in chromosome pairs often exchange segments and pairs separate randomly giving rise to different combinations The formation of haploid cells or gametes occurs during the second division cycle as each chromosome is divided into two chromatids Sexual reproduction results in the formation of haploid sexual spores J 390 t J as r A g r ascospores or basidiospores depending on their source arrangement and means of formation Signi cance importance of fungi Fungi are significant to us and to other organisms in a variety of ways some of which are listed below 1 Many fungi are saprotrophs or decomposers that break down dead decaying materials releasing nutrients into soils Imagine what this world would be like if dead organic materials wood leaves grass etc did not break down Although the random decomposition of organic materials is sometimes irritating detrimental for humans the benefits of this activity far outweigh the costs 2 Many fungi form mycorrhyzae that aid forest trees and other plants with the uptake of minerals and water In some forest regions the harvesting of mushrooms by humans has depleted the fungus populations so much that trees are dying 3 Some fungi serve as a food source for other organisms including humans 4 Various types of fungi are used in food processing eg the production of cheese beer wine bread etc 5 Some fungi are a source of antibiotics e g Penicillins and Cephalosporins chemicals used to control pathogenic bacteria inside the human od 6 Many fungi form enzymes that can be used for industrial processes 7 Many types of fungi produce organic acids and solvents as fermentation products and these have multiple uses Some fungi form organic compounds equivalent to jet fuel 8 Fungi can be genetically engineered to produce complex proteins originating in other organisms 9 Some fungi are pathogens or diseasecausing agents and some produce hallucinogenic substances such as LSD Though these features are not beneficial to humans they are still significant Note Various types of bacteria are significant in all the ways listed for fungi and in addition produce oxygen and methane Bacteria are also being investigated as a source of hydrogen and may be incorporated into computer technology Medical Mycology Although most fungi are saprotrophs and not damaging to other organisms some fungi parasitize plants or animals and some are pathogens responsible for disease symptoms Some plant diseases caused by fungi include Dutch elm diseased chestnut blight potato blight apple scab peach leaf curl corn smut rusts and mildew Since the majority of students taking this class are interested in the allied health fields some medically significant fungi will be described in greater detail Mycoses Mycoses singular mycosis are fungal induced diseases occurring in humans and other animals Though living standards have improved for many people fungal infections remain a significant problem and the incidence of human mycoses has actually increased over time In addition to the obvious increase in the human population the increase in mycoses can be attributed to three major factors 1 Widespread use of antibiotics and other antimicrobial drugs These are used to control bacteria inside the human body and when bacteria are eliminated the delicate balance between organisms categorized as normal ora is upset Fungi gain access to more resources grow rapidly and begin to cause damage to host cells and tissues 2 Increased use of chemotherapeutic agents in the treatment of cancer and organ tissue transplant patients These cause can cause significant damage to immune cells 3 HIV infection and damage to the immune system resulting in Acquired Immune Deficiency Syndrome AIDS All of these factors weaken the immune system either by creating an inlbalance in normal microbial populations or by damaging the host cells involved in defense The primary factor determining if or not a person will be infected by fungi is the state of that individual39s immune system If the inlmune system is functioning normally fungal infection is unlikely but if the immune system is damaged or weakened any fungus can become a pathogen Since fungi are abundant in the enviromnent this is a significant problem Fungal infections can be divided into categories based on what portions of the body are involved For simplicity we will divide the mycoses into three categories as follows 1 Superficial mycoses Superficial mycoses occur on the body surface and are caused y fungi infecting skin hair and nails These tend to be chronic long term and often cause considerable irritation but are generally not life threatening The fungi responsible for superficial mycoses are often called dermatophytes and include genera such as Er 391 r 1 Jim 39 1 r 1 J and M39 r Infection is not limited to hulnans and the symptoms caused by these fungi are often worse when the fungi are transmitted from nonhulnan anilnals to people Some commonly encountered superficial mycoses include a Tinea pedis or athlete39s foot caused by Epidermophyton accosum b Tinea capitis or ringworm of the scalp caused by Microspomm or Trichophyton c Tinea corporis or ringworm of the body caused by Microspomm gypseum or Trichophyton species Fungal infections commonly called ringworm were named for the puffy redcolored ringlike lesions formed on the skin surface These are caused by fungal waste products irritating to the skin but were once believed to be formed by wormlike organisms living under the skin Because dermatophyte fungi are widespread and produce nulnerous tiny asexual spores these are often present on gym oors and pool decks in locker rooms and laundry facilities and sometilnes in other locations frequented by hunlans 2 Subcutaneous mycoses Subcutaneous mycoses involve damage to the tissue layers located just below the skin and typically appear as small wartlike bulnps on the skin surface The fungi involved are usually organisms found in soil or vegetation introduced by a puncture scratch cut or other wound Though usually limited by inlmune mechanisms superficial mycoses can sometimes spread along lymphatic ducts and result in more severe tissue damage They are rarely if ever life threatening Two types of commonly encountered superficial mycoses include a Sporotrichosis Sporotrichosis is caused by fungi identified as Spam trix schenckii and typically occurs in persons working with plants sphagnuln moss or hay Sometimes referred to as quotrosethorn diseasequot sporotrichosis typically involves lesions on the hands arms or feet as the result of contact with thorns pine needles twigs or wire Over time the wartlike bulnps may become painful boillike lesions eventually forming open sores that are slow to heal Chromomycosis Chromomycosis chromoblastomycosis is naIned for the dark colored lesions associated with infection involving various different types of black molds dematiaceous fungi This type of infection is often chronic but not life threatening Deep or systemic mycoses Deep or systemic mycoses involve fungi that enter deep tissues heart liver brain etc by becoming associated with the lymphatic and or cardiovascular systems and traveling throughout the body These fungi typically enter their host through the respiratory system as spores are breathed in They can cause in uenza or pneulnonialike symptoms initially but if not controlled by immune mechanisms can spread into the circulatory system and cause daInage throughout the body a Coccidioidmycosis Coccicioidomycosis also called desert rheulnatism valley fever or San Joaquin Valley fever is caused by a type of soil fungus identified as Coccidioides immitis These fungi occur abundantly in certain regions including the American Southwest and in the southern portion of California39s central valley Arthrospores fragments of hyphae produced by these fungi are abundant in certain soils and can readily be picked up and transported by the wind Most people exposed to arthrospores are asymptomatic ie experience no disease symptoms but ulike symptoms occur in some individuals Between 1 and 10 of those experiencing ulike symptoms later develop a red rash indicative of an allergic or hypersensitivity reaction and this is sometjlnes accompanied by joint pain In persons with poor immune function the fungus is not controlled and spreads throughout the body causing lesions in various organs potentially resulting in death Coccidioidomycosis is usually restricted to specific regions where the fungus is prevalent in soil but during the drought years of 1977 winds carried soil particles and fungus spores as far north as SanFrancisco A nulnber of cases were also reported in the Los Angeles area following the Northridge earthquake of 1994 Histoplasmosis Histoplasmosis is caused by a type of fungus identified as Histop lusmu cupsu la tum These fungi prefer soils rich in nitrogen so are often abundant in areas containing bat and or bird droppings People frequenting caves spelunkers and those working in guano mines are at high risk of developing histoplasmosis Fungus spores are breathed in and the resulting infections cause varying degrees of respiratory damage Some individuals experience chest pain and a dry cough some develop pneulnonialike symptoms and some develo chronic tuberculosislike infections resulting in tissue damage and scarring If not controlled by the ilnmune system these fungi can enter the bloodstream become widely disseminated and cause potentially fatal lesions throughout the body Fungi as Opportunistic Pathogens Fungi not usually associated with disease but able to cause disease symptoms under certain circulnstances are referred to as opportunistic pathogens and the diseases they cause are called opportunistic mycoses Some of the fungi involved are part of our quotnormal oraquot ie organisms normally found living in or on the hulnan body but many are fungi common to soil and vegetation Opportunistic mycoses occur in individuals with compromised immune function and for these individuals virtually any fungus is a potential pathogen Some fungi frequently associated with opportunistic mycoses are listed below a Candida albicans Candida is a type of yeastlike fungus commonly encountered as an inhabitant of the human body forms part of our normal ora Candida populations are usually kept in check by bacteria competing with them for space and nutrients When bacteria populations are reduced as occurs during treatments involving antibiotics these fungi can reproduce rapidly and cause tissue damage resulting in disease symptoms Candida is commonly associated with thrush in the mouth vaginal and eye infections but in severely iminunocompromised individuals this fungus can cause potentially fatal septicemia infection in the bloodstream resulting in disease symptoms b Cryp tacoccus neoformans Cryptococcas is a type of yeastlillte fungus commonly associated with bird droppings Spores from this fungus gain access to the body through the respiratory system enter the bloodstream and migrate to brain where they can cause a potentially fatal form of meningitis C ryptococcas lillte Candida is most dangerous to severely iinmunocompromised individuals F V Aspergillus famigatus Fungi in the genus Aspergillas including A famigatas A niger and others are commonly associated with lung infections in iminunocompromised individuals The conidiospores formed by these fungi are small readily airborne and easily breathed in Various species of Aspergillas have also been found to cause deep or systemic mycoses when introduced through severe tissue trauma associated with lawn mower and chain saw accidents d Rhizopus sto lonifera Though rarely considered as pathogenic Rhizopas stolonifer and other species can sometimes form fungus balls in lung tissue They enter the lungs through respiration and because they are aerobic find the environment well suited to their growth Fungus balls typically occur in lung tissue damaged by prior infection conditions and in individuals lacking normal immune function e Pneumocystis carinii Fungi in the genus Pneumocystis are opportunistic pathogens commonly associated with pneumonia in AIDS patients Pneumocystis carinii pneumonia or PCP Previously categorized as protozoa these fungi initially cause symptoms including dry cough fever and breathing difficulty If left untreated they can cause potentially fatal pneumonia Mycotoxins and Intoxication Various types of fungi produce toxic substances collectively referred to as mycotoxins mycotoxins toxic substances produced by fungi Ingestion of fungus toxins can cause intoxication poisoning due to ingestion of toxic substances sometimes resulting in reduced liver function hallucination or death depending on the type of fungus involved Some examples of fungi known to produce mycotoxins are listed below a E F V Amunitu phalloides Amanita phalloides is a type of eshy fungus often found growing in damp wooded areas This and other species within the Amanita genus produce potent mycotoxins known to cause severe liver damage and sometimes death when ingested Although some species of Amanita are brightly colored and easily avoided others are not and intoxication due to ingestion of Amanita occurs with surprising frequency Aspergillus fluvus Aspergillus avus is a type of mold known to produce a potent mycotoxin called A atoxin A Aspergillus fla avus toxin Fungi in the genus Aspergillus are very common in the environment growing in association with soil and vegetation and A atoxin is often found in association with grain products and peanuts A atoxin is one of the most potent carcinogens cancercausing agents known to man and ingestion of foods contaminated with it can result in sever liver damage C luviceps purpum Claviceps purpum is a type of mold commonly associated with rye grain and responsible for causing a condition called ergot These fungi also produce hallucinogenic toxins lysergic acid derivatives LSD which when ingested can cause convulsive ergotism and hallucination The consumption of bread made with fungus infected rye grain and the resulting hallucination has been implicated as a major factor in uencing witnesses involved in the Salem Witch Trials of the 1600s Stuchybo trys utm S tachybotrys atm is a type of fungus sometimes referred to as black mold and commonly found growing on surfaces rich in cellulose eg wood dead leaves and the paper backing of wall board Detection of S tachybotrys inside buildings wetted by leaky roofs flooding or plumbing problems is problematic because exposure to this fungus can cause health problems Spores produced by S tachybotrys can cause hemorrhaging in infant lungs and inhalation of airborne toxins has been reported to cause headache dizziness and reduced immune function Note In addition to being pathogenic and producers of toxins many fungi release spores that are among the leading causes of atopic allergy hay fever in humans Host Defense Mechanisms Innate Immunity Microorganisms are abundant in the environment and though most of them are not pathogenic some of them are or potentially so depending on circumstances Fortunately the human body is well equipped to defend itself against a wide variety of potential pathogens Despite the quothypequot proliferated on television and radio normally functioning humans even young ones do not require that disinfectants and antiseptics be used regularly to quotprotectquot them from the multitude of quotgermsquot lurking in the environment In fact raising children in ultra clean environments is now recognized as having detrimental consequences e g increasing the incidence of allergic reactions Humans have been living with microorganisms for thousands of years and we are well adapted to their presence Immunity Immunity can be defined as resistance to or the ability to resist infection and disease Although immunity can be though of as being maintained by an immune system in actuality it is dependent on a variety of structures and mechanisms involving many different types of cells tissues and organs Humans vary considerably with respect to their ability to resist disease so for this section assume the information presented applies to a normal healthy individual with a fully functional immune system For convenience immune mechanisms are often divided into two categories innate non specific and adaptive or acquired specific immunity as described below Innate immunity Innate immunity is resistance to or ability to resist infection and disease that is built in or present at birth All immune mechanisms involve cells and tissues that are present when an individual is born but adaptive or acquired immunity typically involves some type of interaction with an external agent an antigen while innate immunity does not Innate immunity is generally nonspecific ie it provides defense against multiple different types of pathogens while adaptive or acquired immunity ends to be quite specific The cells tissues and immune substances involved in innate immunity are more varied and wide spread than are those associated with adaptive or acquired immune responses at lease superficially and often not considered part of the immune system proper though they are here Some important structures and defense mechanisms associated with innate immunity are described below 1 Skin and Mucous Membranes Dry skin and mucous membranes mucosa cover all external surfaces of the body and form an effective barrier between our cells tissues and the external environment These structures provide our first line of defense against infectious agents or potential pathogens Dry skin covers the external surfaces of our bodies and connects with mucous membranes in specific regions e g the oral and anal openings of the gastrointestinal tract urethral openings vaginal openings eyes nostrils etc while mucous membrane cover the internal surfaces Although the concept may seem foreign the gastrointestinal system is actually a tube running through the body and can be thought of as a long complex donut hole Material in the stomach or intestine in the lumen of the tract is technically outside the body Skin and mucous membranes provide both mechanical physical and chemical barriers against potential pathogens Mechanical aspects of dry skin include the following a The skin is multilayered ie it includes an epidermis composed of epithelial cells supported by an underlying dermis a layer of dense connective tissue The epidermis is composed of stratified squamous epithelium so is itself a multilayered structure with attened surface cells b Cells at the skin surface are highly keratinized ie contain high levels of keratin proteins These are tough insoluble fibrous proteins that interconnect to help make skin surfaces effective mechanical barriers Cells at the skin surface are dead and constantly being shed taking microorganisms with them Many of the bacteria commonly found on air plates are inhabitants of human skin shed regularly by their human hosts d The dermis is composed of dense connective tissue and forms a tough leatherlike barrier that is difficult to penetrate Collagen a long fibrous protein with great tensile strength is one of the prilnary components Note Leather is typically made from anilnal hides dermis layers so accurately represents dermis structure F V Chemical aspects of dry skin include the following a The skin surface is salty due to the evaporation of water at the skin surface during thermoregulation and the natural salt content of sweat perspiration Salt in association with keratin makes the skin surface hypertonic and inhospitable for many types of microorganisms b The skin surface is often acidic pH around 55 and this also tends to inhibit microbial growth as most bacteria prefer a pH around 7 This acidity is due primarily to the keratinization of epithelial cells as they move toward the skin surface c Oils and waxes produced by the sebaceous glands help waterproof the skin and prevent it from drying and cracking Some of these also inhibit microbial growth Mechanical aspects of mucous membranes include the following a Mucous membranes are multilayered ie like dry skin they always include an epithelial layer supported by an underlying layer of connective tissue The type of epithelium is variable b Mucous membranes are often covered with a thick sticky material called mucus Within the respiratory tract mucus traps dust and microorganism entering with inspired air and prevents them from reaching the lungs Mucus moistens and lubricates the mouth and esophagus allowing food materials to be readil masticated and swallowed Within the stomach mucus provides a protective layer preventing infection and damage to epithelial cells potentially caused by the acidic environlnent present Cervical mucus also helps prevent infection c Within the respiratory system the epithelium is ciliated and the cilia sweep potential pathogens trapped in mucus up and out of the airways Since smoking causes damage to cilia smokers are more likely to experience lung infections as bacteria are more likely to enter their lungs Chemical aspects of mucous membranes include the following a Mucus and other secretions often associated with moist surfaces eg tears and saliva contain lysozyme enzymes Lysozyme kills bacteria especially Gram N F V positive cells by causing the hydrolysis of peptidoglycan ie by breaking the covalent bonds linking N acetyl muramic acid with N acetylglucosalnine Lysozyme also acts as an opsonin ie a substance causing opsonization makin particles more attractive to phagocytes It can bind to bacteria making them more readily engulfed by phagocytic WBCs Mucous membranes and their secretions are conunonly equipped with antibodies inununoglobulins in the isotype IgA capable of binding with and sometjlnes immobilizing pathogens causing some opsonization or neutralizing toxic bacterial products IgA does not activate compliment proteins The pH in specific regions lined by mucous membranes is sometimes quite low acidic For example within the stomach it may 1 or 2 while within the vagina it is commonly 38 45 These acidic conditions tend to inhibit bacterial growth because most bacteria prefer a more neutral enviromnent Phagocytic white blood cells leukocytes Though many types of eukaryotic cells are capable of taking in materials through endocytosis certain white blood cells or leukocytes found within the human body are particularly adept at this activity These cells are commonly referred to as phagocytic white blood cells and include primarily monocytes and neutrophils a Monocytes Monocytes are large agranular leukocytes agranulocytes forming between 3 to 8 of the WBCs circulating in the bloodstream They are phagocytes produced by bone marrow stem cells called monoblasts They typically stay within the bloodstream for one to three days and then pass through the vessel walls by means of a process called diapedesis When they have entered the tissues monocytes may be generically referred to as histiocytes or macrophages but are commonly given specific different naInes depending on their anatomical location For exaInple those entering the liver are called Kupffer cells and those entering lymphatic tissues are called dendritic macrophages Although monocytes typically consume or ingest cells and particles coated with opsonizing proteins such as compliment factors or antibiodies they are also capable of recognizing certain pathogens by means of patternrecognition receptors toll like receptors or TLRs on their cell surfaces Following phagocytosis ingested materials are digested within phagosomes or food vacules by enzymes supplied by lysosomes Monocytes observed in prepared slides of blood smears are recognized by their large size bilobed horseshoe or kidney shaped nuclei and by the presence of phagosomes within their blue colored cytoplasm clearly visible as white spots Monocytosis the condition of having higher than normal numbers of monocytes in the peripheral bloodstream is often indicative of a disease state since monocytes tend to increase in number during chronic infections and stress responses Neutrophils Neutrophils also called polymorphonuclear leukocytes are medium sized granular leukocytes granulocytes forming around 70 of the circulating white blood cells They are the most commonly observed WBCs in a blood smear and are easily recognized by their dark staining polymorphic nuclei and pale lavender colored cytoplasm Since their cytoplasmic granules are not stained by commonly used reagents they are not readily visible f1 Although neutrophils act as phagocytes within the bloodstream they also undergo diapedesis and enter into tissue spaces especially during in ammation associated with bacterial infections Within tissues they display positive chemotaxis being attracted by chemicals including interleukin8 IL 8 gammainterferon interferon gamma or INF y and compliment C5a Neutrophils attracted to infected tissues not only consume bacteria and damaged cells but also secrete substances that trap and kill bacteria extracellularly Neutrophils are short lived cells with a half life of 4 10 hours in the bloodstream and surviving only 1 2 days after entering tissue spaces Neutrophilia an increase in the number of neutrophils within the bloodstream can occur in association with acute bacterial infections and acute in ammatory responses eg resulting from a heart attack or other infarct Reticuloendothelial tissues The reticuloendothelial tissues also called reticular connective tissues are sponge like tissues composed of reticular fibers surrounded by endothelial cells These are commonly inhabited by lymphocytes and phagocytic white blood cells monocytes that have left the circulation and form an important part of the immune system Reticuloendothelial tissues include the liver spleen lymph nodes thymus gland and a variety of cell accumulations associated with the gut Peyer39s patches and respiratory tract Reticuloendothelial tissues have two major functions 1 They filter body fluids blood and lymph removing dead cells pathogens and debris that are then readily consumed by phagocytes They allow for rapid communication between the phagocytes of the innate immune system and the lymphocytes of the adaptive or acquired immune system As we shall see later this communication often plays an essential role in the initiation of humoral immune responses 2 3 Inflammation in ammatory response ln amination an inflammatory response is usually the immune system39s first response to trauma and is characterized by an increase in redness rubor swelling tumor and temperature calor within an area of traumatized tissue typica accompanied by pain dolor Although in ammation is a complex process involving a variety of different cells and chemical substances it can be summarized as follows a b V Traumatized tissue cells including mast cells release in ammatory substances into the surrounding area Among these are histamine and prostaglandins Histamine is a powerful vasodilatory substance ie it causes relaxation of precapillary sphinctors allowing increased blood ow into capillary beds resulting in increased redness and heat within the area it also increases the permeability of capillary walls Prostaglandins are lipids with a variety of functions but in this case responsible for vasoconstriction beyond downstream from the area of trauma and increased capillary permeability Increased capillary permeability coupled with an increase in hydrostatic pressure due to vessel dilation upstream from and vasoconstriction downstream from traumatized area causes uid to move from the capillary into the tissue spaces resulting in swelling Increased capillary permeability also allows proteins including antibodies complement factors and fibrin to exit the bloodstreaIn and accumulate in the traumatized area This also changes capillary membrane dynamics resulting in uid movement into the tissue and additional swelling The swelling puts pressure on nerve endings causing pain Substances called leukotrienes are also released by mast cells in the area of trauma and have a variety of functions They cause vasoconstriction especially in venules downstreaIn from the trauma site increase capillary permeability and attract phagocytic white blood cells to the area Phagocytes including neutrophils and monocytes attracted to the area by leukotrienes pass readily through the vessel walls due to their increased permeability and begin to consume bacteria and dead damaged body cells Neutrophils also release lysozyme enzymes that kill bacteria e Antibodies entering the area lgG and lgM with blood can immobilize pathogens neutralize their toxins cause opsonization and activate complement factors f Complement factors proteins entering the area with blood and produced by monocytes cause opsonization and make holes in the cell membranes of pathogens bacteria fungi protozoa etc and sometimes in host cells as well Complement factors also attract phagocytes and stimulate mast cells to release more histamine g Pyrogens from various sources stimulate an increase in temperature fever Exogenous pyrogens are substances associated with pathogens eg the lipopolysaccharide material from bacterial cell walls or various toxins produced by cells Endogenous pyrogen also known as interleukin 1 is a protein produced by monocytes and by macrophages in reticuloendothelial tissues It causes fever by acting on the thermoregulatory center within the hypothalaInus Raising the body temperature often inhibits microbial growth and sometimes causes cell death F V E When functioning as a protective response in ammation increases blood ow to the traumatized area and improves access for phagocytes antibodies and complement factors The phagocytes then consume pathogens kill them with lysozyme and release endogenous pyrogen while antibodies and complement factors interact to immobilize neutralize opsonize and or blow holes in pathogens Fibrin serves to wall off the area preventing potential pathogens from spreading into other regions of the body and the infection remains localized If the initial trauma involves the entry of foreign objects eg splinters of wood rock fragments etc the in anunatory response may lead to an accumulation of puss dead bacteria and neutrophils around the offending object If interleukin 1 is released in sufficient quantity fever results and this also kills some types of pathogens Innate immune proteins Proteins involved in defending the body against a variety of different pathogens and potentially present in the body from the time of birth are generally considered as innate immune proteins Several different proteins fall into this category including interferons interleukins and complement factors a Interferons Interferons were initially named for their ability to interfere with the life cycles of cytolytic viruses but have a variety of other functions within the body They are produced by several different types of cells including lymphocytes monocytes macrophages endothelial cells and others usually in response to some type of infection Type I interferons INF0L and INF 5 have antiviral action and also act against tumor cells Type II interferon INF y attracts phagocytes to areas of infection and increases their activity ingestion and digestion of bacteria Recombinant DNA technology allowing researchers to transfer genes encoding interferons into E coli cells has made it possible to mass produce these proteins They are currently being used in the treatment of several different types of cancer Interferon alpha INF0L is also used in the treatment of hepatitis C and interferon beta INF 5 is used to treat patients with multiple sclerosis an autoimmune disease b Interleukins Interleukins are cytokines released by lymphocytes macrophages and a variety of other cells They occur as multiple different types IL l through IL 23 so far and have a variety of functions Interleukin 1 IL 1 sometimes called endogenous pyrogen acts on the hypothalamus to elevate body temperature induce fever among other actions Other interleukins lL 2 4 and 5 stinlulate the growth and proliferation of inunune cells including B and T lymphocytes attract neutrophils IL 8 or induce the production and release of other cytokines and or interferons c Complement factors Complement factors are serum proteins produced constitutively by macrophages and released into the circulation as inactive proteins When activated they become proteases that break peptide bonds within other complement proteins thus activating them ie they act on one another in a sequence Since each active complement factor can act on multiple others the initiation of complement activity typically results in a massive reaction called a complement cascade Complement activity is commonly initiated by the binding of antibodies lgG or lgM with antigens but can also be initiated by certain molecules such as lipopolysaccharides on the surfaces of bacteria and indirectly by cytokines In the classical pathway C3 is cleaved to form C3a and C3b C3a then causes opsonization ie binds to cells or other particles making them more attractive to phagocytes while C3b interacts with other complement factors to bring about the formation of membrane attack complexes C78 and multiple C9 units that insert into cellular membranes forming holes These cause cell lysis and death Although human cells are generally protected from the potentially destructive activity of C78 and 9 this is not always the case and these factors sometilnes cause considerable daInage within the human body The Role of Normal Flora in Host resistance Microorganisms commonly referred to as normal flora include a variety of different bacteria fungi protozoa and other organisms living on and within the human body They are not all present at birth but gain access to the body shortly thereafter many passed from mother to infant and colonize the various regions or habitats available Although we typically view ourselves as autonomous beings we are actually walking ecosystems supporting a plethora of prokaryotic cells there are more of them than there are eukaryotic cells within all our various tissues organs and systems Fortunately most of the bacteria living on our skin and within our gastrointestinal urinary reproductive and respiratory tracts are not pathogenic Instead these organisms help to defend us against other organisms that are Organisms living as normal ora associated with skin and mucous membranes protect our cells and tissues in a variety of ways as described below 1 They provide protection by competing against potential pathogens for available nutrients When non pathogenic bacteria are abundant they use up most of the nutrients and lilnit the growth of pathogens 2 They provide protection by competing against potential pathogens for binding sites on cell surfaces Most pathogens must bind with cellular surfaces in order to infect or cause daInage If the binding sites available on cellular surfaces are occupied by normal ora pathogens can39t bind They provide protection by producing chemical substances called bacteriocins that kill other closely related cells Humans typically have Escherichia coli cells living within their guts but do not normally carry highly pathogenic strains The E coli comprising the normal ora of most individuals form toxins called colicins that kill other more pathogenic strains such as the E coli 0157zH7 responsible for causing hemolytic uremic syndrome DJ V Be nice to your normal ora they are helping you survive and most of us need all the help we can get Genes and Mutations As explained earlier genes are segments of DNA and DNA is the hereditary material of cells Although this is clearly understood now it was not obvious when biologists first used the terms gene and genetics for this reason the definitions of many of the terms associated with this section have undergone considerable change over time Because not all references change at the same rate significant variation exists with respect to terminology and definitions used by various sources Providing current definitions for some commonly used terms as appropriate for this course is therefore useful Definitions for some important terms 1 Gene A gene is a unit of heredity The term gene is usually applied to segments of DNA however since the genetic material within some viruses is RNA viral genes may be segments of RNA Most genes encode proteins ie code for m RNA that is translated into amino acid sequences For our purposes these will be designated as structural genes In eukaryotic cells structural genes carry regions not expressed as amino acid sequence these regions include promoter sequences and the introns removed during post transcriptional modification Prokaryotic genes are often associated with operons When they are a single promoter is involved and multiple structural genes are transcribed as one long m RNA molecule The process and the genes are polycistronic Not all genes are structural genes Segments of DNA encoding t RNA r RNA s RNA etc are transcribed to produce RNA molecules but these are not translated Some authors consider promoter sites operator sites attenuator sites etc to be regions within genes but according to the Sequence Ontology Project definition these regions with their own distinct function are separate genes Alternate forms of a gene are called alleles These are nucleotide sequences coding for polypeptides with the same function in general but with slightly different amino acid sequences For example normal hemoglobin and sickle cell hemoglobin or the various different agellin proteins made by bacteria N Genetics Genetics is the science or study of genes heredity and factors that in uence the variations observed within organisms Heredity has been studied at multiple levels since humans first began to domesticate animals and breed plants for food production Classical genetics Mendelian inheritance is based primarily on the work of Gregor Mendel and his studies involving pea plants 1860s Modern geneticists focus more on the structure and function of genes at the molecular level ie DNA and RNA and their interactions with various proteins Needless to say there is considerable variation in the field of genetics 3 Genome The term genome can be defined as the total DNA content of the chromosome or multiple chromosomes within eukaryotic organisms or as the total DNA content of the organism Since the term genome was generated from the terms gene and chromosome the first definition is more accurate The genome includes all portions of the chromosome including non coding regions such as promoters operators and introns It does not include plasmids mitochondrial DNA chloroplast DNA or viral DNA H Genotype The genotype is the genetically determined characteristics of an organism or the genetic potential of that organism As we have already seen genes may be present within an organism but not be expressed ie transcribed and translated to produce proteins because transcription is being repressed Some bacteria carry genes that are not expressed for long periods of time eg E coli cells living inside adult cows and some carry genes that are never expressed due to slight changes in their DNA ie due to mutation E quot Phenotype The phenotype is the observed characteristics of an organism or the genotype expressed The environment both inside and outside cells has a significant in uence on phenotype as we have seen in the laboratory Recall the variation in morphology of E coli colonies grown on MAC EMB T 7 and nutrient agar Bacteria often express certain genes only under certain circumstances and gene expression may or may not result in an observable characteristic even when it is occurring 6 Locus The term locus pleural loci usually refers to the location of a gene on a chromosome but can also be applied to other structures The locus is a fixed location on a chromosome and is usually occupied by a specific gene or codon etc The genome of an organism and therefore the genotype is not necessarily static over time Within populations organisms can appear with novel characteristics encoded by genes not previously present There are two basic mechanisms involved in bringing about such changes these are mutations and genetic exchange mechanisms sexual reproduction Under some circumstances it is difficult to distinguish between the two Mutations A mutation is any change in the nucleotide sequence of DNA within a cell or RNA within certain types of viruses Though some references define mutations as heritable changes in DNA most mutations are not heritable because they cause cells to die The majority of mutations are lethal to the cells they occur in In multicellular organisms such as ourselves these changes frequently go unnoticed but in the case of single celled organisms they end existence Mutations can be caused by a variety of factors including various types of chemicals and radiation When the specific cause of a mutation cannot be identified the mutation is said to occur spontaneously Though exact numbers undoubtedly change over time the spontaneous mutation rate within living organisms is around 1 per 100 million copies of DNA This means polymerase enzymes can copy a single DNA molecule about 100 million times incorporating only one error Though this seems initially like a very low rate for mutations remember that bacteria growing under optimal conditions often reach an m concentration of 109 cells ml of culture medium in less than 24 hours This means a typical population of bacteria will experience one mutation every day Mutation rate can be significantly increased by specific factors chemicals and physical factors called mutagenic agents or mutagens Since mutations can sometimes lead to tumor development mutagens are also often carcinogens ie cancercausing agents Types of Mutations In multicellular eukaryotic organisms mutations are often divided into two categories based on the types of cells or tissues involved Germ line mutations occur within cells giving rise to gametes sex cells and can be passed to future generations while somatic mutations occur within cells or tissues making up the body of the organism and cannot be passed on When dealing with single celled organisms this distinction becomes meaningless because any mutation that does not kill the cell is likely to be passed to the next generation although there are exceptions For this section we will concentrate on the types of mutations occurring within single celled organisms specifically bacteria 1 Point mutations Point mutations are those involving single base changes ie changes in just one nucleotide within a gene or chromosome These single base changes can involve a Additions The addition of one extra base within the nucleotide sequence b Deletions The removal of one base from the nucleotide sequence c Substitutions The replacement of one base in the sequence with a different one Of these mutation types substitutions are least likely to be lethal Substitution type point mutations usually involve the replacement of a purine with a different purine or a pyrimidine with a different pyrimidine but can sometilnes involve purine for pyrimidine substitutions or vice versa Substitution mutations may or may not result in changes to polypeptides this is because the genetic code contains considerable redundancy Consider the following nucleotide sequences DNA nucleotide sequence TAC CCG GTT AAA CTG CGG TTT ACT m RNA nucleotide sequence AUG GGC CAA UUU GAC GCC AAA UGA A polypeptide encoded by this m RNA sequence would contain methionine glycine glutamine phenylalanine aspartic acid alanine and lysine The last codon quotUGAquot is quotUmberquot a terminator codon so would not code for any amino acid If we allow a substitution type point mutation to change the third base in the second codon any letter will do the mutation will have no effect on the polypeptide being formed This is because all codons containing GG as the first two bases code for glycine A mutation like this having no effect on the amino acid sequence being formed is called a silent mutation Changing the last base in fifth codon would also result in a silent mutation Substitution type point mutations are not always silent Sometimes a single base change will cause the aInino acid encoded to change for example if we substitute a quotCquot for the quotAquot in the third position of the third codon the amino acid at that position will be changed from glutamine to histadine Changing a single aInino acid may or may not be significant but the difference between normal hemoglobin and sickle cell hemoglobin involves a single amino acid change N If a substitution type point mutation occurred within the initiator codon AUG translation could not occur because the fmet t RNA could not bind to m RNA at the ribosome In this case if the protein being encoded was essential to cell survival the cell would die So although substitution type point mutations can be silent they can also be lethal to the cell Addition and deletion type point mutations always have a significant impact If one base is added or one base is removed the result will be a shift in the codon quotreading framequot either to the left or right This is called a frame shift mutation In either case the amino acid sequence is very likely to be changed A substitution type point mutation can change a single amino acid but an addition or a deletion will likely change all the amino acids being encoded beyond the mutation point Under such circumstances it is highly unlikely that a functional protein will be formed but it is not impossible Novel proteins with unique functions do sometimes arise within populations and this is one mechanism allowing such changes If a mutation allows an individual within a population to gain an advantage over others within the population it can be maintained and passed to future generations For example consider a population of bacteria living within a host organism A point mutation occurring within one of the cells allows it to produce a novel protein an enzyme called S lactainase This will have little effect under most circumstances but if S lactain antibiotics are present within the environment as they would be within a host taking oral penicillin the organism producing S lactamase suddenly has a significant advantage over other cells because it is resistant to the drug Cells susceptible to the drug will die and the resistant organism will reproduce freely having lost competitors using common resources The result will be a new population of organisms all resistant to the S lactam drug Organisms do not develop resistance to drugs because they are exposed to the drugs They develop resistance because they mutate and the presence of the drug in the environment exerts selective pressure on the population The process is called natural selection and it is a major factor in uencing evolution Unfortunately the current widespread use of antimicrobial drugs is exerting selective pressure on existin bacterial populations and many important pathogens are becoming resistant Bacteria mutate recall mutation is a characteristic of life and humans are exerting selective pressure on their populations by trying to control them Control measures are necessary under some circumstances but exceedingly foolish when applied without considering the consequences Nonpoint Mutations Non point mutations involve regions of DNA containing multiple nucleotides sometimes one or more genes in length A variety of such changes can occur but some of the most common types include a Translocation or transposition the movement of a segment of DNA from one location to another within or between chromosomes or plasmids b Deletions The removal of segments of DNA one or more genes in length c Inversions The reversal of a segment of DNA within a chromosome Translocations or transpositions sometimes involve segments of DNA called r 39 t or t r These are segments of DNA that can initiate their own translocation ie movement from one location to another within a chromosomes between chromosomes or between a chromosome and a plasmid Transposons also called quotjumping genesquot or quotmobile genetic elementsquot were first observed by Barbara McClintock during her studies involving corn plants Though their mechanism for movement is variable some transposons initiate movement by producing an enzyme called transposase that recognizes and cuts DNA within regions containing inverted repeat sequences The enzyme breaks phosphodiester bonds within these regions and allows the DNA segment to exit the chromosome and then insert itself in a new location Some types of transposons reproduce themselves and only the new copies move This allows them to increase in number within the genome Bacterial transposons often carry genes encoding antibiotic resistance so are involved in the development of new drug resistant strains A DNA sequence called Alu associated with the human genome is a transposon about 300bp long This sequence occurs with variable frequency within humans but is typically repeated between 300000 and one million times Translocation also occurs during meiosis when eukaryotic chromosomes line up in homologous pairs Sections of chromosome cross one another break and then reattach in new locations This process adds considerable genetic variation to eukaryotic genomes Deletions can be initiated by a variety of means but sometilnes result from transposon activity They can also be caused by errors in chromosome separation during meiosis and by ionizing radiation Inversions can occur when segments of DNA form loops break and then attach themselves in an inverted position In this case no DNA is lost but genes may be significantly altered Mutations involving the rearrangement of large sections of DNA are often lethal to cells but not always Different types of proteins often contain regions with conunon amino acid sequences just as different words often contain conunon letter sequences consider the number of words containing the prefix quottransquot If the beginnin sequence of one gene is moved and attached to the ending sequence of a different gene the new combination is more likely to result in a functional protein than is a completely novel aInino acid sequence as would be produced as the result of a frame shift mutation Non point mutations are probably responsible for much of the variation currently present within fully functional genomes Some Examples of Mutagenic Agents A thorough coverage of mutagenic agents is beyond the scope of this class because they are too numerous and their mechanisms of action are too varied A brief presentation including a few specific examples will be sufficient Mutagenic agents mutagens can be categorized as either chemical agents or physical factors as indicated below 1 Chemical mutagens Chemical mutagens are chemical agents known to increase mutation rates or to cause changes in nucleotide sequences within DNA molecules Two categories of chemical mutagens are a Base analogs Chemicals resembling naturally occurring nitrogenous bases N b Alkylating agents Chemicals that add methyl or ethyl groups to molecules among other things Base analogs are chemicals that resemble naturally occurring nitrogenous bases A T C and G so can be incorporated into nucleic acids in place of these When the DNA containing a base analog replicates the analog codes for the wrong complimentary base and causes a substitution type point mutation Examples of base analogs include 539 bromouracil 539 uorouracil Zidovudine also called Azidothymidine AZT or Retrovir and many other chemicals Analogs of uracil a pyrimidine base with a single ring in its structure typically resemble thymine 539 methyl uracil and are incorporated into DNA in the place of thymine When the DNA replicates these chemicals code for thymine instead of adenine so cause the substitution of a pyrimidine for what would normally have been a purine Some base analogs cause chain termination during replication recall the dideoxynucleotides described in lab Alkylating agents can cause the addition of methyl and ethyl groups to various types of organic compounds When this involves nucleotides the result can be substitution type point mutations or deletions Alkylating agents can also cause purine bases such as guanine to form cross links between complimentary strands in DNA and these prevent the strands from separating If strands cannot separate replication and transcription cannot occur Sometimes alkylating agents cause DNA repair enzymes to fragment DNA basically destroying it Physical mutagens Physical factors recognized as mutagenic agents include various forms of electromagnetic radiation eg ultraviolet light X rays and gamma rays Ultraviolet light causes damage to DNA by inducing the formation of dimers between adjacent thymine bases ie thyminethymine dimers When a DNA strand containing a T T dimer replicates the dimer will code for only one adenine rather that two resulting in a deletion type point mutation Since deletions cause frame shifts ultra violet light is often lethal to cells Many types of bacteria are exposed to sunlight for prolonged periods of time and have developed resistance to ultra violet rays The DNA within these cells can still be damaged by ultra violet radiation ie thymine thymine dimers are still formed but some cells also have mechanisms for repairing this type of damage Some bacteria use quotlight repairquot mechanisms to repair damage caused by ultra violet light In these organisms enzymes activated by visible light locate the thymine thymine dimers and break them apart Other organisms use quotdark repairquot mechanisms that do not require light The enzymes involved in this type of repair can locate thymine thymine dimers remove regions of DNA containing these and replace the damaged regions with new correct bases Ionizing radiation including X rays and gamma rays are recognized as powerful mutagens These can cause ionization of various molecules within cells including nucleic acids proteins lipids etc The effects of ionizing radiation on DNA vary but may include loss of or damage to bases breaks in one or both strands resulting in deletions and or chromosome rearrangements or cross linking within DNA strands inhibiting replication and transcription This type of radiation is usually lethal to cells but some bacteria eg Deinococcus radiodumns and D radiopugmms can repair damage caused by ionizing radiation Researchers are very interested in the enzymes formed by these organisms as they may have potential use in the clinical setting Characteristics of Life and Biochemistry Microbiology is the science or study of microscopic life forms and these like all other living organisms have a number of characteristics in common that can be used to distinguish them from nonliving materials These characteristics of life include the ability to reproduce both sexually and asexually the ability to carry out metabolic processes and to grow by assimilation the ability to respond to environmental stimuli both external and internal the ability to mutate and the ability to maintain a high degree of organization Reproduction is a characteristic common to all living organisms because living cells arise only from preexisting living cells biogenesis as opposed to abiogenesis Most microorganisms reproduce themselves by a process called fission that involves the division of one cell into two new cells daughter cells This form of reproduction is considered to be asexual because it does not involve a reorganization of the genetic material present The daughter cells formed are genetically identical to the original cell Most if not all organisms also engage in some sort of sexual reproduction This involves the reorganization of genetic materials and results in the formation of genetically unique individuals Growth by assimilation is another characteristic common to living organisms Assimilation is the process organisms use to take in materials from their environment break them down and then reorganize them into new cellular components Assimilation involves metabolism another characteristic of living organisms Metabolism can be defined as all the chemical reactions that take place within living organisms and includes both catabolism breakdown reactions and anabolism building reactions Although growth typically involves an increase in size and weight the growth of living organisms also involves an increase in cell numbers so involves reproduction Microorganisms such as bacteria and yeasts when maintained under certain conditions typically on solid media grow from single cells into masses of cells called colonies These represent populations of cells in the millions or billions Although an individual bacterium or yeast cell is not visible to the naked eye a colony is readily visible is macroscopic Another characteristic of living organisms is response to environmental stimuli or changes in their environments both internal and external Responses that are rapid may be referred to as irritability or behavior and typically involve movement Many microorganisms move through their environment in response to stimuli such as light temperature chemicals magnetic fields and or gravity This type of response is often clearly visible to the naked eye Response to environmental change that occurs slowly may be referred to as adaptation and may be less readily observed Many microbes form specialized cell types that allow them to survive unfavorable conditions such as the heat and dryness of summer or the freezing temperatures of winter Adaptations that appear as changes in populations over many generations involve reorganization of genetic materials due to sexual reproduction and mutation changes in the composition of nucleic acids DNA and RNA These adaptations are often visible as specialized structures behaviors or abilities and are indicative of evolution ie changes in populations over time The complex structure of even the simplest living organism represents a high degree of organization The material that cells are made of contains billions of atoms elements arranged into simple and complex molecules compounds that interact continuously in metabolic processes Organisms must expend a tremendous amount of energy to maintain their organization and without it their life processes will stop The Composition of Protoplasm Protoplasm may be defined as living substance or as the chemical and physical basis for life It is a dynaInic changing material and is what all cells are made of One way to visualize protoplasm is to think about a boiling soup that is constantly being added to and taken from Imagine the contents of that soup The smallest particles of protoplasm we are concerned with are the atoms or elements There are thirteen different elements that make up approximately 99 of all living organisms by weight These can be arranged into a pattern that makes them easy to remember as follows C H O P K I N S Ca Fe Mg Na and Cl These may be remembered as C HOPKINS CaFe Mg mighty good but you also need salt NaCl Though handy this sequence is not always accurate since many cells also use other elements such as copper zinc manganese silica and molybdenuln though these are present in very small amounts Of those listed above certain elements are more important than others The ve elements C H O N and P are said to make up about 96 of the weight of living organisms Living organisms cannot convert one type of element into another but they do carry out metabolic processes in which elements combine to form a wide variety of molecules or compounds This is where a high degree of organization comes into play Molecules are combinations of atoms joined together by chemical bonds Water an essential compound Water is an inorganic compound that is essential to life as we know it It is a major component of protoplasm in all types of cells A molecule of water contains two atoms of hydrogen and one atom of oxygen HZO Water has a nulnber of features making it essential to living organisms including 1 Water is made up of polar molecules those having an unequal distribution of charge and so is an important solvent many types of solute will dissolve in it It is involved in many chemical reactions Reactions that involve the splitting of organic compounds by adding water to them are called hydrolysis reactions hydro water lysis to split Hydrolysis can be used to break down a wide variety of organic compounds Reactions that involve the formation of larger molecules by removing water are called 1 39 1 io J quot 39 or J quot quot Macromolecules such as proteins polysaccharides and nucleic acids are formed in this way DJ Water is liquid so is essential to transport It allows materials to be moved readily within cells and allows cells to move through a variety of habitats Water helps to maintain cell size and shape It moves freely through most cell membranes and has a high surface tension due to cohesion between water molecules Water resists temperature change and plays an important role in temperature regulation in macroscopic organisms such as ourselves It also in uences weather and climate Water with ions in it can conduct electricity This feature is important in various life processes and is useful in the laboratory during electrophoresis experiments Water as a solid is less dense than it is as a liquid so ice floats Imagine what our world would be like if it didn t Water accounts for the bulk of protoplasm usually around 70 and provides a background matrix for the support of other materials Certain substances electrolytes will dissociate in water to form charged particles called ions Table salt NaCl is one of these When table salt is dissolved in water each sodiuln atom gives up one electron to form a positively charged particle called a cation Na and each chlorine atom takes on one extra electron to form a negatively charged particle called an anion Cl These charged particles play important roles within cells Some important cations include Na K Ca Mg and Fe while some important anions include Cl HCO3 and OH Some Important Types of Organic Compounds Organic compounds are those containing carbon with the exception of C0 C02 and HCO3 and are generally larger and more complex than inorganic compounds Some organic molecules are very large macromolecules being composed of one thousand or more atoms Such molecules are generally composed of repeating smaller units and so are called polymers poly many For note taking purposes the major groups of organic compounds are indicated in different colors This will make it easier for students to keep track of them as they occur in cellular structures Carbohydrates sugars and starches Color these pink or red The carbohydrates are organic compounds made up of the elements C H O and sometimes N A variety of specific molecular structures are shown in the text If they are simple sugar molecules they are called monosaccharides and may contain three four ve six or seven carbon atoms These are referred to as triose tetrose pentose hexose or heptose monosaccharides respectively For our purposes the pentose and hexose monosaccharides are the most important These can exist in cyclic forms and are often represented visually as rings Hexose monosaccharides typically have the molecular formula C5H1206 and include glucose fructose galactose and mannose The pentose monosaccharides typically have the molecular formula C5H1O5 and include arabinose rhamnose ribose and deoxyribose note that the last one does not have the typical formula because it is missing an oxygen When sugar units bond to other molecules having hydroxyl groups they form glycosi des and the bond between the two molecules is called a glycosidic linkage When two monosaccharides bind together via a glycosidic linkage they form a pair of sugar units called a disaccharide The process involves the removal of a water molecule so is a dehydration synthesis reaction The three most common of these are lactose milk sugar which is glucose galactose maltose two glucose molecules and sucrose table sugar which is glucose fructose If three monosaccharides are joined together they form a short chain known as a trisaccharide two water molecules are removed Raffinose is a trisaccharide made up of glucose galactose and fructose All sugar molecules have some characteristics in common They are sweet to the taste and soluble in water Chains of four to six sugar units may be referred to as oligosaccharides but more commonly these join to form long chains containing many sugar units or polysaccharides These are macromolecules and are sometimes huge The most common polysaccharides are starch the storage product of plants glycogen the chief storage product in animals cellulose the primary structural component of wood and agar which we use as a solidifying agent in our culture media Polysaccharides are not sweet to the taste and are not soluble in water so are ideal for storage Carbohydrates are quantitatively the most common biochemicals found They serve as an important source of energy to maintain metabolic functions They are also involved as structural components supporting materials within cells and can bind with proteins to form glycoproteins involved as receptor sites on cell surfaces Proteins enzymes antibodies flagellins etc Color these blue Proteins contain the elements C H O N and sometimes 5 The are macromolecules or polymers composed of many small repeating units called amino acids There are twenty different types of amino acids typically found in nature although bacteria contain a few unusual types not found in proteins Amino acids are all similar in structure in that they all possess a carboxyl group COOH at one end and an amino group NH2 at the other The remainder of the amino acid structure is variable Amino acids are bounded to one another by covalent bonds called peptide bonds The formation of each peptide bond requires the removal of one water molecule so again involves dehydration synthesis When many amino acids are bound together the result is a long string of peptide bonds or a polypeptide This may or may not form a complete protein All proteins are made of amino acids but they are not all the same The most important factors making one protein different from the next is the number of amino acids present and more importantly the sequence in which they are arranged This is referred to as the primary structure of the protein The type and arrangement of amino acids in proteins allows for tremendous variation just as the type and arrangement of letters makes up variation in words Proteins are considerably longer than words however with some of the smallest being around 500 amino acids in length Proteins do not usually remain as long strands but instead tend to roll up in very specific ways This gives them additional structure as follows Primary structure sequence of aInino acids present Secondary structure development of a helix or pleated sheet due to hydrogen bonding between amino acids Tertiary structure folding of a protein into a globular form Some amino acids contain thiol groups SH capable of forming disulfide bonds or crossbridges SS between amino acids The tertiary structure of a protein is loosely stabilized by hydrogen bonds and held more firmly by the disulfide bridges Much of its surface structure is changeable a factor important to enzyme function Such changes are called allosteric changes The difference between a normal brain protein and an infective agent known as a prion etiological agent of mad cow disease is a change in threedimensional configuration or tertiary structure Quaternary structure when a protein contains more than one polypeptide chain Many proteins are functional only as complexes of several polypeptide chains The quaternary structure is this complexing The various chains are held together by disulfide bridges and by hydrogen bonds Because of their globular structure most proteins tend to have polar surface groups and so are externally hydrophilic water loving while their internal portions are hydrophobic water fearing Proteins then are like phospholipids in that they are aInphipathic or amphiphilic see lipid section below Proteins are important structural components of cells occuring in cell membranes microtubules fibrils ribosomes agella etc Other types are functional proteins such as enzymes the catalysts for chemical reactions within cells hemoglobin hormones antibodies etc Proteins may also be catabolized and so serve as a source of carbon and energy Lipids fats oils and waxes Color these yellow Lipids always contain C H and 0 but often include smaller aInounts of elements such as P and N as well Lipids as a group are soluble in organic solvents rather than water so are said to be hydrophobic water fearing Some important groups of lipids include fats oils waxes phospholipids and steroids Fats and oils are structurally triglycerides complexes composed of three fatty acid molecules attached to a single molecule of glycerol The fatty acid chains are either saturated or unsaturated depending on the nu1nber of hydrogen atoms they contain Saturated fatty acids contain a maximuln nu1nber of hydrogen atoms while unsaturated forms are missing some The reduction in hydrogen is due to the presence of double or sometimes triple bonds between carbon atoms If there are two or more double bonds formed between the carbons present within a fatty acid it is said to be polyunsaturated Fats tend to be saturated and are usually solid at room temperature while oils are unsaturated and are liquid Most fats and oils are actually mixtures of various types of triglycerides Waxes have a structure similar to triglycerides but the alcohol involved is much larger Some examples of waxes include beeswax carnuba and lanolin Phospholipids are similar to triglycerides except that in these molecules one fatty acid chain is replaced by a polar phosphate group This is a very important factor since it in uences how the phospholipid molecules react with water The polar phosphate is hydrophilic water loving while the fatty acid chains are hydrophobic water fearing The result is a molecule that loves and suffers in two different types of enviromnents Such molecules are said to be amphipathic or amphiphilic amphi two ways or both ways pathos suffering and phil love Phospholipid molecules play a major role in the structure of cell membranes where they form an interface between water and lipid layers Steroids are lipids with ringform structure They do not contain fatty acid chains Cholesterol and cortisone are examples of steroids Lipids serve as important structural components of cells as in cell membranes as insulation and as hormones Lipids can also be broken down and utilized as an energy source Nucleic Acids DNA and RNA Color these violet The nucleic acids are the molecules that carry the genetic information within cells and also within viruses and viroids There are two major types deoxyribonucleic acid DNA and ribonucleic acid RNA Like proteins and polysaccharides the nucleic acids are long chain molecules polymers made up of many smaller units The smaller units in this case are called nucleotides Each nucleotide contains a pentose monosaccharide either ribose or deoxyribose a phosphate group and one nitrogenous base The nitrogenous bases vary and may be adenine guanine cytosine or thymine in DNA or uracil in RNA Adenine and guanine are called purine bases or purines while cytosine thymine and uracil are called pyrimidine bases or pyrimidines The structural differences between DNA and RNA as well as their function within cells will be covered extensively later Nucleotides have a number of important functions within cells as follows 1 When joined within polymers they form the nucleic acids that carry genetic information DNA and RNA 2 Individually they can be used as a form of energy currency Nucleotides that ave three phosphate groups instead of one are called nucleoside triphosphates NTPs or activated nucleotides These highenergy compounds are essential to cell function The most commonly encountered nucleoside triphosphate is adenosine triphosphate ATP but it is not the only one 3 Nucleotides can be used to form molecules known as coenzymes These serve as electron carriers in association with metabolic processes Two important coenzymes are NAD nicotinamide adenine dinucleotide and FAD avin adenine dinucleotide both of which are Bcomplex vitaInins 4 Ringform nucleotides sometimes serve as regulatory molecules They interact with proteins and regulate a variety of cellular functions Two important regulatory nucleotides are cyclicAMP and cyclicGMP There are many types of organic compounds not specifically included in this section Some of these will be covered later in the section on metabolism In addition to the major groups of organic compounds as described above there are complexes formed by joining different types Lipids and carbohydrates can join to form lipopolysaccharides or glycolipids proteins and lipids can join to form lipoproteins and proteins and carbohydrates can join to form glycoproteins Eukaryotic Cell Structure and Function Part 2 ll Nucleus 7 The nucleus is a relatively large centrally located organelle surrounded by a double layer of membrane called the nuclear envelope and containing the nucleoplasm Though separated from the cytoplasm by two layers of membrane the nucleus maintains contact by transportng materials through nuclear pores regions with greater permeability The nuclear envelope is also connected to the endoplasmic reticulum Sometimes called the quotbrain of the cellquot the nucleus is a control center involved in regulating physiological processes Most of the DNA contained within eukaryotic cells is located here and the associated genes determine which proteins the cell can make The nucleoplasm includes a diffuse threadlike material called chromatin and one or more darkstaining bodies called nucleoli Chromatin is a threadlike material made up of DNA and proteins some of which are homogeneous all of the same type and others heterogeneous of different types Groups of homogeneous proteins called histones help maintain the structural stability of DNA and in uence gene expression by binding with DNA in structures called nucleosomes Each nucleosome contains a histone octomer wrapped with DNA and multiple nucleosomes are bound together by other histones to form beadlike chromatin strands Heterogeneous proteins include enzymes of various types and are involved in processes such as DNA replication and transcription During cell division the chromatin threads are highly folded into discrete structures called chromosomes These are clearly visible with a light microscope in some cell types Nucleoli singular nucleolus 7 Nucleoli are darkstaining bodies sometimes visible within the nucleoplasm of certain cells They are composed of ribosomal RNA rRNA and protein and are sometimes referred to as the quotpacemakersquot of the cell because they in uence protein synthesis These bodies are the site of r RNA synthesis and the assembly of ribosomal subunits In essence they make the ribosomes responsible for all protein synthesis Spliceosomes 7 Spliceosomes are small granular bodies made up of small or shortRNA s RNA and protein The sRNA molecules present contain high levels of uracil uridine nucleotides and there are several different segments of sRNA involved The function of Spliceosomes within the nucleus is to modify RNA molecules as they are transcribed from DNA The process is called post transcriptional modi cation and involves cutting out some regions of RNA and splicing the remaining pieces together this will be explained in greater detail later Microtubules 7 Microtubules are microscopic tubelike structures cylinders made up of proteins called tubulins or tubulin proteins Each tubulin unit is actually a heterodimer composed of one alpha 0L and one beta 5 tubulin an a heterodimer The tubulin dimers polymerize connect together in long chains to form linear structures called protofilaments each with an OLsubunit at one end and a Ssubunit at the other This orientation is signi cant and consistent within each microtubule The Ssubunit end is called the positive end while the OLsubunit end is called the negative end The wall of a typical microtubule is composed of thirteen protofilaments arranged as linear strands that spiral around the tubule forming imperfect helixes In cross section these appear as thirteen globular protein dimers arranged in a circle Microtubules can be lengthened or shortened by adding or removing tubulin dimers and so exist as dynamic structures changing as necessary for cellular function Addition of tubulin dimers occurs most readily at the positive end while removal of dimers occurs most readily at the negative end Microtubules function as support structures forming part of the cytoskeleton and are also involved in intracellular motion Proteins known as microtubule associated proteins or MAPs interact with them in various ways Two types of MAPs known as motor proteins are Kinesin and Dynein Kinesin proteins kine movement attach to and quotwalkquot along protofilaments toward the positive end and can carry cellular components such as small organelles vesicles and other cytoskeletal elements along the microtubule Dynein is also capable of moving along microtubules but carries materials toward the negative end Dynein is also involved in the movement of cilia and agella Both motor proteins require ATP as an energy source have ATPase activity Together they can allow materials to travel along microtubules in opposite directions at the same time forming an intracellular highway Cilia and agella 7 Cilia singular cilium and agella singular agellum are locomotor structures found on the surfaces of some eukaryotic cells Cilia are typically short and hairlike while agella are longer and whiplike Both cilia and agella are attached to basal bodies surrounded by the cell membrane and supported by microtubules arranged in a specific pattern typically nine groups of two around the periphery and two at the center The center two microtubules are attached to the outer nine pairs by quotspokesquot made of dynein and this motor protein with its ATPase activity provides the motion required to bend the cilium or agellum The complex formed by microtubules and MAPs within a cilium or agellum is called an axoneme and is formed from the basal body a centriole that has moved to the cell surface though how this occurs in not thoroughly understood Flagella are typically less numerous than cilia and most agellated cells have only one or two Cilia are usually quite numerous and often cover the entire cell surface A single agellum can pull a cell through its environment or agella in pairs can move together like the arms of a person 39 39 the 39 quot one agellum in a pair pulls the cell forward while the other trails along at rest Cilia move in a highly coordinated fashion creating wave like patterns that ow over the cell surface or rotate around foodgetting structures Some cilia move singlecelled organisms through their watery habitats while others move materials along the surfaces of stationary cells as occurs within the human respiratory system Many types of protozoa use cilia to sweep food materials toward and into a cellular quotmout quot or cytostome Centrioles 7 Centrioles are cylindrical bodies made up of microtubules arranged in nine groups of three They typically occur as a single pair oriented at right angles to one another within a region of the cell called the centrosome In addition to forming the basal bodies producing cilia and agella centrioles give rise to a structure known as the spindle apparatus involved in chromosome separation during cell division mitosis and meiosis Some of the microtubules forming the spindle apparatus attach to chromosomes at their center the kinetochore and then pull the chromosomes apart by shortening a process involving the removal of oc dimers as described above Other microtubules move the centrosomes toward opposite sides of the cells which also serves to pull the chromosomes apart In addition to the tubulin protofilaments found in microtubules eukaryotic cells carry micro laments made up of actin a protein associated with muscle bers These laments are involved in a variety of cellular processes such as cytokinesis separation of the cytoplasm during cell division endocytosis and the movements of cells involving pseudopodia Inclusions 7 Inclusions are bodies of material stored within the cytoplasm of eukaryotic cells and sometimes considered to be nonliving They frequently include such materials as crystals fat droplets pigment granules or glycogen Some inclusions contain materials produced by the cell while others contain materials taken from the outside environment Cell Walls and Skeletons 7 Cell walls and skeletons are rigid layers found outside the cell membranes of certain types of cells including algae fungi and certain protozoa Cell walls are generally associated with plantlike cells such as algae while skeletons occur on animallike protozoa Fungi were originally classified with plants so their rigid coverings are also called walls Cell walls are typically made of polysaccarides such as cellulose chitin glucan pectin or agar but sometimes contain glass They give cells a characteristic shape and provide protection against changes in osmotic pressure e g in hypotonic environments and potential predators Skeletons occur on some animallike single celled organisms protozoa and are typically made of glass or calcium carbonate They also give organisms a characteristic shape and provide protection against potential predators Most skeletons do not provide protection against the osmotic pressure exerted by hypotonic environments because they are perforated by numerous holes Control of Microorganisms Microorganisms are abundant in the environment living in soil in water on the surfaces of plants and animals and inside many types of multicellular organisms including humans Fortunately most microorganisms live freely in the environment without causing harm to humans or other organisms in fact most of them are beneficial in a variety of ways For these reasons humans seek to control only certain types of microorganisms and under relatively few specific circumstances We exert control over microorganisms associated with food materials so that foods can be stored and made available for human consumption without being consumed by microorganisms first We seek to control microorganisms that might cause damage to agricultural crops forest trees and ornamental plants maintained for our purposes and we seek to control potential pathogens that threaten our health or the health of animals we consider important for various reasons Microbial control methods vary considerably depending on where and on what types of microorganisms they are being applied Some of the more commonly used control methods will be presented below Control methods can be divided into categories based on their effects and the types of factors involved Control methods or treatments that kill cells are given the prefix cidal for example bactericidal treatments methods kill bacteria while fungicidal treatments methods kill fungi Most people are familiar with the prefix cidal as it is applied to insecticides and herbicides used frequently and often without consideration for potential consequences throughout this country Control methods or treatments that inhibit microbial growth but do not kill cells are given the prefix static Static treatments eg freezing are effective only as long as they are maintained but within the body the inhibition of microbial growth for even a short time period often improves the effectiveness of immune mechanisms ultimately resulting in elimination of potential pathogens Sterilization When used with reference to microbial control the term sterilization refers to any treatment or method rendering an object or material virtually free of any viable cells Skin surfaces cannot be sterilized because the skin is composed of living cells even though the surface epithelial cells are mostly dead Liquids air metal instruments and other materials can be sterilized by a variety of methods as described below Physical Factors Effective in the Control of Microorganisms A variety of microbial control methods involve physical factors such as temperature pressure and radiation Variations exist within these categories and in some cases more than one factor can be applied simultaneously Some specific examples are listed below 1 Temperature Freezing exposure to low temperatures Freezing is a bacteristatic physical control method applied frequently in a wide variety of settings Freezing is used extensively to control microorganisms associated with food materials drugs research chemicals etc Freezing effectively inhibits the growth of most microorganisms by stopping metabolic processes but is rarely cidal to bacteria E 9 because most of them are psychroduric Bacteria culture collections are typically maintained in ultra low freezers at temperatures of minus 72 to minus 800 C and remain fully viable It is important to remember when thawing large frozen food items eg turkeys that the external surfaces can reach temperatures suitable for microbial growth long before the center is thawed Microorganisms and nutrients are typically abundant on skin surfaces and growth will occur if moderate temperatures are maintained for any length of time Cold temperatures commonly maintained within refrigeration units slow metabolic processes and effectively prevent the growth of some types of microorganisms but not all Potential pathogens including C ampyl obacter and Listeria can grow on food materials maintained at temperatures conunon within most refrigerators Pasteurization Pasteurization is a control method developed by Lewis Pasteur and first tested in 1862 It involves the application of heat at a specified temperature for a specific period of time and is intended to bring about a logarithmic reduction in the number of viable cells present so that spoilage due to microorganisms is prevented or at least slowed Heat will denature enzymes essential to metabolism if applied appropriately In our laboratory we Pasteurized soil samples by placing them into boiling water 1000 C for one minute lf lower temperatures are used the exposure time is increased and if higher temperatures are used the exposure time is decreased Pasteurization was used initially to control microorganisms associated with fruit juices but can also be used to control potential pathogens in milk beer vinegar and other food products Although Pasteurization kills the vegetative cells of many pathogenic bacteria Mycobacteriam tuberculosis Escherichia coli Salmonella cholerasais and others it cannot be used to sterilize liquids Pasteurization does not kill thermoduric endospores or hyperthermophilic bacteria Subjecting materials to boiling water for more than one minute eg five minutes ten minutes etc is sometimes considered sufficient for sterilization though technically it is not because hyperthermophiles are not killed Fortunately hyperthermophiles are rarely if ever pathogenic so controlling them is not an issue Tyndallization Tyndallization also known as fractional sterilization was developed by John Tyndall 1875 76 and was used to sterilize liquids before the development of autoclaves Tyndallization involves subjecting liquids to alternate periods of boiling and cooling extended over three days Liquids cannot be sterilized by simple boiling even when it is applied for 30 minutes but boiling for 30 minutes followed by cooling to room temperature overnight and then repeating this procedure for the next two days will result in sterilization Maintaining liquids at room temperature typically causes endospores to germinate and the resulting vegetative cells can then be killed during the next phase of boiling Autoclaving moist heat An autoclave is a device essentially a sophisticated pressure cooker used to sterilize liquids glass containers pipette tips and other materials by subjecting them to steam heat under pressure Autoclaves allow for the application to two physical factors heat and pressure simultaneously and will effectively kill all types of cellular pathogens within minutes Although modern autoclaves can typically be set to run different cycles sterilization is usually accomplished by raising the temperature to 121 C with steam under 15 17 pounds per square inch psi pressure for 15 20 minutes The endospores of Bacillus stearothermophilus can survive 121 C for about 13 minutes but not longer and are sometilnes used to test the effectiveness of autoclaves Although autoclaves effectively kill pathogens they involve considerable moisture steam that is potentially damaging to some metal instruments Materials sensitive to moisture can be sterilized by subjecting them to dry heat D Dry heat Dry heat as would be applied within an oven is sometilnes used to sterilize glassware or metal instruments potentially damaged by exposure to steaIn although dry heat will also cause surgical instruments to loose sharpness Dry heat applied at 160 C will sterilize materials in about 2 hours f Incineration Incineration ie burning or exposing to open ame can be used to sterilize materials such as wire loops and glass spreader rods and is used routinely in the microbiology laboratory Organic materials are vaporized during incineration and no living cells can withstand this treatment Incineration was once commonly used as a method for disposing of corpses associated with large epidemics and is still used for elilninating body parts animals dead due to anthrax and microorganisms associated with large structures such as the filters used inside laminar ow hoods 2 Pressure As described above autoclaves use heat and pressure silnultaneously to kill microorganisms and sterilize various types of materials Pressure is also a major factor used to kill and break open Gram positive bacteria in preparation for the PCR activities carried out in our laboratory When subjected to sufficient pressure eg being slanuned repeatedly by 2mm glass beads bacterial cell walls will break According to some sources nanotechnology is being investigated as a means of applying pressure to the control of microorganisms on commonly used surfaces such as computer keyboards instrument control panels light switches etc within clinical settings If such surfaces could be equipped with nano spines the pressure exerted by human hands would result in cell death as the bacteria on skin surfaces were punctured Exactly how the skewered bacteria would be removed from such surfaces is difficult to unagine 3 Radiation a Ultra violet light As described earlier ultra violet light electromagnetic radiation with a wavelength shorter than visible light can cause the formation of thymine thymine dimers resulting in deletion type point mutations but can also cause chromosome distortion and inhibition of replication Ultra violet light with a wavelength of 260 270n1n is particularly effective in controlling bacteria and is often used in research laboratories clinical settings and to reduce the number of viable bacteria in water exiting wastewater treatment facilities b Ionizing radiation Ionizing radiation such as X rays and gamIna rays can be used to sterilize heat sensitive materials such as plastics vaccines drugs important spices and other materials that would be damaged or destroyed by exposure to heat Ionizing radiation is very effective but used less commonly than other control methods because it is expensive and because it is potentially harmful to persons applying it Treating food materials with ionizing radiation does not render them radioactive 4 Filtration Filtration is a static control method used to sterilize liquids and gases The organisms removed during filtration may eventually be killed by exposure to heat or some other treatment but the filtration itself merely inhibits their growth by removing them from the potential growth medium In our laboratory we filter sterilize certain media such as urea agar arabinose and xylose as well as chemicals to be added to autoclaved media eg ampicillin and CaClz The air owing through the laminar ow hood is filter sterilized to provide a clean space for the preparation of plate media Beer is typically filtered to remove yeast cells prior to bottling Chemical Factors Effective in the Control of Microorganisms Many different chemicals are used to control microorganisms in a variety of settings but when used in the control of potentially pathogenic microorganisms outside the body chemicals are frequently divided into two categories on the basis of the types of surfaces they are applied to These two categories are antiseptics and disinfectants Antiseptics Chemicals categorized as antiseptics are those used to control potentially pathogenic microorganisms on living surfaces Sepsis means infection and chemicals designed to prevent sepsis must be mild enough to be used on skin surfaces or mucous membranes without damaging the eukaryotic cells present Disinfectants Chemicals categorized as disinfectants are those used primarily to control potentially pathogenic microorganisms on nonliving surfaces Many chemicals used as disinfectants are not safe for use on living surfaces and should not be applied to them Although this distinction is handy it is not always accurate because some types of chemicals are used on both living and non living surfaces eg alcohols some halogens and certain detergents The distinction between antiseptic and disinfectant may be a matter of concentration with higher concentrations being used only on non living surfaces If they are to be used routinely chemicals both antiseptics and disinfectants are expected to meet certain criteria including They should be effective within a reasonable time period They should not be toxic or hazardous to persons applying them They should be readily soluble and able to penetrate into small openings because that39s where the microorganisms are They should not damage the surfaces or materials they are being applied to They should be biodegradable within a reasonable period of tilne WNH 01H Although mercury compounds effectively control microorganisms they are not biodegradable and tend to accumulate within human tissues causing severe neurological daInage Mercury compounds are used less extensively than they once were Although many different types of chemicals are used to control potentially pathogenic microorganisms outside the body certain groups of chemicals are applied more commonly than others Some frequently encountered chemical groups are listed below H Surfactants The term surfactant is an abbreviation for surface active agent and applies to a group of chemicals that decrease the surface tension of water Soaps and detergents fall into this category and are conunonly mixed with other chemicals to increase their penetrating abilities When used in high concentrations surfactants can also disrupt cell membranes and cause cell lysis Halogens The elements chlorine bromine iodine and uorine are halogens and are all powerful oxidizing agents that can inactivate cellular proteins Chlorine is used most extensively in water treatment and as a disinfectant while iodine is often used as an antiseptic Bromine gas is sometimes used as a fumigating agent but is highly toxic Metal Ions The ionic forms of heavy metals including copper silver zinc mercury and lead have been used as both antiseptics and disinfectants These typically interact with proteins rendering them inactive Silver nitrate was used extensively to prevent daInage from sexually transmitted pathogens in the eyes of newborn infants while mercury compounds were previously used as antiseptics Merthiolate and mercurochrome and as antifungal agents on seed corn Copper sulfate is used to prevent hoof rot in sheep cattle and other livestock and lead nitrate is an ingredient in Desitin an ointment used to prevent diaper rash 4 Alkylating agents Alkylating agents can cause the addition of methyl or ethyl groups to organic compounds and tend to disrupt protein function Ethylene oxide is an alkylating agent most conunonly used as a fumigant 5 Formaldehyde Formaldehyde reacts with chemical groups carboxyl amino and sulfhydryl and so tends to disrupt protein function 6 Alcohols Alcohols including ethanol and isopropanol are conunonly used as both disinfectants and antiseptics When used in high concentrations they denature cellular proteins cause coagulation and kill most types of cells 7 Phenol derivatives Phenol also called carbolic acid was used by Joseph Lister as a means of preventing sepsis When used in high concentrations it disrupts cellular membranes and kills cells Phenol is found in Lysol and Hexachlorophene 8 Hydrogen peroxide Hydrogen peroxide is a powerful oxidizing agent effective against catalase negative organisms ie those lacking the ability to produce catalase enzymes It is often used as an antiseptic and sometimes as a mouth rinse N DJ When used in high concentrations chemicals such as those listed above are generally bactericidal however their effectiveness can be decreased by accumulations of organic material debris and by biofilms Biofilms are formed by populations of bacteria growing on the surfaces of objects and coating themselves with polysaccharide glycocalyx The plaque forming on tooth surfaces and the shine common on river rocks are two exaInples of biofilms Biofillns often form on catheters inside tubing and on other wet surfaces present in clinical settings When present they can significantly reduce the effectiveness of chemical control agents Antimicrobial Chemotherapy Chemicals used to control potentially pathogenic microorganisms systemically ie inside the body are called antimicrobial drugs or chemotherapeutic agents In order to be effective these chemicals must be able to control pathogens without doing damage to host cells or tissue recall the quotmagic bulletquot concept popularized by Paul Ehrlich during the early 1900s Although many types of chemicals can be used to control microorganisms outside the body those available for controlling potential pathogens inside the body are somewhat limited Antimicrobial drugs can be divided into categories based on the types of pathogens they control e g anti fungal drugs anti viral drugs anti helininth drugs anti parasitic drugs and antibiotics Antibiotics Antibiotics are antimicrobial agents originally produced by some type of living organism and used to control bacteria Most antibiotics were originally produced by bacteria or fungi but recently chemicals with antibiotic activity have been found in association with multicellular organisms including trees and frogs Differential toxicity The ability of a chemical to control pathogens inside the body without doing damage to host cells or tissues is referred to as differential toxicity and is in uenced primarily by the drug39s concentration and its mechanism of action The concentration of a chemotherapeutic agent necessary to provide clinical control of a pathogen is called the therapeutic dose for that drug Since many drugs are metabolized or actively excreted by the body and since human physiology is variable between patients there is no single concentration capable of providing clinical control in all individuals Instead there is a therapeutic range within which a drug can be expected to be effective The concentration at which drugs are applied must be carefully monitored because if the concentration is too low the drug will be ineffective and if it is too high the drug may have toxic side effects or cause damage within the body The lowest concentration of an antimicrobial drug that will effectively inhibit the growth of a specific type of microorganism in vitro is called the minimal inhibitory concentration MIC and can be determined by a variety of methods one of which will be demonstrated in the laboratory Knowing the Mle for specific drugs and their targets can significantly improve the ability of clinicians to effectively treat and cure their patients Antimicrobial drugs vary significantly with respect to their mechanisms of action and it is not uncommon for drugs to have more than one effect on pathogens Differential toxicity is most readily maintained if drugs act on cellular features unique to prokaryotes because then they are less likely to damage human cells Some example mechanisms of action and some specific drug types displaying these actions are listed below 1 Inhibition of enzymatic pathways Some types of antimicrobial drugs exert their in uence by inhibiting metabolic pathways unique to prokaryotic cells For example the Sulfa drugs act as competitive inhibitors blocking the activity of enzymes involved in the conversion of para aminobenzoic acid PABA into folic acid a compound essential for growth Since human cells cannot form folic acid the sulfa drugs have good differential toxicity Sulfa drugs are static antimicrobial drugs but not antibiotics and some types of bacteria have developed resistance to them 2 DJ 4 U1 Inhibition of cell wall synthesis Antimicrobial drugs collectively referred to as 5 lactam antibiotics Penicillins and Cephalosporins prevent the formation of peptidoglycan and so effectively inhibit cell wall synthesis These drugs are bactericidal to actively growing cells because growing cells partially degrade their peptidoglycan walls during elongation and if they cannot replace this material they die Since human cells do not form peptidoglycan walls drugs with this activity have good differential toxicity Penicillins and Cephalosporins are produced by fungi in the genera Penicillium and Cephalosporium respectively Although initially very effective in controlling pathogens they are now losing effectiveness because many bacteria have developed resistance to them by acquiring genes encoding enzymes that degrade them e g penicillinases or cephalosporinases collectively called 3lactan391ase enzymes Inhibition of Protein synthesis Several different types of antibiotics control pathogens by inhibiting protein synthesis but not all of them exert their in uence in the same manner Tetracyclines antibiotics produced by bacteria in the genus Streptomyces inhibit translation by preventing the binding of aminoacyl tRNA molecules to ribosomes Since these drugs only prevent protein synthesis while present in effective concentrations their action is not permanent and they are static rather than cidal When used to treat young children they can cause permentent discoloration darking of the teeth Other complications can include liver damage skin photosensitivity and damage to 30S ribosomal subunits within mitochondria Antibiotics called Arninoglycosides also inhibit protein synthesis but bind permanently to ribosomes either the 30S or 50S subunits and block the transfer of peptidyl t RNA molecules from the A site to the P site Though their exact mechanism of action is uncertain the effect is permanent and so these drugs are cidal ie cause cells to die Aminoglycosides including Streptomycin neomycin and kanainycin were originally produced by bacteria in the genus Streptomyces while Gentamicin Tobrainicin and Amikacin were made by a bacteria in the genus Micromonospom Aminoglycosides can sometimes cause damage to kidneys so their concentrations and effects must be carefully monitored Inhibition of membrane function Drugs that interfere with membrane function may act either on the cell membrane or on the outer membrane of the Gram negative cell wall Two drugs that interfere with membrane function are Bacitracin and Polymyxin antibiotics produced by bacteria in the genus Bacillus Bacitracin inhibits the formation of peptidoglycan by acting on membrane transporters involved in moving materials required for wall synthesis across the cell membrane Polymyxins interact with phospholipids and disrupt membrane structure as well as function Both of these antibiotics are cidal Inhibition of nucleic acid synthesis mRNA synthesis Two chemicals that inhibit nucleic acid synthesis are Rifampin Rifainpicin and Actinomycin D Rifampin is an antibiotic produced by bacteria in the genus Streptomyces and binds with the beta subunit of prokaryotic RNA polymerase inhibiting transcription It can have adverse effects on liver tissue Actinomycin D also called Dactinomycin binds to DNA molecules and inhibits both replication and transcription Because it is highly toxic to mammalian cells as well as bacteria it is used primarily as a chemotherapeutic agent in the treatment of certain cancers Antimicrobial drugs are sometimes referred to as narrow spectrum or broad spectrum depending on their range of effectiveness Narrowspectrum drugs effectively control only a few or sometimes only one type of pathogen while broadspectrum drugs control many types usually both Grain positive and Gram negative forms Formerly broad spectrum drugs were preferred because physicians did not appreciate the role played by normal ora Currently some attempt is being made to limit treatments to narrow spectrum drugs and to develop drugs with more specific mechanisms of action This would help prevent damage to bacteria beneficial to the body and would also help reduce the development of drug resistant strains Not surprisingly antibiotics are not effective against viruses because viruses do not have metabolic pathways do not form cell walls do not synthesize proteins and do not have cell membranes Antiviral drugs typically act to inhibit the binding of viruses with their host cells or to inhibit viral replication within cells Harriet tare Notes 39 7w 0 0 B10 ijci Control of Metabolic Processes As described earlier the metabolic processes occurring within living organisms glycolysis respiration photophosphorylation etc are dependent upon the enzymes present within cells and these are determined by the genes present or the information carried within DNA molecules Whether or not a specific cell is using one or another metabolic process is determined by regulatory mechanisms functioning at various levels In this section two mechanisms for controlling metabolism will be described one functioning at the enzyme level and the other functioning at the gene level Feedback inhibition Most enzymes are proteins though some are RNA molecules so are composed of multiple amino acids connected together in long chains They typically have tertiary structure are 3dimensional and many are quaternary are composed of multiple polypeptide chains As described earlier enzymes are specific in their action and bind with their substrate or reactant molecules through regions on their surfaces called reactive sites or binding sites Enzymes can also be inhibited ie the catalytic activity of enzymes can be blocked through either competitive or allosteric inhibition Feedback inhibition is a regulatory mechanism involving the allosteric inhibition of one or more enzymes usually the first or second involved a common metabolic pathway The inhibitor is usually the endproduct of the pathway so this mechanism can also be called endproduct inhibition ln bacteria the biosynthesis of isoleucine an amino acid involves threonine also an amino acid as a substrate and a metabolic pathway with five steps catalyzed by five different enzymes The enzymes involved are represented by the letters AE and the metabolic intermediates by the letters WZ in the diagram below A B c D E Threonine w gt X gt Y gt Z gt 18016110116 Allosteric inhihih39rm of enzyme A When isoleucine begins to accumulate within the cytoplasm it acts as an allosteric inhibitor of the first enzyme in the pathway enzyme A and effectively shuts down isoleucine synthesis Because the endproduct of the pathway acts to reduce its own production this is an example of negative feedback Feedback or endproduct inhibition occurs within both eukaryotic and prokaryotic cells and allows organisms to control metabolic processes relatively quickly exerts rapid control This mechanism is also reversible because when the concentration of endproduct is decreased the first enzyme is no longer inhibited Though used extensively this mechanism is not efficient in terms of energy conservation because when metabolic pathways are inhibited enzymes are inactive This means the cell had to expend considerable energy in the formation of m RNA molecules and polypeptides to establish a metabolic pathway no longer being used A more efficient means of controlling metabolism can be exerted at the gene level Genetic control In prokaryotic cells the genes coding for enzymes involved in a common metabolic pathways are often arranged together within specific regions of the chromosome called operons An operon is a segment of DNA a nucleotide sequence containing a series of structural genes and the control elements regulating the transcription of those genes The quotcontrol elementsquot typically include a promoter site or sequence and a region known as the operator site an attenuator site may also be present but will not be included here Recall that promoter sites are nucleotide sequences recognized by the sigma factors of RNA polymerase Promoters interacting with sigma factors determine where transcription will begin and in which direction it will proceed along DNA molecules The operator site functions like an quotonof quot switch and is in uenced by DNAbinding proteins called repressers When a represser protein is bound to the operator transcription is blocked repressed but if the represser is not bound transcription is allowed to proceed There are many different metabolic pathways controlled by operons in different types of bacteria but two such systems found within E coli cells are commonly used as examples of genetic control mechanisms Tryptophan biosynthesis Control involving a repressible operon The operon controlling tryptophan biosynthesis in E coli is commonly used as an example of a repressible operon ie one in which transcription is usually occurring but can be repressed or quotturned offquot Tryptophan is an amino acid synthesized from glutamine and chorismic acid by means of a metabolic pathway involving five enzymes as diagramined below Chorsmic acid A D E W gt X gt Y gt Z gt Tryptophan Glutamine In this pathway enzymes are designated by the letters quotAEquot and metabolic intermediates formed within the pathway are designated by the letters quotWZquot The genes coding for the enzymes above are arranged together within the tryptophan biosynthesis operon This operon includes a promoter site an operator site and an attenuator site but only the first two control elements will be described here Promoter Operator Attenua Gene A Gene B Gene C Gene D Gene E The promoter site is the nucleotide sequence sigma factor binds with to start transcription The operator site is the nucleotide sequence the represser protein binds with to block or repress transcription In this case the gene coding for the represser protein is not part of the operon but is located in a different region of the chromosome The represser protein associated with the tryptophan biosynthesis operon is inactive alone so cannot bind with the operator site Under most circumstances this operon is quotonquot ie transcription is allowed to proceed While the operon is active transcription results in the formation of m RNA molecules that travel to the ribosome and code for the production of enzymes AE The biosynthesis is then allowed to proceed and tryptophan levels within the cell increase Tryptophan serves as a corepresser ie a compound that can bind with the inactive represser protein and activate it much like coenzymes and cofactors activate enzymes The tryptophanrepresser complex can then bind with the operator site and block transcription of the genes present within the operon This mechanism is very efficient in terms of energy conservation because when tryptophan concentrations become high the cell will not just inhibit the enzymes involved in tryptophan synthesis it will stop making the mRNA molecules necessary to form the polypeptides needed to run the tryptophan biosynthesis pathway Lactose utilization Control involving an inducible operon Lactose utilization in E coli is controlled by an inducible operon ie one in which transcription is usually repressed or quotoffquot but can be induced or quotturned onquot This operon often referred to as the lac operon includes three structural genes a promoter and an operator A regulatory gene near the lac operon promoter site codes for a represser protein that is active alone The represser protein is constitutive so is always being made consequently under most circulnstances the structural genes within the operon are not being transcribed Note When E coli cells are living within the intestines of adult cows there is no lactose available so making enzymes to catabolize it would be a waste of energy The E coli cells conserve energy by repressing the transcription of the lac operon genes Promoter I lacl l Promoter l Operator l lacZ l lacY l lacA In this diagram the lacl gene is the regulatory gene coding for the represser protein promoter sites are nucleotide sequences where sigma factors bind to begin transcription the operator site is where the active represser protein binds to block repress transcription and the structural genes lacZ lacY and lacA code for enzymes associated with lactose utilization Since the represser is constitutive and active alone transcription of the three structural genes is usually being repressed but not entirely Why not The structural genes within the lac operon code for enzymes Two of these lacY and lacZ encode enzymes directly involved in lactose utilization ie Sgalactoside permease an enzyme allowing lactose to enter the cell and galactosidase an enzyme that breaks lactose into glucose and galactose respectively The third gene lacA codes for the enzyme thiogalactoside transacetylase This enzyme catalyzes a chemical reaction converting lactose into allolactose Allolactose is significant because it serves as the inducer for the lac operon When allolactose is abundant it binds with the represser protein and inactivates it ie changes its configuration so it can no longer bind with the operator site With the represser removed transcription is allowed to proceed At this point one might well ask the following question If the enzymes encoded by the genes of the lac operon are not being made lactose cannot enter the cell and allolactose cannot be formed therefore how can this operon ever be induced Apparently the operon is quotof quot like a leaky faucet some transcription occurs even when the operon is bein repressed This is because although the represser protein binds tightly to the operator site it only interferes with RNA polymerase and doesn39t completely block transcription Although inducers eg allolactose partially control the transcription of inducible genes inducible operons are also regulated by a mechanism called catabolite repression This mechanism involves cyclicAMP as a regulatory molecule Note The lac operon is not the only inducible operon in E coli cells other operons have similar control mechanisms Catabolite repression Catabolite repression is a mechanism allowing bacteria such as E coli to utilize constitutive enzymes in favor of inducible ones This mechanism adds a second layer of control and ilnproves the efficiency of inducible operons eg the quotleakyquot systems as described above A catabolite is any substance a cell can catabolize break down to release energy Glucose is a common catabolite and as described earlier can be broken down by various metabolic pathways Since E coli cells are facultatively anaerobic they can use either fermentation or respiratory pathways to catabolize glucose In either case when glucose is available ATP is being made and energy is available to drive cellular processes The ow of energy through E coli cells is vastly simpli ed in the diagram presented below Glucose ATP Energy released Major source of gt I gt to drive cellular chemical energy AMP PP processes In this diagram the covalent bonds within glucose molecules are being broken through catabolic processes recall fermentation and cellular respiration and the energy released is being used to convert AMP PP into ATP Since ATP is the energy currency of the cell it is constantly being broken down and the energy released is used to drive cellular processes e g active transport agellar motion replication transcription etc The catabolism of ATP yields AMP PP This diagram is not accurate because as was explained earlier ATP is formed from ADP Pi adenosine diphosphate and inorganic phosphate however the diagraIn is accurate to this extent When ATP levels increase c AMP levels decrease When associated with glucose catabolism this occurs because the transport of glucose into cells inhibits the activity of adenylate cyclase the enzyme responsible for converting ATP into cyclicAMP In its cyclic form 339 539cyclic adenosine monophosphate cyclicAMP AMP serves as a regulatory molecule ie as a quotsecond messengerquot or a molecule involved in signal transduction CyclicAMP can bind with a protein called catabolite activating protein CAP also called cyclicAMP receptor protein CRP to form a complex that can interact with DNA The cyclicAMPCAP complex binds to DNA at a site very near the promoter site on the lac operon and makes it easier for the sigma factor of RNA polymerase to bind ie it enhances the promotor site makes it more attractive to sigma factor When cyclic AMP levels are high the cAMPCAP complex is bound to DNA and the structural genes of the lac operon are transcribed transcription is allowed to occur assuming lactose is available and some of it has been converted into allolactose When the lac operon promoter site is not quotenhancedquot sigma factor is only weakly attracted to it So if E coli cells are placed into a TSI slant which of the three sugars present will they use first and why Although the lac operon is quotleakyquot in terms of the control exerted upon it by the represser protein very little transcription of the lac operon structural genes will occur as long as glucose is available to the E coli cells The enzymes involved in glucose catabolism are constitutive and not under control of a represser The E coli will use glucose first and only when glucose is no longer available will they use lactose Prokaryotic Cell Structure and Function Prokaryotic organisms are much less complex than eukaryotic cells but have some features in common This presentation will begin with prokaryotic structures found outside the cell membrane and will work inward from there Structures visible on the cell model are similar in appearance to those associated with some eukaryotic organisms though not usually found together on a single cell however although the long slender appendages are agella the shorter hairlike structures are not cilia Prokaryotic cells do not have cilia A Structures found outside the cell membrane 1 N Flagella The agella singular agellum of prokaryotic cells bacteria specifically are made up of proteins called agellin proteins or agellins These globular proteins polymerize to form linear strands that wrap around one another forming the walls of the agellum the core is hollow Prokaryotic agella are not surrounded by the cell membrane and are not supported by microtubules Instead each agellum is attached to the cell membrane and the cell wall by a series of complex ringlike structures That portion of a agellum associated with the rings extends through the membrane and the wall like an axel attached to several quotwheelsquot Outside the wall it makes a 900 bend and then extends lengthwise away from the cell The motion of bacteria agella is rotary ie they spin Movement is powered by ion ow usually Ht but sometimes Na across the cell membrane Prokaryotic agella are not visible when live bacteria are viewed using light microscopes because they are two thin however they can be observed if treated with stain reagents increasing their thickness Flagellar number and arrangement varies between bacterial species and is sometimes useful in identification Some commonly encountered arrangements are indicated below and include the root word trich meaning hairlike Atrichous Cells having no agella a without Monotrichous Cells having only one agellum mono one Peritrichous Cells having agella arranged more or less uniformly all over their surfaces peri around Amphitrichous Cells having one or more agella at both ends amphi both ways or two ways Flagella allow bacteria to swim through their environments in search of food materials light etc so are involved in taxis directed movement If the bacteria are pathogens agella can aid their dispersal so may increase their ability to cause disease symptoms Certain viruses may access bacteria through their agella so agella can also serve as virus entry ports The ability to form agella is dependent on environmental conditions therefore even though cells are genetically capable of forming agella they may not do so Fimbriae and Pili 7 Fimbriae singular f1mbria are short hairlike appendages found outside the cell walls of certain types of bacteria especially Gramnegative cells They are made of E 4 fimbrin proteins and are o en quite numerous covering the entire cell surface Although frmbriae look similar to cilia in scanning electronphotomicrographs they are not used for swimming Fimbriae are used primarily for attachment They allow bacteria to bind to the surfaces of rocks sticks leaves etc in their environments or in the case of pathogenic forms they allow for attachment to host cells Pathogenic bacteria such as Neisseria gonorrhoeae cannot cause disease if they are unable to bind to the surfaces of their host cells Pili singular pilus are longer and less numerous than frmbriae and are made of pilin proteins Pili allow bacteria to bind other cells of the same species and facilitate genetic exchange ie the passage of DNA from one cell to another Pili involved in this gene transfer activity are called quotFquot fertility or sex pili Glycocalyx 7 The glycocalyX is a layer found outside the cell wall of certain types of bacteria It is usually made up of polysaccharide but may contain protein and can be thick or thin depending on the availability of nutrient materials in the environment If the glycocalyX is dense and well organized it is called a capsule while if it is loose and poorly organized it is called a slime layer The colonies of bacteria forming glycocalyxes are often wet looking slimy and will sometimes drip from agar surfaces into the lids of inverted culture plates The primary function of the glycocalyX is food storage Because bacteria live in environments prone to quotfeast or faminequot conditions they frequently collect reservoirs of stored food materials outside their walls when conditions are favorable and then use these to sustain themselves during long periods with poor nutrient availability Capsules cause the surfaces of bacteria to be sticky facilitating their attachment to smooth surfaces such as tooth enamel The capsules formed by Streptococcus mutans a common inhabitant of the human mouth are largely responsible for the formation of dental plaque a soft whitish gooey material readily removed with dental oss Capsules provide a certain amount of protection to cells forming them both from predators in their environments and desiccation Because capsuleforming bacteria are generally ignored by phagocytic WBCs involved in defending us against pathogens the presence of a glycocalyX also increases the pathogenicity diseasecausing ability of bacteria Cell wall 7 The cell wall is a rigid layer usually found outside the cell membrane of most bacteria cells In the case of bacteria commonly encountered in the clinical setting it is composed of a unique material called peptidoglycan described in detail during the Gram stain lab The peptidoglycan layer is thick in Grampositive cell walls and much thinner in those of Gramnegative cells The walls of Gramnegative cells are also equipped with an outer membranous layer absent from Grampositive cells Differences in wall composition made evident by stain reactions Grampositive purple Vs Gramnegative pink in uence the responses of bacteria to environmental conditions Gram positive bacteria with their thick peptidoglycan walls are more resistant to damage from physical factors such as heat radiation desiccation and pressure than are Gramnegative cells therefore pasteurization will kill Gramnegative cells more readily than Grampositive forms Gram negative cells protected by an extra membrane layer outside their thin peptidoglycan wall are more resistant to chemicals such as basic dyes Slactam antibiotics and lysozyme Media containing basic dyes are frequently used to select for Gramnegative bacteria because Grampositive cultures will not grow on them 5 Periplasmic space 7 The periplasmic space peri around is a potential space existing outside the cell membrane but inside the outer membrane of the Gramnegative cell wall though a similar potential space exists outside the cell membrane and inside the peptidoglycan layer of the Grampositive cell wall It is a region where enzymes and wall components released by the cell membrane tend to accumulate so is involved in storage of these materials 0 Periplasmic agella 7 Periplasmic agella also known as endo agella or axial laments are agella found within the periplasmic space of Spirochetes long slender spiralshaped bacteria with exible peptidoglycan walls Periplasmic agella arise from both ends of a typical spirochete and wrap around the cell surface inside the outer membrane The rotary motion of these agella causes the entire cell to spin creating a swimming motion similar to a corkscrew The Cell Membrane described in detail in an earlier section serves here as a boundary between external and internal cell structures Recall that the prokaryotic cell membrane is involved in ATP synthesis and contains enzymes involved in other physiological processes The cell membranes of some prokaryotic cells fold inward forming regions with extensive surface area and therefore high concentrations of enzymes Two of these structures are described below 1 N 55 N Mesosomes 7 Mesosomes are membranous folds extending into the cytoplasm of some prokaryotic cells They contain enzymes involved in ATP synthesis Via oxidative phosphorylation so are analogous to the cristae of mitochondria Mesosomes also apparently attach to the covalently closed circular DNA molecules of prokaryotic cells and aid in chromosome separation during cell division This is significant because prokaryotic cells do not contain microtubules and so cannot form a spindle apparatus Thylakoids 7 Thylakoids are long slender membranous structures formed by the inward folded cell membranes of certain bacteria capable of using light as an energy source ie cyanobacteria green and purple sulfur bacteria and others The pigments involved in capturing light energy as well as the enzymes involved in photophosphorylation are bound to the membranes of thylakoids Structures found inside internal to the cell membrane Cytoplasm The cytoplasm of a prokaryotic cell includes all the protoplasm surrounded by the cell membrane Because prokaryotes are not nucleated there is no nucleoplasm surrounded by a nuclear envelope The cytoplasm forms the bulk of the cell but is not compartmentalized by membranous organelles Structures common in eukaryotes such as the endoplasmic reticulum Golgi apparatus lysosomes peroxisomes vacuoles vesicles and mitochondria are lacking A variety of structures are suspended in the cytoplasm but these are not surrounded by membranes made of lipid bilayers with integral and peripheral proteins some may be surrounded by thin membranes made up of protein or other materials Nucleoid 7 The nucleoid nucleoid nucleuslike or nuclear region is the control center of the prokaryotic cell and contains an extremely dense accumulation of cccDNA sometimes more than one segment accompanied by small amounts of RNA and protein It is not surrounded by a membrane or nuclear envelope so is not a true nucleus Like the nucleus of eukaryotic cells E 4 V39 the nucleoid contains most of the cell s DNA and controls physiological processes by determining which enzymes the cell can make It contains homogeneous proteins known as nucleoid proteins that are functionally similar to the histones of eukaryotic cells but nucleosomes are not formed within prokaryotes and neither are nucleoli Most of the RNA present within the nucleoid is messengerRNA mRNA Plasmids 7 Plasmids are small extrachromosomal loops of DNA cchNA carrying genes not essential to cell function under most circumstances Plasmids replicate themselves independently of the cells chromosome and frequently occur in relatively large numbers sometimes more than 100 per cell Their size is variable and the genes they carry code for a variety of different proteins Some examples of frequently described plasmids include F factor plasmids 7 Plasmids known as F factor F fertility carry genes coding for the production of sex pili and the transfer of DNA through conjugation R factor plasmids 7 Plasmids known as R factor R resistance carry genes coding for resistance to various antimicrobial drugs For example many bacteria carry genes coding for 5 lactamase an enzyme that degrades Slactam antibiotics such as penicillins Bacteria carring R factor plasmids are resistant to antimicrobial drugs ie are not harmedcontrolled by them Col plasmids 7 Plasmids called Colplasmids found in Gramnegative bacteria identified as Escherichia coli carry genes coding for the production of proteins called colicins These proteins can be released by E coli cells and will kill other closely related cells The E 001139 living in your gut produce colicins that can kill other strains of E coli thus protecting you from infection The ability of Pseudomonas species to catabolize a wide range of unusual organic compounds is determined by genes carried on plasmids Ribosomes 7 The ribosomes of prokaryotic cells like those of eukaryotic cells are small granular bodies made up of ribosomal RNA r RNA and protein Unlike eukaryotic ribosomes those in prokaryotic cells are 708 instead of 808 and are made up of SOS and 308 subunits Ribosomes are the site of protein synthesis and are numerous within actively growing cells Most of the RNA present in prokaryotic cytoplasm is rRNA Inclusions 7 Prokaryotic cells may contain a variety of structures known as inclusions Although eukaryotic inclusions are usually considered to be nonliving this description seems less appropriate for those found in prokaryotes Some example inclusions are listed below a Carboxysomes 7 Carboxysomes are inclusions containing enzymes involved in quotfixingquot carbon dioxide C02 into organic compounds Although not all prokaryotes capable of using C02 from the air contain carboxysomes many of them do b Poly hydroxybutyrate granules PHB granules 7 PHB granules contain nutrient materials stored as polymers during periods when cells are not actively growing Although important to cells as nutrient reserves PHB granules are used by researchers to capture recombinant proteins produced within genetically engineered cells and are also being considered for use in the production of biodegradable plastics 0 3 1 D quot1 Metachromatic granules 7 Metachromatic granules are accumulations of stored phosphate reserves polyphosphates Since phosphates are required for the production or ATP metachromatic granules are sometimes considered to be energy reserves They tend to stain red in the presence of methylene blue Gas vacuoles 7 The gas vacuoles gas vesicles found within certain prokaryotic cells are not true vacuoles but are hollow cylinders surrounded by a thin layer of protein They are found in aquatic bacteria and are used to regulate buoyancy within water environments Magnetosomes 7 Magnetosomes are inclusions made up of crystalline metals usually iron or magnetite contained within folds of the cell membrane They are involved in the detection of magnetic elds in bacteria capable of magnetotaxis Sulfur granules 7 Sulfur granules are particles of elemental sulfur formed within cells capable of using hydrogen sulfide HzS as an electron donor Electrons pulled away from HzS can be passed to pigment molecules replacing those removed by exposure to light a phenomenon explained later The remaining sulfur atoms can bind to form elemental sulfur SS C Specialized cell types A number of structures associated with prokaryotic cells are most accurately considered as specialized cell types even though they may be formed within or from other cells Some examples of specialized cell types are included below 1 Endospores 7 Endospores are dormant structures produced within certain types of bacteria including the genera Bacillus and Clostridium They are not reproductive structures but are highly resistant to damage and allow cells to survive periods of unfavorable conditions Endospores are unlike vegetative cells the cells forming them in a number of ways including 999 D quot1 They contain little or no water and are metabolically inactive They contain higher levels of DNA than active cells and almost no RNA They contain high levels of calcium and dipicolinic acid pyridine26dicarboxylic acid Each endospore is surrounded by two layers of membrane and two layers of wall like material a cortex of modified peptidoglycan and a spore coat formed of multilayered hydrophobic protein They are highly resistant to damage caused by physical factors such as heat pressure radiation desiccation and are also resistant to toxic chemicals including antimicrobial drugs Endospores can remain inactive but potentially viable for long periods of time e g 500 1000 years in lake sediments millions of years in amber Endospores are formed inside vegetative cells through a process called sporulation A cell containing an endospore or forming an endospore is called a sporangium The sporulation process can be divided into a number of stages or steps as indicated below a b The cells DNA is replicated copied and condensed The cell membrane invaginates forming a septum between the chromosomes toward one end of the cell This divides the cytoplasm into two unequal parts or protoplasts the smaller one being the forespore or prespore N E 4 c The larger protoplast engulfs the smaller one endocytosis occurs and the resulting forespore is surrounded by two layers of membrane d A modi ed form of peptidoglycan is deposited outside the inner spore membrane forming the cortex e Layers of hydrophobic protein are deposited outside the outer spore membrane forming the spore coat At this time also calciumdipicolinate dipicolinic acid accumulates within the developing endospore f The endospore matures developing its characteristics as described above Eventually the sporangium deteriorates and an exospore is released A sample of Bacillus cells stained with malachite green or carbol fuchsin as practiced in the laboratory will typically contain mature spores within cells as well as numerous exospores Endospore shape and location within the sporangium is variable and can be useful for identi cation purposes The formation of an endospore sometimes causes swelling of the sporangium and sometimes not The process involved when an exospore develops into a new vegetative cell is called germination It requires a triggering event or activation of the spore that may involve aging temperature change pH change or exposure to certain chemicals During activation the exspore undergoes internal changes in molecular con guration but does not lose its resistance to environmental stress During germination the exospore takes on water and becomes metabolically active The levels of calcium and dipicolinic acid inside decrease outer coverings are degraded and a new cell grows out The outgrowth phase occurring after the cortex and spore coat are degraded is somewhat similar at least in appearance to the growth of a new seedling from a germinating plant seed hence the name Heterocysts and Akinetes 7 Heterocysts and akinetes are specialized cells formed by certain types of bacteria called cyanobacteria formerly known as blue green algae Heterocysts are modi ed vegetative cells that function as anaerobic compartments rich in enzymes involved in nitrogen xation They are typically somewhat larger have thicker coverings than vegetative cells and are internally modi ed to fix nitrogen most ef ciently They communicate with vegetative cells through their cell membranes and the two are interdependent Heterocysts cannot reproduce Akinetes are specialized cells that often develop adjacent to or near heterocysts They are larger than vegetative cells have thick walls granular cytoplasm and are less active metabolically Akinetes are more resistant than vegetative cells to extreme temperatures and desiccation allowing cyanobacteria populations to survive cold winters and possibly dry summer months Conidia conidiospores 7 Conidia are reproductive structures produced by lamentous bacteria the term is also applied to reproductive structures produced by certain fungi They appear as cocci arranged in long chains at the ends of threadlike laments The colonies of bacteria forming conidia are typically dry and powderlike in surface texture like a powdered sugar doughnut Sphaeroplasts and Protoplasts 7 Sphaeroplasts and protoplasts are spherical fragile cells formed by removing the peptidoglycan walls from bacteria maintained in isotonic environments Sphaeroplasts are formed from Gramnegative cells and maintain a partial wall ie the outer membrane of their cell wall Protoplasts are formed from Grampositive cells and lack cell walls These specialized cell types are sometimes developed for research purposes but require careful handling because they are sensitive to changes in osmotic pressure Eukaryotic Cell Structure and Function Part 1 The science or study of cell structure and function is called cytology but courses dealing with this topic frequently come under the heading of cell and molecular biology Cytology has undergone extensive change over time The term cell cella a small room was first used by Robert Hooke 1665 with reference to an empty space or chamber like a prison cell Hooke was observing the cell walls of dead cork cells from the bark of cork oaks and not living cells We now know cells are far from empty spaces According to the cell theory as articulated by Matthias Schleiden and Theodor Schwann 1839 the cell is the basic unit of structure and function in all living organisms When first written the cell theory indicated that living cells could arise spontaneously through abiogenesis but experiments conducted by Louis Pasteur and others invalidated this concept Instead it is now recognized that all cells arise from preexisting cells and that they carry hereditary information DNA that is passed from one generation to the next through cell division Cells are currently divided into two types Prokaryotic and Eukaryotic The term karyon karyon nucleus appears in both names and is preceded by either pro meaning before or eu meaning well or truly Fossil and molecular evidence indicates that prokaryotic cells evolved first and that the larger nucleated cells evolved later Some of the distinguishing features of these two cell types are outlined below A typical prokaryotic cell Before a nucleus Does not contain a nucleus surrounded by a nuclear membrane or envelope Contains one or more loops of covalentlyclosed circular DNA cccDNA Is not compartmentalized by membranous organelles Contains 705 ribosomes ls surrounded by a membrane lacking cholesterol and involved in ATP synthesis wall synthesis taxis and other physiological activities ls usually smaller than a typical eukaryotic cell Bacteria and Archaea are prokaryotic organisms however the Archaea have membranes unlike other cells A typical eukaryotic cell Well or truly nucleated Contains one or more true nuclei each surrounded by a nuclear envelope Contains two or more linear chromosomes ls compartmentalized by many membranous organelles Contains SOS ribosomes ls surrounded by a membrane with cholesterol and not involved in the synthesis of ATP ls usually larger than a typical prokaryotic cell Microorganisms categorized as Protozoa Algae and Fungi are made up of eukaryotic cells as are all plants and animals including hulnans Since eukaryotic cells are more faIniliar to many students than are prokaryotic cells these will be covered first Each eukaryotic cell is surrounded by a cell membrane and most have one or more true nuclei though there are some exceptions The region of protoplasm between the cell membrane and the nucleus is referred to as cytoplasm while the protoplasm inside the nuclear membrane or envelope is called nucleoplasm all is living substance The cytoplasm is often highly compartmentalized by internal structures called organelles organelles little organs and makes up the bulk of the cell Organelles occur in a variety of shapes and sizes and typically carry out specific functions like the organs found within the hulnan body A nulnber of the structures found within eukaryotic cells are outlined in the lecture syllabus Keep in mind that not all of the structures listed are found within all types of eukaryotic cells 1 Cell membrane The cell membrane surrounds and limits the cell and has the structure and functions covered in an earlier section N Endoplasmic reticulum The endoplasmic reticululn endo inside plasmic cytoplasm reticululn network is a membranous organelle extending from the nuclear envelope into the cytoplasm and occasionally connecting to the cell membrane In composition it is like the cell membrane but includes two membrane layers separated by a thin space In some cells the endoplasmic reticululn is highly folded and takes up most of the cytoplasm Functions associated with the smooth endoplasmic reticulum include storage transport and the synthesis of lipids Materials can be moved from place to place between the membrane layers and portions of membrane can be transferred from the ER to other structures Some regions of endoplasmic reticululn are covered with small granular bodies called ribosomes This is called rough endoplasmic reticulum and is involved in protein synthesis DJ Ribosomes Ribosomes are small granular bodies composed of ribosomalRNA and protein ribo ribose the pentose monosaccharide found in RNA soma body Eukaryotic ribosomes are SOS the S referring to Svedburg units a measure of density and include two subunits of 605 and 405 Ribosomes are the site of protein synthesis so are essential to cell function The two subunits come together only when proteins are being made and those ribosomes associated with the rough endoplasmic reticululn are attached to the membranes by the proteins they are forming Proteins formed by ribosomes bound to the rough ER are usually exported from the cell while those formed by ribosomes free in the cytoplasm stay inside the cell Protein synthesis will be described in detail later 4 Golgi complex apparatus or body The Golgi complex is an organelle composed of at membranous sacs arranged in a stack and is essentially modified smoo endoplasmic reticululn Like the ER the Golgi is involved in transport and storage but is also the primary site of polysacchari de synthesis Membranous components and other materials passing from the ER both smooth and rough to the Golgi provide rawmaterials for the assembly of complex organic compounds such as lipopolysaccharides lipoproteins and glycoproteins The Golgi is also involved in F 1 00 o packaging and secretion as segments of it can be pinched off to form vesicles and these can move to the cell surface where their membranes fuse with the cell membrane The materials contained within these vesicles can then exit the cell through exocytosis Vacuoles and vesicles Vacuoles and vesicles are membranous organelles containing a variety of materials and formed in various ways The term vacuole suggests an empty space a vacuum and in some cells plants and algae refers to a large centrally located region filled with what appears to be clear uid the central vacuole This structure helps maintain tugor pressure within cells and stores toxins that might interfere with metabolism Vesicles are generally smaller than vacuoles but the terminology associated with these membranous quotbubblesquot is inconsistent Protozoa taking in food materials through endocytosis form food vacuoles or pinocytic vesicles depending on what was ingested Both types of structures essentially store nutrients until they can be digested The membranous fragments quotpinchedquot from the Golgi are most commonly referred to as vesicles and these carry various materials waste products digestive enzymes etc toward the cell surface where they can be released through exocytosis Lysosomes are formed by the Golgi complex Contractile vacuoles Contractile vacuoles are organelles found commonly in fresh water protozoa They vary considerably in size and complexity but often appear as circular structures that swell and shrink in a repeating pattern The swelling occurs when the contractile vacuole is relaxed and filling diastole while the shrinking occurs when it contracts pumping water out of the cell systole Contractile vacuoles pump excess water out so function as osmoregulatory structures preventing cells from bursting due to osmosis They are connected to the smooth endoplasmic reticulum so also aid uid circulation within the cell Since liquid wastes may be eliminated along with the water contractile vacuoles may also have an excretory function Lysosomes Lysosomes lysis to split soma body are membranous organelles containing a variety of digestive enzymes hydrolases involved in the hydrolysis catabolism of organic compounds Their primary function within microorganisms is the digestion of food materials taken in through endocytosis however they also degrade worn out organelles and help the cell recycle membrane components The hydrolase enzymes carried within lysosomes are activated after the lysosome membrane binds with that of a food vacuole and hydrogen ions are pumped in lowering the pH Bacteria able to prevent this acidification can avoid digestion and may take up residence within the phagocytic cell Lysosomes are formed and released by the Golgi complex Peroxisomes Peroxisomes are membranous organelles containing enzymes involved in hydrogen peroxide metabolism Some of these generate hydrogen peroxide by oxidizing organic compounds while others called catalase enzymes break down the hydrogen peroxide produced Since hydrogen peroxide is toxic the overall function of catalase enzymes is neutralizing toxins Peroxisomes are not formed by the golgi complex but are apparently selfreplicating Mitochondria Mitochondria singular mitochondrion are selfreplicating organelles surrounded by double layers of membrane Each mitochondrlon has an outer membrane typical of eukaryotic cells and an inner membrane lacking cholesterol and carrying enzymes involved in ATP synthesis The surface area of the inner membrane is greatly increased by folds called cristae and these are the site of oxidative phosphorylation ATP synthesis involving the oxidation of coenzymes Since quanti es of ATP are produced by mitochondria they are sometimes referred to as the quotpowerhouses of the cellquot In addition to their unique inner membranes mitochondria have a nu1nber of other interesting features they carry 705 ribosomes have closed loops of DNA cccDNA and are daInaged by antibacterial drugs These features provide strong evidence that mitochondria evolved from prokaryotic cells taken in by larger eukaryotic organisms through endocytosis Having avoided digestion possibly by the mechanism mentioned above they formed a permanent symbiotic relationship beneficial to both them and their host organisms Chloroplasts Chloroplasts like mitochondria are selfreplicating organelles surrounded by double layers of membrane Within each chloroplast the inner membrane is folded into nu1nerous sacklike structures called thylakoids containing lighttrapping pigments and enzymes involved in ATP synthesis through photophosphorylation a process dependent on light energy Chloroplasts are only found in certain types of cells such as algae and green plants Like mitochondria chloroplasts carry 705 ribosomes have cccDNA and are damaged by antibacterial drugs They apparently evolved from several different types of cyanobacteria ingested by eukaryotes on more than one occasion because not all chloroplasts carry the same types of lighttrapping pigments Chloroplasts represent another fine exaInple of mutually beneficial symbiosis end of Part 1


Buy Material

Are you sure you want to buy this material for

25 Karma

Buy Material

BOOM! Enjoy Your Free Notes!

We've added these Notes to your profile, click here to view them now.


You're already Subscribed!

Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'

Why people love StudySoup

Steve Martinelli UC Los Angeles

"There's no way I would have passed my Organic Chemistry class this semester without the notes and study guides I got from StudySoup."

Jennifer McGill UCSF Med School

"Selling my MCAT study guides and notes has been a great source of side revenue while I'm in school. Some months I'm making over $500! Plus, it makes me happy knowing that I'm helping future med students with their MCAT."

Bentley McCaw University of Florida

"I was shooting for a perfect 4.0 GPA this semester. Having StudySoup as a study aid was critical to helping me achieve my goal...and I nailed it!"

Parker Thompson 500 Startups

"It's a great way for students to improve their educational experience and it seemed like a product that everybody wants, so all the people participating are winning."

Become an Elite Notetaker and start selling your notes online!

Refund Policy


All subscriptions to StudySoup are paid in full at the time of subscribing. To change your credit card information or to cancel your subscription, go to "Edit Settings". All credit card information will be available there. If you should decide to cancel your subscription, it will continue to be valid until the next payment period, as all payments for the current period were made in advance. For special circumstances, please email


StudySoup has more than 1 million course-specific study resources to help students study smarter. If you’re having trouble finding what you’re looking for, our customer support team can help you find what you need! Feel free to contact them here:

Recurring Subscriptions: If you have canceled your recurring subscription on the day of renewal and have not downloaded any documents, you may request a refund by submitting an email to

Satisfaction Guarantee: If you’re not satisfied with your subscription, you can contact us for further help. Contact must be made within 3 business days of your subscription purchase and your refund request will be subject for review.

Please Note: Refunds can never be provided more than 30 days after the initial purchase date regardless of your activity on the site.