Introductory Microbiology MMG 301
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Lecture 3 Microbial diversity and microbial evolution Yeast cell Eukarya 8 um In diameter Virus eukamotes Cytoplasm Nucleoid Ribosomes 739 7 Cell of Escherichia coli Bacteria Cytoplasmic quot 1 x 3 Mm Cell wall membrane a Prokaryote Endoplasmic Cytoplasmic reticulum membrane l l Ribosomes 1000 nm 1 pm Nucleus NUCIBOIUS 39 gt Mitochondrion f Nuclear membrane Golgi PROKARYOTES ARCHAEA Methanogens Chloroplast Extreme halophiles Cytoplasm thsioloqic diversity of microorqanisms Energy for production of highenergy Ener source bonds 6399quot ATP Chemicals Light Phototrophs obtain energy from light Chemotrophs obtain energy by oxidizing electron donors in their Chemotrophv trlmo wvvw w environments Organic Inorganic chemicals chemicals 0 Chemoorganotrophs obtain their glucose acetate etc H2HZS Fe NH4 etc energy from organic compounds Chemoorganotrophs Chemolithotrophs l glucose oz gt co2 H20 H2 02gt H20 light Chemolithotrophs obtain their energy from inorganic compounds only found among prokaryotes a iiiiiii All nnnnnnnn a ATP ATP Advantages of chemolithotro hs over chemoorganotro hs no com etition for organic compounds and inorganic compounds used a sources of energy are often waste products from chemoorganotrophs CH2005N02 thsioloqic diversity of microorqanisms Carbon Autotrophs use 002 as sole C source self feeders Primary producers Heterotroph use several to many different preformed organic carbon molecules eg glucose as C source majority of microorganisms Chemoorganotrophs are by definition heterotrophs All phototrophs and most chemolithotrophs are autotrophs Oxygen Strict aerobes only grow in the presence of O2 Strict anaerobes only grow in the absence of O2 Facultative anaerobes can grow in the presence of absence of 02 Food chain Habitat diversity Microorganisms are present anywhere on Earth where life is possible Familiar habitats soil water animals plants Extreme environments extremes of temperatures extremely salty waters extreme pHs high pressure low water content Classes and examples of extremophiles Extreme Descriptive Organism Domain Habitat Min Opt Max term name Temperature High Hyperthermophile Pyroobusfumarii Archaea Undersea 90 C 106 C 113 C thermal vents Low Polaromonas vacuoata Bacteria Sea ice 0 C 4 C 12 C It Low Picrophius Archaea Acidic hot 006 07 4 oshimae springs High Natronobacterium Archaea Soda lakes 8 5 1O 12 gregoryi Pressure Moritela yayanosii Bacteria Deep ocean 500 atm 700 atm gt1000 atm sediments NaCl Haobacterium Archaea Salterns 15 25 32 salinarum saturation Microbes are the most abundant biomass on Ea h Carbon Nitrogen Phosphorus Plants are a much larger reservoir of carbon than animals Whitman et al PNAS 1998 956578 6583 Evolution of life on Earth throuqh time Conditions needed for life Origin of Earth Essential elements 46 bya components of organic Oceanformation mOIeCUIeS Continual source of energy Mild temperature range allowing liquid water First water on Earth was steam Earth N203 First sedimentary rocks 0 4am 6 date from 386 billion years oxygenated Conyrlght 2009 Pearsmi Education Inc publishing as Pearson Banlamln Cummings Evidence for microbial life on early earth Microbial fossils in ancient rocks Stromatolites fossilized microbial communities microbial mats can be over 36 billion years old Modern stromatolites in Australia Frances Westall Subsurface hypothesis for the origin of life Current hypothesis Life arose on Earth in hot oxygenfree conditions probably on the ocean floor Mixing of hydrothermal water and ocean water causes precipitates Formation of porous mounds that trap minerals Acidic oxidized Fe and Ni catalyze the formation of mm quoton amino acids peptides sugars and 7 nitrogenous bases Organic molecules get concentrated in the pores ofthe mounds and can start interacting System powered by ow of reduced inorganic compounds and pH gradient Copyrlgm 2009 Psavsm Eaucai on in puhllshiilg as Pearson Bantam Cummings Evidence for this theorv on the oriqin of life Presentday deepsea vent PROKARYOTES A REA Marianasans EUKARYA Eu karyotic Crown speciesquot Slime molds Flagellates Giardia Root of the tree cwmmmum mum u EmitMian Mm emu Root of the evolution tree close to many hyperthermophilic organisms Presentday hyperthermophiles can be found in presentday deepsea vents What type of life came first RNA as a primitive form of life Early Bacteria Early Archaea Dispersal to other habitats The dilemma Divergance of cell wall biochemistry DNA provides the genetic information for proteins but Divergance 0 leId V biosynthesisv DNA requires proteins for 3 replication E 395 Diversification gt 2 How to reconcile this A primitive 3 334323 39 RNA world 5 DNA 7 39 L RNA can act both as gene and mm enzyme a way around the quotchickenor eggquot problem RNA Some RNAs known today can Preblotlc chemistry carry out enzyme reactions Self replication Synthesize nucleotides nanyrigmems Pearson mean m pubiishmg as Pearson Esmzmin Cummings Metabolism of primitive cells Primitive environmental conditions No 02 in the atmosphere or in the oceans Not many organic compounds Environment rich in CO2 H2 and H28 Deepsea environment Reminder Definitions Energy Phototrophs obtain energy from light Chemotrophs obtain energy from chemicals Chemoorganotrophs obtain their energy from organic compounds Chemolithotrophs obtain their energy from inorganic compounds Carbon Autotrophs use 002 as sole C source Heterotroph use organic molecules as C sources Oxygen Aerobes grow in the presence of O2 Anaerobes grow in the absence of O2 Metabolism of primitive cells 0 Alternative source of H2 Anaerobic energygenerating metabolism uv Chemolithotrophic and autotrophic 24n12 2 Possible mechanisms 1 2 Primitive 1 ATPase I Primitive I 2 H2 002 6 H20 CH2O iiiscat iv With H2 as electron donor sulfur SO could have been the electron acceptor H2 and S0 were abundant on early Earth V Accumulation of organic compounds including acetate 9 l Development of methane production by archaea from H2 CO2 or from acetate Development of phototrophv and oxvqenic metabolism Eon Billion Evolutionary Oxygen Metabolic and years ago event level other highlights li ca n 1f oxides l 20 Fi rsteukaryo tesl 1 onne shield V uu pnu Gremxida m My The common ancestor of most quot event bacteria not hyperthermophlles Cyanobacteria Oxygenic photosynthesis is likely an anaerobic phototroph 2H20 gt02 4H quotchmquot Devt of oxygenic photosynthesis Purple and green Anoxygenlc photosynthesis 35 bacteria Has gt502H AnOXIc quotquot Fistceua Methanogenesis O2 in the atmosphere ozone shield H d 40 life 0024H2gtCH42H20 a ean Formation of crust and ocean 45 Formation of Earth Devt of aerobic metabolism and of m life on surface of the Earth Oriqin of eukarvotic cells endosvmbiosis Eukaryote organism with a membrane enclosed nucleus and other organelles Mitochondrion site of respiration contains its own circular DNA Chloroplast site of photosynthesis contains its own circular DNA Mitochondria originate from endosymbiosis between an archaeum the host and an aerobic bacterium the symbiont Chloroplasts originate from endosymbiosis with an oxygenic phototroph a c anobacterium the s mbiont The eukaryotic cell is a chimera with attributes of both bacteria lipids and archaea transcription and translation apparatus Bacteria Eukarya Archaea Ancestor of chloroplast I lAnnestor of mitochondrion Hypothesis 1 Bacteria Eukarya Archaea Animals Ancestor of chloroplasl Nucleus formed y Ancestor of mitochondrion Hypothesis 2 Hydrogen hypothesis Taxonomy Taxonomv science of classification Usually thought of as a discipline in biology Three core activities Identification Classification Nomenclature Two types of classification systems Traditional artificial based on superficial characteristics phenotype and may not reflect true relationships between species Modern phylogenetic based on evolutionary relationships based on genotype Definitions Phenotype The observable characteristics of an organism such as morphology biochemical or phy U Genotype The complete description of an organism s genetic information Classification methods Classical approaches use physical tests that are applied in a pre determined order Artificial classification systems Select phenotypes that differentiate among different groups Examples Gram stain Ability to grow on various carbon sources Limitations of artificial classification phenotype may not reflect evolution Require application of the appropriate test in proper sequence Phenotypic tests are weighed based on the order in which they are used Currently accepted method of qenebased phyloqenv Small Subunit 16S rRNA sequences see next lecture Example of classical phenotvpic identification Obtain pure culture Based on observable Gram reaction characteristics phenotype of the organism an Gramnegative Gram positive artificial classification l39 Rodshaped Non rodshaped Facultative Obligate aerobic aerobic l I I Ferme nts lactose Does not producing acids ferment and gas lactose Perform biological tests Positive for indole methyl red and mucate Negative torcnrate vosgesHrOSKauer H28 Escherichia coli Taxonomic units Higher taxa I uunlalll Genus genera gt Phylum Species gt Class I The fundamental unit in biology Biovars varieties Within a speCIes gt Genus Serovar serological varieties gt Species Pathovar pathogenic varieties gt Variety Strains and Clones Population of cells descended from a Single cell of a speCIes NomencIature Daughter Put Catsup Formal process of naming organisms on Fathers Good Sunday Vest Latinized binomial names Each species has a unique name the Approved List At present there are approximately 9000 validly named species of prokaryotes Phylogenetic tree of bacteria All known prokaryotic pathogens are Bacteria but many many bacterial species are nonpathogenic The domain Bacteria has more than 40 phylogenetically distinct subdivisions fquot 4x or phyla 9 a z x g E 3 Splrochetes 39 I quot Green sulfur Planctomyces Demococcus bacteria Obligate green nonsu ur 4 intracellular acteria Cyanobacteria paras39tes Never cultured Thermotoga existence Grampositive deduced from bacteria rRNA sequences Aquifex Largest Proteobacteria4 lineage of bacteria Not all phyla are shown Proteobacteria The phylum Proteobacteria represents the largest lineage of Bacteria and includes 1 Many chemoorganotrophic species such as Escherichia coli the Gram negative model organism of bacterial physiology 2 Several chemolithotrophs and phototrophic chemolithotrophs including some using sulfur in their metabolism 5 Many important pathogens including salmonella gastroenteritis typn0id fever Neisseria gonorrhea and Rickettsia typhus Rocky Mountain spotted fever 4 Many of the bacteria that live in water soil animals and plants including species that can be casual opportunistic pathogens or degrade toxic organic compounds particularly Pseudomonas 5 Organisms that can fix nitrogen including Azotobacter MMG 301 Lecture 1 Milestones in Microbiology Discovery of microorganisms 1660 s Robert Hooke First to describe eukaryotic microorganisms He used early compound microscopes to describe fruiting structures of mold fungi Named the unit of living material cell after monk cells 168039s Antoni van Leeuwenhoek First to describe bacteria He made superior quality simple microscope with 300X magnification He described wee animalcules in pond water bacteria of various shapes and their motility 1850 s Ferdinand Cohn used early microscopes to study photosynthetic algae and bacteria He laid the foundation for bacterial classification system Binomial nomenclature system Genus species Staphylococcus aureus Staphylo bunch of grapes in Greek W Coccos granule in Greek Aureus gold Thiomargarita namibiensis Thio sulfur in Greek Margarita pearl in Latin Namibiensis bacterium found in the ocean sediments of the continental shelf of Namibia Pseudomonas aeruginosa Pseudo false in Greek Monas single unit in Greek Aeruginosa copper rust in Latin also bluegreen color of Pseudomonas aeruginosa cultures Names of bacteria provide characteristic information eg spherical gapelike clusters produce a golden pigment in culture Pioneers in Microbiology The defeat of spontaneous generation 320 BC Aristotle theory of spontaneous generation life arises from inanimate objects 168039s van Leeuwenhoek believed that his wee animalcules arose from preexisting organisms rather than by spontaneous generation 168039s Francesco Redi experiment with maggots Rotting meat carefully kept away from flies does not spontaneously produce maggotsbut meat st putrefies 1790 s Lazzaro Spallanzani boiled medium in sealed flasks did not putrefy Proponents of spontaneous generation argued that fresh air was necessary 186039s Louis Pasteur Father of Microbiology Observation microbes in air resemble those found in putrefying food Hypothesis microbes in putrefying food originate from microbes in air that deposit on the food Therefore if food were treated to destroy microbes it should not putrefy Brilliantly designed the classic Mnneck flask experiment heated food liquid nutrient medium will not putrefy with microbial growth even while exposed to Tresn air details on next page Significance of this experiment provided definitive proof of his hypothesis putting an end to the theory of spontaneous generation as the origin of life Airf r d t u ope g d Pasteur s Swan Neck Experiment Preparation of the liquid culture medium cook meat in water cool filter a Nonsterile liquid Neck of flask Liquid sterilized poured into drawn out in by heating flask flame Air contains dust and microbes that get Dust and trapped in the bent but open swan neck microorganisms Open trapped in bend end Long time c Flask tipped so Microorganisms b Liquid cooled LiqUid remains microorganismladen grow in liquid slowly Sterile for dust contacts sterile many years liquid Other accomplishments of Louis Pasteur Determined that yeasts are responsible for consuming the sugar and producing the alcohol in beer fermentations Invented a method to stop milk and wine from causing sickness pasteurization Created the first vaccines for chicken cholera anthrax and rabies First use of a rabies vaccine in a human Young boy Joseph Meister was bitten by a rabid animal Injected child with 15day old spinal chord sample from a rabies infected rabbit The spinal cord had been preserved for fifteen days in a flask of dry air Continued inrections with increasing IV virulent spinal chord samples every day for two weeks The Germ Theory of Disease Robert Koch investigated the cause of anthrax and tuberculosis Koch s Postulates set the experimental criteria required to establish t e between the presence of a microbial pathogen and the disease it causes Koch provided the experimental proof of the Germ Theory of Disease Observation Many microbes can be isolated from an infected or dead animal Question How do you test and prove which if any of the microbes present actually caused the disease 7 2 E2 KOCH S POSTULATES l E emafd 1 39II39he suspetted pathogenie Red Microscopy reveals organism should be present blood bs39ervelilbod l bacterial cells in blood in all uses of the disease 9quot I tissue under the Red and absent from healthy suspected 39 39 i msmpe 2 0f diseased animal animals pathogen l i 5 k H 7 Bacteria added to 39 r a quot z 21ujfgg quot withasantplrepae l fl gregamsms solidified culture medium culture 533223 a 3233 grow into visible colonies Path 9en 53quot Vamma39 39 groups of cells with l Inoculate healthy animal with d n ctive Ch a ra cte ristics cells of suspected pathogen 3Cells from a pure culture of a m the suspected organism t I l should ause disease in a Disased animal Expe mentally teSt the My quot m l l ability of the isolated 1 Remove blood or tissue sample bacterial culture to cause and observe by microscopy 439I39he rgaaismdshlould be b Laboratory 39 Pure culture disease When inoculated reiso ate an s own to e s t d culture the same as the original pgi zgeen same In a healthy animal and organism r as before later be recovered from it The 1880 s 1890 s was a period of great discovery in microbiology Using Koch s Postulates as a guideline in experimental studies many investigators established the primary role of bacteria in causing infectious diseases Pathogenic microorganisms able to cause infectious disease Koch anthrax tuberculosis Asiatic cholera Other microbiologists diphtheria typhoid fever gonorrhea bacterial pneumonia staphylococcal and streptococcal infections tetanus botulism bubonic plague bacterial meningitis bacterial dysentery Theobald Smith First to discover that an arthropod can transmit an infectious disease Texas cattle fever to mammals Presaged the eventual discovery of insects as important vectors in a number of diseases Ferdinand Cohn discovery of heatresistant bacterial endospores made by bacteria that cause anthrax botulism tetanus and gas gangreen Koch Hesse and Petri isolation of bacteria in pure cultures by growing them on solid medium potato slices gelatin then agar Petri invented the l transparent plates Streakplate method for isolating bacterial colonies on agarsolidi ed culture media Importance of nonpathogenic microbes in the biosphere Many microbes are beneficial carry out key steps in nutrient cycling essential to sustain life on Earth 1880 s Martinus Beijerinck established the central role of microbes in the nitrogen cycle needed to sustain life on Earth developed enricnment curture technique to isolate metabolically diverse bacteria Also defined the concept of virus 188039s 189039s Sergei WinograasKy discovered autotrophic bacteria use CO2 as sole carbon source and importance of chemolithotrophy in biogeochemical cycles Cyanobacteria perform oxygenic photosynthesis We will return to some 5quot Water of these topics in Purple sulfur bacteria perform subsequent lectures anDXygeniC Photosynthesis in this course split hydrogen sul de Sulfatereducing bacteria produce hydrogen sul de and precipitate iron Mnogradsky column Contrast APRIL 1355 m MARCH 1856 a 1850 s Florence Nightingale used statistical methods to demonstrate the high mortality rate of soldiers due to diseases during the Crimean war kv mm mm Milestones of the modern era of microbiology 1920 s Alexander Fleming discovery of penicillin antibiotic from a fungal culture and its use in chemotherapy 1930 s Gerhard Domagk discovery of sulfa drugs that saved tens of thousands of lives during WWII 1940 s Selman Waksman discovery of streptomycin antibiotic from an actinomycete 4014 gnaw it Soil 0 O O 0 i3 FISH L32 Key Points Soil communities affected by I pH moisture weather climate nutrient input and physical disturbance Important for carbon amp nitrogen cycling I Carbon decomposition mineralization of biomass I Nitrogen fixation or release denitrification Microbial life has been detected at 15km below seafloor I Could use and may even catalyze the production of hydrogen gas fuel product of the oxidation of basal FeO I Likely eat hydrocarbon that formed abiotically from C0 water and rock Geosmin produced by Streptomyces bacteria gives soil musty odor Microniches ampgradients a great diversity ofmicrobes and physiological processes can occur Soil Subsidence I Phenomenon wh ere soil organic matter is mineralized to C02 gas 0 Greatest when a wetland is drained before drainage the productive wetland community adds soil organic matter which is not fully degraded in the anaerobic water saturated environment Draining the wetland allows for increased gas exchange and aerobic respiration of organic matter to CO2 Gene sequences analyzed to infer microbial diversity Used to correlate a 165 gene sequence with a microbial cell type using oligonucleotide probes that hybridize or base pair to 16S rRNA sequences can label general or specific type of bacteria depending on if the probe binds to sequences conserved by many microbe types oris specific it ZonesSpheres of 3 Plant 0 O O Phyllosphere above soil part ofplant I Must adapt to high UV radiation and low humidity stresses Rhizosphere zone around roots below soil Rhizoplane surface of the plant root it Promotion of Plant Growth by Microbes O Acquire nutrients produce hormones that stimulate growth inhibit pathogenic microbes through antibiotic production it RhizobiaLegume Symbiosis O O OO Rhizobia quotquot into 39 that fix quot 39L g in L g for sugar Infection thread tube of plant that allows rhizobia to migrate through to infect and cause nodule development 4 steps of rhizobialegume symbiosis development I Roots secrete flavonoid compounds I Rhizobia sense flavonoid signal I Rhizobia make and secrete Nod Factor in response to flavonoid sensing I Plant responds to Nod by curling root hair to begin nodule formation Bacteroids Differentiated rhizobial cells surrounded by plant membrane that fix nitrogen Inputs amp Outputs I In 9Sugar input from plant that feeds rhizobia Leghemoglobin w 02 input delivered to ETC I Out Glutamine output NH3 rich amino acid is source ofammonia for plants it Crown Gall Disease o A tum senses phenolic compounds produced after wounding o VirG activates transcription of virulence genes 0 TDNA transferred into theplant cell byconiugal horizontal gene transfer I Important for making genetically modified plants 0 TDNA encodes opine biosynthesis genes that bacterium uses for food and genes that cause gall to form it Plant Biotechnology 0 Foreign DNA cloned in TDNA in Ecoli Disarmed Ti plasmid helps transfer TDNA into plant horizontal transfer TDNA integrate into plant chromosome Transgenic cell grown into a transgenic plant gives seeds 9 new crops 000 L33 Key Points i3 Rickettsia ampWhiteflies 0 Evolution by symbiosis Rickettsia endosymbiotic association caused increased fitness of whiteflies so now most whiteflies in a population have it when 10 years ago few contained Rickettsia endosymbionts i3 Symbiosis o A phenomenon in which dissimilar organisms live together or symbiosis o Symbiosis is present and often operating in cognito in healthy animals and plants I Functions more than niche exclusion of pathogens 0 Types I Parasitism host is harmed I Commensalism theoretically neutral effects for host and bacteria I Mutualism both bacteria and host benefit it Eukaryotes Resulted from Endosymbionts 0 Prevalence essentially every organism is symbiotic 0 Function symbiotic bacteria provide important novel functions to the host I Endosymbionts can biosynthesize amino acids nutrients and vitamins for host 0 Primary have an essential function for the host 0 Secondary have benefits that are facultative o Ourselves and the plants and animals that we consume are symbiotic i3 Vertical Transmission 0 Transovarian transmission from mother to offspring down the line of descent i3 Buchnem aphidocola 0 Function provides essential amino acidsrequired for aphid reproduction and efficient growth that are lacking from the plant phloem on which the aphids feed 0 Bacteriocyte insect cell that harbors Buchnera symbionts i3 Leaf Cutter Ant Symbiosis o Ants in symbiosis with fungi ants feed leaves to a fungal garden which they later harvest for food 0 Streptomyces symbionts present on abdomen of ants and produce antibiotics used to ward off pathogenic fungi i3 SquidVibrio Symbiosis 0 Function squid s light organ has bioluminescent Vibrio fischeri that function incounter illumination eg makes the squid bright against the star filled sky hides silhouette from predators O 0 Good Model System of Symbiosis I Measureable obvious and easy to detect phenotype I Simple due to a single symbiotic bacteria instead of thousands I Bacteria related to pathogenic V cholera that has genetic techniques established I Genomics available in both organisms Ciliated appendages I Used to acquire symbiont cells present in seawater these degrade after symbiosis develops by the production of tracheal cytotoxin component of peptidoglycan cell wall and lipopolysaccharide endotoxin 0 Function signals for symbiosis NOT AS ATOXIN 0 Steps are similar to disease progression but function for mutualism and not disease in this system it NematodePhotorhabdus Symbiosis O O O In a Mutualism Nematode is host for Photorhabdus symbionts In a Parasitism Insect is host for Photorhabdus pathogens and nematode parasites Photorhabdus infected insects are bioluminescent Nematode Functions provided by Photorhabdus luminescens I Pathogenicity to insect production of antibiotics production of bioluminescence Photorhabdus symbionts functionkill the insect and produce insect toxins inclusion proteins and antibiotics to preserve insect after death so the pair can reproduce at the expense ofinsect hosts Stage of Nematode Growth Infective juvenile offspring develop only inside the bodies of maternal nematodes Pulse chase and green fluorescent protein can be used to detect symbiontsthat persist in nematodes vs the symbionts only transiently present The mother nematode acquires the symbionts in a complex process where the symbiont first establish a persistent biofilm L34 Key Points it Normal Microbiota O O O The 10005 of diverse microbe types that inhabit nearly all parts of the body exposed to the environment I We have 10x more microbial cells in our body than we do human cells I 40 bacteria in or on each human being I Types ofmicrobes may vary depending on location ofniche on or inside human body Blood is a human environment that is normally devoid of human microbiota Benefits I Bacterial interference amp niche exclusion presence of normal microbiota inhibits the colonization by pathogens Mammals develop in utero in a sterile environment and then acquire skinvaginal bacteria after birth Approx 1yr after birth they acquire a normal microbiota that resembles that of an adult it Pathogenicity vs Virulence O O Pathogenicity is the ability ofa pathogen to damage the host Virulence is the quantitativemeasurement of pathogenicity eg of cells it Gnotobiotic animals 0 O 0 Known life animals born in germfree environment and monitored for microbes Allows for the investigation of complex microbial communities on human health Conventionalized animals Germfree animal that has been associated with normal microbiota it Tissue Tropism O Microbes have a preference to colonize specific tissues often mediated by the production of adhesins including fimbrial surface organelles that bind to host cell receptors often glycoproteins i3 Opportunistic Pathogenicity amp Normal Microbiota 0 Some members ofnormal microbiota function as opportunistic pathogens and cause disease in immunocompromised individuals I eg Cystic Fibrosis i3 Skin Microbiota o Inhospitable for the growth of many microbes I Periodic dryingacidicpH and high osmolarity o Apocrine sweat glands most skin microbes grow here because it s less exposed to environment than bare skin 9 provides niches for bacteria to grow I Sweat itselfis odorless it s the microbial activity in the gland that produces the odor i3 Mouth ampTeeth Microbiota 0 Oral microbiota depend primarily on saliva and ingested food for growth 0 Stresses Lactoperoxidase produces antibacterial compound that bacteria need to resist to live Saliva also contains lysozyme which breaks down the peptidoglycan cell wall 0 Plaque thick biofilm layer formed when Streptococcus primary colonizers binds to and grows on tooth surface and is followed by binding of secondary colonizers I Sugar from diet influences plaque formation 0 Dental carries cavities anaerobic environment near tooth surface influences production of lactic acid from sugar which causes the demineralization of tooth surface it General Microbiota of GI Tract o Microbes are prevalent throughout entire GI tract even though stomach has very acidic pH 2 o Bacteria produce butyrate and other acids and vitamins important for nutrition 0 Human Gut Diversity I Community composition can vary from individual to individual Much of the diversity if withdifferent strains more than species or genus if Large Intestine Microbiota o Anaerobic fermentation chamber consists ofmore than 700 types varies greatly between people 0 Functions modulate the immune system and produce organic acids vitamins odors and steroidmetabolism 0 Altered gut microbiota Correlated with several diseases inflammatory bowel disease Type 2 diabetes necrotizing enterocolitis i3 Respiratory Tract Microbiota 0 Defense mechanism mucous also contains lysozyme flow out of the respiratory tract by ciliated epithelia cells 0 Lungs are normally free of a microbiota so oxygen can be exchanged i3 Urogenital Tract Microbiota o Lactobacillus abundance in vagina indicateshealth acidic bacteria inhibits STDs and ferments glycogen to lactic acid which maintains the low pH 0 Bacterial vaginosis diagnosed as an altered community with several disease characteristics such as increased transmission of STDsamp inflammation i3 Probiotics o Livemicroorganisms that confer health benefits when administered in sufficient quantities clinical evidence is lacking usually administered to the GI tract 0 Potential mechanisms for health I Enhance immune system I Inhibit pathogenic microorganism by producing antibiotics I Uptake and removal of toxins L35 Key Points i3 Pathogen Types Opportunistic infects immunocompromised host Accidental accidental infection ofa host can still live inside host Obligate cannot exist outside a host organism eg gonorrhea o Facultative able to live outside a host organism eg E coli it General Mechanism of Pathogenesis 0 Exposure Adherence Invasion Colonization amp Growth Toxicity amp Invasiveness cause 0 Tissue Damage amp Disease it Adherence 0 Persistent attachment of pathogen to host cells 0 Adhesins proteins that recognize receptors on host cells often glycoproteins or glycolipids and may be attached to fimbriae or pili polymeric protein fibers radiating from cell 0 Lectins sugarbinding adhesins on host cell it Colonization Growth Toxicity and Invasiveness o Bacteremia disease caused by bacterial growth I eg plague amp pneumonia caused by S pneumoniae o Toxicemia disease caused by toxin production 0 Septicemia bacteremia with sepsis blood infection I Severe lifethreateninginflammation triggered by bacterial components like LPS it Virulence 0 Quantitative measure of pathogenicity I LD50 lowest lethal dose to cause 50 mortality 0 Low LD509 high virulence o LD90 is also used LD90gt LDso I LT50 lowest time for a dose to cause 50 mortality Virulence factor a pathogenproduced factor that is important for the development or maintenance of a disease aids in the ability ofa pathogen to cause disease I eg toxins or factors involved in invasiveness I Siderophores Fechelating molecules bacteria use to acquire iron have very high affinity for Fe and can steal it from its animalbound host transferrins o Siderophores as virulence factor keeping free Fe concentration low limits bacterial growth since high concentrations in blood lead to increased infections Siderophores increase Fe concentration in blood and therefore allow infections to grow COO 0000 O O Virulence gene gene that encodes the virulence factors Virulence plasmid independently replicating structures that contain virulence genes may provide bacteria with additional capabilities such as pilus formation Fe transport systems toxin production and antibiotic resistance it Exotoxins o Toxic proteins usually released from pathogen cells that carry out specific reactions 0 Types I Cytolytic toxins Lyse host cell and release nutrients for bacterial cell use 0 Hemolysis RBC lysis detected by a zone of clearing on culture medium around colonies due to the lysis of blood cells I AB exotoxins two subunits one involved in host cell binding B and the other A causes a variety of toxic activities 0 Diphtheria toxin stops translation by ADPribosylating EF2 o Botulism toxin blocks release of acetylcholine neurotransmitter 9 flaccid paralysis o Tetanus toxin block release of glycine from inhibitor neurons 9 rigid paralysis O o Cholera toxin activates adenylate cycase increase cAMP and release of Cl amp HCOZ I Superantigens cytokine release causes hyperstimulation ofimmune cells leading to inflammation and tissue damage it Endotoxin o Lipid Aportion of LPS present in many Gram cell walls 0 Toxicity is due to its effectiveness for stimulating the innate immune system 0 Released only when cell yses causes Inflammation and septicemia L36 Key Points i3 Epidemiology Vocab o Epidemiology the study of occurrence distribution and determinants of health and disease in a population 0 Prevalence fraction or percent of the population with disease 0 Outbreak sudden increase of diseased individuals 0 Incidence number of disease cases in a population it Disease Occurrences Location 0 Endemic normal or background incidence ofdisease in a population 0 Epidemic high incidence of disease in a population 0 Pandemic widespread often worldwide epidemic eg most recent is H1N1 now is endemic i3 Disease Occurrences Time 0 Sporadic epidemics emerge sporadically from endemic low levels 0 Seasonal epidemics emerge seasonally indicate indirect hosttohost transmission caused by variations in vector populations eg higher Insect transmitted diseases in summer it Mortality vs Morbidity Rates 0 Morbidity rate of diseased both ivingamp deadtota in population I Represents the incidence of disease in a population 0 Mortality rate of deaths due to disease of infected persons i3 Carriers 0 Individuals with asymptomatic or subclinical infections usually chronic that can expose others to a disease eg HIVinfected prostitutes 0 Can reinfect over and over again it Acute vs Chronic Infections 0 Acute Carrier state lasts a short time eg when developing flu disease I Transmits quickly not more than 1 or 2 days 0 Chronic Carrier state lasts a long time eg HIV virus I Transmits over and over again chronic infections enhance the spread of a virus it Reservoir 0 Place where disease is maintained between outbreaks can be inanimate or living 0 Reservoir Types I Soil diphtheria tetanus anthrax I Animals H1N1virus I Humans 0 Transmission I Common source single source for the epidemic eg contaminated foodwater o Affect lots of people at once taper off faster I Host to host spread directly from person to personperson to animal to person 0 Affect over longer period of time but persist i3 Direct Transmission 0 Human to human STDs or respiratory disease I Transmission by sneezing feces blood mucous 0 Animal to human bovine tuberculosis SARS rabies i3 Indirect Transmission 0 Vectors liveorganism such as insects rodents ticks and fleas I Eg plague malaria o Fomites inanimateobjects such as toys or bedding 0 Vehicles nonliving sources such as food or water it Herd Immunity o Immunity of members of a population limits its transmission and spread o Keeps endemics diseases at low prevalence o Mediated by vaccinated individuals I A group is resistant only when a high number of people are resistant required number varies related to the transmission of the agent I An individual s choice to be vaccinated or not affects the outbreak potential for the entire population in addition to the health ofthe single individual it Stages of Disease 0 Infection initial exposure and entry 0 Incubation period where the agent multiples before disease I Transmission is most likely after some incubation but before acute disease because this period corresponds to high numbers of infectious disease agent without the host being severely ill 0 Acute Disease symptoms are evident o Decline Survival of host often acquired resistance to infectious agent it Nosocomical Disease 0 Hospitalacquired infection 0 Transmission I Person to person by healthcare workers and patients I Airborne to exposed surfaces eg open wounds or surgical procedure I Device associated contaminated medical devices ventilators catheters syringes 0 Prevalence of Antibiotic Resistance I Overuse of antibiotics I Rapid transfer of antibiotic resistance by conjugal gene transfer of plasmids o Majorinfection sites urinary tract surgical sites lung pneumonia blood bacteremia i3 HIV Virus 0 Cause of the AIDS epidemic I HIV1 causes 99 ofinfections HIV2 is less virulent 0 Basis for Antimicrobial Treatment I Reverse transcriptase inhibitors base analogs prevents the copy of RNA into DNA I Protease inhibitors inhibits cleavage of polyprotein 0 AIDS is theoretically preventable I Advances in technology will lead to a cure I Avaccine will become available I Transmission is largely due to human behavior that can be altered i3 Infectious Disease Epidemics 0 Strategies to Prevent the Spread of Infectious Disease I Reservoir control monitor disease prevalence in animals immunize uninfected amp euthanize infected I Transmission control eliminate or modify insect vectors improve water quality I Immunizations successful in elimination and prevention of small pox diphtheria tetanus pertussis measles rubella amppolio I Quarantine restricted movements ofinfected individuals I Surveillance monitor disease spread eg CDC 0 Reasons for New Emerging Diseases I Ecologicalenvironmental changes agriculture dams deforestation global warming I Human demographics population growth migration drug use I International travel globalization of economy and food I Microbial adaptation acquisition ofnovel virulence characteristics I Poor public health lack of vector control or water treatment it SARS Severe Acute Respiratory Syndrome o Causecoronavirus likely originated from animal reservoir Symptoms Severe coldlike upper respiratory infection Treatment Antivirals ineffective Prevention Fast recognition and containment on a global scale may have prevented a more serious epidemic outbreak it Multidrug Resistance 0 Emerging infectious diseases ofnew multidrug resistant strains I Cause for concern strains are developing resistance to multiple antibiotic treatments leaving no effective treatment to prevent the diseases from spreading through the population it Category A Characteristics 0 Easily transmitted or persontoperson transmission High mortality rate potential for major health impact Cause panic or social disruption Special action to prepare public GOO 000 L37 Key Pomts it Outdoor vs Indoor air 0 Outdoor Contains soil and other environmental microbes 0 Indoor Higher abundance of human associated microbes more likely to have opportunistic pathogens it Air is inhospitable for microbial growth 0 Dry any moisture dropletsquickly evaporate o Enveloped viruses and Gram easily dry out it Respiratory tract defense 0 Air particles are trapped by mucous that is pushed out of the respiratory tract by ciliated epithelia cells it Cystic Fibrosis o SymptomsThick mucus that is not removed and thus becomes colonized with opportunistic pathogens it Streptococcus 0 Causes strep throat scarlet fever rheumatic fever impetigo and necrotizing fasciitis 0 Types I Group A S pyogenes common w many asymptomatic carriers I Group B S agalactiae septicemia and meningitis are major causes of infant mortality 0 Virulence factors capsule hemolysins and superantigens it Meningitis 0 Cause Neisserl39a meningitides o Mechanism Infection of the membranes and cerebrospinalfluid surrounding the spinal cordand brain 0 NotesSpread rapidly in closed quarters it Diphtheria 0 Cause Corynebacterl39um diphtheria o SymptomsDiphtheria toxin can cause formation of pseudomembrane and block airway it Whooping cough O 0 Cause Bordetella pertussis Virulence factors I Filamentous hemaggluttinin antigen surface adhesive factor for colonizing epithelial cells of upper respiratory tract Pertussis exotoxin induces synthesis of CAMP that creates osmotic imbalance and tissue damage Tracheal cytotoxin i3 Tuberculosis 0 Cause Mycobacterium tuberculosis o Virulence factors I Acid fast waxy mycolic acid of cell wall causes them to retain carbolfuchsin red dye I Invasiveness I Dormancy TB can entera dormant state and requires lengthy antibiotic treatments 0 Mechanism Bacterium is phagocytized and triggers nodule formation in lungs Nodules are detected by Xrays 0 Treatment lsoniazid prevents mycolic acid synthesis 0 Notes currently is one of the most significant infectious diseases i3 Leprosy 0 Cause M leprae o SymptomsBulblikelesions of the skin 0 NotesDoes not grow in lab only hostis armadillo i3 Common Cold amp Flu o Caused by too many virus types cannot use vaccine i3 Influenza o TypeAis most common I Has unusual genome that consists of 8 different RNA fragments that can be reshuffled ifa cell is infected by more than 1 type A virus 0 Major glycoproteins I Hemagglutanin and Neuraminidase 0 Evolution of Flu I Antigenic drift change from mutation and selection during infectious outbreaks and are the main cause of pandemics I Antigenic shift a new antigen gene from another flu type is acquired that may cause a pandemic because the population may be na39I39ve of the new combination ofantigens 0 Flu epidemics happen every year Flu pandemics every 1040 years 0 Pigs are permissive for both avian and human flue and can be a place where antigenic shift occurs L38 Key Points i3 Staphylococcus O 0 Members of normal microbiota of skin and upper respiratory tract Virulence factors I Hemolysins lyse RBC I Coagulase causes blood to clot and blocks the infection site from immune system I Leukocidin destroys leukocytes of the immune system I Superantigens activates Tcells i3 Toxic Shock Syndrome o Mechanism Hyper and systematic inflammatory immune response caused by superantigens o SymptomsSerious fever rash vomiting diarrhea and sometimes death it Helicobacter pylori o Discovered by Warren and Marshal for causing ulcers that are treatable with antibiotics I Wasn t accepted at first because the bacteria were not known to grow in the low pH environment of the stomach o Virulence factors I VacA cytotoxin I Urease makes alkaline environment I Lipopolysaccharide causes inflammation that likely benefits bacteria i3 Hepatitis o Mechanism Disease of the liver often caused by viruses I Cirrhosis destruction of liver cells and tissue damage 0 Treatment Vaccines against types A and B have lowered prevalence i3 Gonorrhea 0 Cause Neisseria gonorrhoeae o SymptomsPainfu in males asymptomaticin females 0 TreatmentAntibiotic resistance to penicillin and some others 0 Continued prevalence is likely because I No immunity due to antigenic variation of Opa surface proteins I Oral contraceptives decrease Lactobacilli that inhibit N gonorrhoeae I Symptoms in females are weak or aymptomatic i3 Syphilis 0 Cause spirocheteTrepanemapallidom 0 Types I Primary syphilis localized chancrelesion which can spread I Secondary skin rash caused by the spread of the primary lesion I Tertiary Widespread infection ranging from mild to lethal 0 Treatment penicillin is effective disease at lowest levels on record in US it Chlamydia 0 Cause Chlamydia tracomatl39s an obligate intracellular pathogen o Mechanism Has differentiated elementary body stage for transmission and reticulate body for intracellular growth 0 Notesmost prevalent STD i3 Herpes 0 Cause herpes simplexvirus 0 Types cold sores caused by HSV1 genital herpes caused by HSV2 I Trichomoniasis urethritis caused by a protozoa I HPV can cause cervical cancer vaccine available and genital warts L39 Key Pomts i3 Vectorborne diseases 0 Routes include blood sucking insects and animals I Animal transmitted diseases nonhuman vertebrate hosts 0 Zoonosis animal disease that can be transmitted to humans rabies o Enzootic endemic zoonosis o Epizootic epidemic zoonosis i3 Rabies 0 Transmission bites or saliva ofinfected animals 0 Mechanism Infects CNS and almost 100 fatal if untreated I Negri virus inclusions are visible in infected nerve cells 0 Prevention control of disease in animals 0 Treatment Postinfection vaccination is effective due to the slow disease progression antirabies immunoglobulin allows an immune response before host acquired immune response 0 Notes in US rabies is endemic highly virulent and enzootic i3 Hantaviruses 0 Transmission inhalation of rodent droppings and urine 0 Mechanism Related to hemorrhagic fever viruses like Ebola o NotesEndemic epidemic outbreaks can be caused by population booms of rodents i3 Rickettsial Diseases 0 CauseRl39ckettsiabacten39a closely related to mitochondria obligate intracellular parasites 0 Transmission ticks lice or flea I Rocky Mt Spotted Fever and Ehrlichiosis cause flulike symptoms i3 Lyme Disease 0 CauseBorreliaburgdorferi 0 Transmission deer ticks o Symptomsnitial circular rash is followed by chronic infections i3 Malaria o CausePlasmodium protl39st 0 Transmission Anopheles mosquito restricted to tropical and subtropical regions 0 Mechanism Sporozoites released in blood an enlarged cell called a schizontis formed in the liver schizonts segment into merozoites that infect RBCs gametocytes are also made that infect the mosquito intestine sporozoites are formed again in the salivary glands ofmosquitoes 0 Prevention insecticides repellent and screens control the mosquito vector 0 Treatment Quinolones i3 West Nile Virus 0 Transmission mosquito vector transmits virus from bird reservoir to human terminal host 0 Symptoms flulike symptoms and 4 mortality 0 Treatment No effective treatment 0 Notes now considered an endemic in US it Plague 0 Cause Yersinl39a pestis 0 Transmission From rodents to humans via flea vectors 0 Types I Pandemic plague caused more deaths except for TB and malaria I Bubonic plague Y pestis infect lymph nodes causing inflammation or bumps I Pneumonic plague transmitted airborne and is highly contagious i3 Fungal soil borne diseases 0 Cause Largely opportunistic pathogens I Tetanus and botulism i3 Polymicrobial disease 0 Cause more than one microorganism o NotesMore difficult to discover Altered communities of the normal microbiota can be considered a polymicrobial disease L40 Key Points i3 Food Spoilage 0 Primary determinant for spoilage moisture content 0 Classes of Spoilage I Perishable meats fish poultry eggs milk most fruits and vegetables I Semiperishable Potatoes some apples nuts I Nonperishable sugar flour rice and dry beans i3 Food Preservation o Refrigeration inhibits microbial growth lower temp slower growth Acidity pH below 5 inhibits growth of most pathogens Drying reducing watercontent inhibits growth Chemical preservatives act as growth inhibitors and are GRAS by the FDA Radiation ionizing radiation kills microbes used a lot for ground beef and spices I Controversial possible breakdown ofnutrients and generation of toxic compounds 0 Heat pasteurization shortterm heating to reduce microbe in food without significant reduction in quality 0 Heat canning heating in sealedcontained often complete sterility is not achieved it Fermented Foods 0 Yeast bread yeast ferments sugars in the flour to produce C02 which raises the dough 0 Dairy products lactose is hydrolyzed to glucose galactose then fermented bylactic acid bacteria into lactic acid low pH inhibits the growth of spoiling organisms 0 Meat products ground meat is fermented using lactic acid bacteria dry sausages to 30 moisture semidry to 50 moisture 0 Vegetables often fermented in salt brine to enhance preservation and flavor fermentation can improve digestibility of vegetables i3 Foodborne Diseases 0 Food infection arises from ingestion of a live pathogen I Symptomsoccurwhenorganism grows after ingestion 0 Food poisoning food intoxication from ingestion of preformed microbial toxins I Microbes that produced the toxins do not need to grow in the host disease due to action of ingestion toxin i3 Food Poisoning Bacterial Toxins o Staphylococcus aureus some produce heatstable enterotoxin in intestine these enterotoxins activate general inflammatory response causes gastroenteritis w significant fluid loss 0 Clostridium perfringens sporulation in intestine accompanied by enterotoxin production enterotoxin alters permeability of intestine epithelium o Clostridium botulinum improperly heated canned foods can contain live spores spores germinate in can and produce a neurotoxin that affects nerves controlling respiration and heartbeat 0 O O 0 if Food Infection Bacterial Pathogens o Salmonellosis I Agent Salmonella I Transmission uncooked food containing fecal contamination Disease strains colonize smalllarge intestines headaches chills vomiting diarrhea fever sometimes septicemia Salmonella Typhi causes typhoid fever 0 Pathogenic E coli I Agent certain strains have virulence factors and cause diarrheal and UTIs I Transmission uncooked food containing fecal contamination I Disease EnterohemorrhagicE coli grow in small intestine and produce verotoxin bloody diarrhea kidney failure 0 EnterotoxigenicE coli causes watery diarrhea o Campylobacter I Most prevalent bacterial foodbome pathogen in the US MlVIG301 Introductory Microbiology Part 2 Ciche portion Study Guide for Exam 3 Lecture 22 Viruses What is a virus phage viron viroid and prion Why are the later 2 not viruses Are viruses alive What kind of genetic material can be present in viruses What is the consequence of the type of genetic material for gene expression transcription and replication 0 Know signi cance of viruses to diversity and function of planet Earth human health and science 0 Parts of simple complex and enveloped viruses Clicker A plasmid is not a virus because it is not surrounded by a protein coat POGIL what is a plaque In detecting phage what are in greater access phage or host cells Most plaques are clear some that have cloudy centers are resistant to phage likely lysogenic Virus binding to host cells starts the infection process Host cell resistance can be obtained by eliminating changing or hiding the receptor Virus penetrate barriers of cell wall and outer and inner membranes inner only for Gram positives Naked DNA should be lytic and make phage if DNA is introduced into host cytoplasm Resistance is possible by identifying the phage DNA as foreign For example a cell might methylate speci c sequences of it s DNA and produce a restriction endonuclease that will cut unmethylated DNA Retroviruses go against the central dogma of DNA9RNA9protein and need the enzyme reverse transcriptase to make DNA from RNA Clicker ssRNA goes against the central dogma Clicker questions 64 codons are possible Replication of lytic viruses involves expression of early genes depends on host RNA polymerase usually Middle genes involved in DNA replication and hijacking host cell and late genes making host proteins What is lysogeny o The two routes to the lysis decision 1 immediately after infection and 2 from a lysogen A sick or damaged cell can trigger lysogen to go lytic like a rat leaving a sinking ship SKIP lambda integration replication What are the two main in uenza antigens and how does this relate to u names e g H1N1 SKIP most details of retrovirus life cycle except 0 Know reverse transcriptase and protease inhibitors to the protease that breaks the polyprotein into single proteins are important What is a genotype phenotype What is a gene Types of genes Forward and Reverse Genetics 0 Know the size of an average gene and how many genes you would guess to be present on a DNA element What is an operon SKIP physical genetic map discussion and Hfr mentioned more clearing in Lecture 24 Clicker a 6 8 Mbp genome would most likely contain 6800 genes 68 Mbp 6800000 bp divided by approx gene size of 1000 bp o Transcription and translation central dogma covered better in Lect 24 A promoter is where RNA polymerase initiates transcription of DNA to RNA and can be terminated by a termination loop What is a codon and codon bias SKIP mutations from this lecture covered again in Lect 24 What is a plasmid and why are they important for human health 0 Know examples of mutant phenotypes especially auxotrophy that was covered extensively Screen vs selection maybe unclear here but covered again extensively Screen you look at mutant individuals for phenotype selection kills any mutant without the desired phenotype you look only at mutants of interest Types of mutations in codons and consequence for gene expression Neutral has no or little effect on phenotype missense more so nonsense and frameshifts likely to eliminate function because they stop reading or change the reading from of the gene covered again Lect 24 Types of reversions Clicker point mutation to be generated at lowest frequency is b same site reversion of a missense mutation because 1 speci c mutation in one position must be made mutations in many genes cause auxotrophy frame shift suppressor can occur after several bases many mutations lead to temperature sensitive lethal Mobile DNA elements Insertion sequences and transposons cause mutations by inserting into genes Transposons often have carry antibiotic resistance that can increase the spread of antibiotic resistance along with plasmids Clicker transposon mutations are easily identi ed because they are marked with antibiotic resistance genes Stopped at POGIL not covered as such SKIP it Lecture 24 What is an ORF An ORF can be encoded in one of 3 reading frames on one of the DNA strands top or bottom 5 to 3 Transcription copies template strand to coding mRNA frame is initiated by a start codon AUG Transfer RNAs have anticodons that base pair with codons of mRN A and bring the appropriate amino acid to the codon39 translation RNA code translated to amino acid code Neutral mutation 9 no change in protein sequence missense 9 1 amino acid change Nonsense 9 stop no protein synthesized after stop codon Frameshifts also bad for protein function if they change the reading frame and change the amino acids in the protein and often lead to stop Clicker which mutation likely leads to a null mutation this was unclear it means to completely abolish protein function answer is 1 bp because 3 bp deletion only subtracts 1 amino acid and maintains reading frame neutral mutation makes no change missense is only 1 change in amino acid sequence Insertion sequence is a basic element of mobile DNA having inverted repeat sequences at the ends that are recognized by a transposase enzyme that recognizes the inverted repeat sequences and inserts the Insertion sequence into target DNA Transposon often derived from two IS one of which degrades eg loses transposase and often contain antibiotic resistance genes between the IS This is important for spread of antibiotic resistance and useful for genetics because the DNA containing the transposon is marked by antibiotic resistance Replica plating useful to screen for nutritional auxotrophy Clicker mutants are screen for auxotrophic phenotypes Ames test39 uses 108 auxotrophs for the amino acid histidine plated on mimimal media lacking his Colonies that grow are his revertants that are selected because they grow when most cells don t grow If more colonies are found around a test substance mutation rate is higher and the substance is mutagenic and possibly carcinogenic cancer causing 24 Gene Exchange 0 Know that bacteria are proli c exchangers of DNA even among distantly related organisms like bacterium and plant To be inherited the exchanged DNA need to be replicated with the cell Replication can be by selfreplication plasmids or by recombination in the cell s DNA genome or plasmid Recombination is the exchange of two genetic elements in a cell 0 Site speci c or homologous later requires stretches of homology DNA of similar sequence usually 100s of bps SKIP mechanism of homologous recombination Know what is transduction transformation and conjugation Natural vs arti cial competence w I39 II I I VS 39139 II I I 1 Conjugation of a plasmid recipient becomes F vs chromosome by Hfr Hfr is a strain with a conjugative plasmid integrated into the genome transfers the genome but not plasmid because transfer originates in the middle of the integrated plasmid and transfer of the entire chromosome is needed for the entire integrated plasmid to be transferred Hfr can be used to map the genes where the time it takes for a marker to be transferred is related to it s vicinity to an integrated conjugative plasmid Antibiotic selection is useful in killing donor cells Complementation tests if two mutants have a mutation in the same gene if function is not restored means mutations in the same gene if function is restored then wt gene is likely in each mutant to the other mutation Homologous recombination is not necessarily required genes can be inherited if present on a selfreplicating plasmid 25 Metabolic Regulation 1 Clicker question A missense is less likely to cause a null mutation because it changes only 1 amino acid 47 bp insertion transposon insertion or 1 bp insertion change reading frame or transposons have stop codons Metabolic Regulation can occurs at the level of protein function activity translation and transcription protein amounts Constitutively expressed genes are not regulated Regulation of enzyme activity by allosteric or feedback inhibition involves an endproduct binding to an allosteric site usually different from the catalytic site because substrate is different than end product Know what is positive and negative regulation effectos attenuation riboswitching Skip types of DNAbinding proteins DNA has a major and minor grove DNA binding proteins bind to the major grove often in inverted repeat sequences Know features of an inducible gene and relate this to the regulation of the lac operon Know that the lac operon encodes lacZ encoding the enzyme beta galactosidase along with two other genes The 11100 operon is in between the promoter and operon Betagalactosidase breaks down lactose to sugars used by the cell for grth amp conversion to the effector allolactose IPTG is a gratuitous inducer of the lac operon effector for LacI repressor Xgal is a compound that turns blue in the presense of beta galactosidase Clicker function of the lac genes can be determined using both a genetic screen blue color in the presence of Xgal and selection grth on lactose minimal media LacI repressor is made independently of lactose binds to the 11100 operator and blocks transcription of ac operon in the absence of lactose or allolactose IPTG Inducers allolatose or IPTG cause LacI to not bind to the lac operator and to allow transcription of the lac operon DiauXic growth covered in POGIL Glucose catabolite repression is actually activation of the lac operon by CRP or sometimes called CAP bound to cAMP effector The presence of glucose inhibits cAMP synthesis with the result of no activation that appears like repression Skip structure of cAMP or other small molecules shown for completeness Skip mechanism of glucose inhibition of cAMP synthesis Know that glucose inhibits cAMP synthesis POGIL exercise Wed dealt with activation Know CAP bound to cAMP binds to the CAP site and activates transcription Lecture 26 Metabolic Regulation 2 0 Regulation of tryptophan biosynthesis Skip biochemical steps in tryptophan and that there is a Trp repressor that blocks transcription like LacI represson Focus on attenuation 0 Know that attenuation is after transcription initiation and involves the termination of transcription or antitermination Ribosome pausing at Trp codons allows antitermintion stem loop to form which prevents termination loop 34 to orm Whe Ribosome stalls at Trp codons because of low amounts of Trp charged transfer RNAs Access Trp means access charged Trp tRNAs and no stalling of the ribosome This causes the ribosome to rapidly go over stem 2 preventing the 23 antitermination loop to form and allowing termination loop 34 to form The Bacillus TrpTrap system showed as an example of another mechanism to use attenuation to regulate transcription Low Trp no TrpTrap binding allowing anti termination loop to form High Trp TrpTRAP binds to A preventing anti termination loop to form and termination loop to form In TrpTRAP the ShineDelgamo ribosome binding site is exposed during antitermination so translation can proceed It is buried in termination stem loop preventing translation of mRNA into protein miRNAs are genes transcribed into small RNAs that bind to ribosome binding or other sites of mRNA and affect translation of those genes e g if it binds to the ribosome binding site it inhibits translation like when the site is double stranded in the termination loop in TrpTRAP miRNA genes are difficult to nd in gene sequences because they don t encode and ORF no protein miRNAs also are known to affect the stability of mRNA Riboswitches can directly sense an effector and cause or inhibit transcription attenuation or translation by exposure of a ribosome binding site Skip SAM as an example of a riboswitch tRNA charged or uncharged is used as another neat example Thus we covered examples of all levels of metabolic reguluation focusing on speci c examples 26 Metabolic Regulation 2 o Revisited lac regulation 0 CRP cAMP Activator LacI allolactose or IPTG effector Repressor Clicker wild type E coli on minimal media with lactose and X gal will yield blue colonies no glucose to inhibit cAMP levels lactose is made to allolactose and de represses LacI repressor Clicker lacZ mutant on glucose and Xgal will yield white colonies for two reasons 1 Catabolite repression from glucose low cAMP 2 no allolactose so LacI repression Clicker lac mutant on glucose plus Xgal will yield blue colonies because glucose will become limiting during colony growth and there is no LacI repressor so lactose is not needed POGIL diauxic growth curve regulation of ac operon Diauxic growth is like two sequential growth curves stationary phaselag between the two slope of each line indicates the growth rate steeper slope is faster growth E coli grows about twice as fast on glucose than lactose Betagalactosidase detectable after glucose is exhausted Why cAMP levels become high and CAPcAMP activate transcription of ac operon Clicker wild type E coli on minimal media with glucose lactose and Xgal will yield blue colonies the bacteria quickly exhaust glucose during colonial growth like in the diauxic growth curve Clicker A gene encoding a small regulatory RNA contains a promoter because it is transcribed from DNA to RNA No ShineDelgamo or ORF because no translation Skip SELEX for exam Clicker false statement about riboswitches is that the can regulate promoter activity This is false because transcription is terminated after transcription is activate riboswitches can regulated transcription translation and can bind to ligand 27 Biotech 1 0 Know cloning molecular cloning genetic engineering recombinant DNA Appreciated the signi cance of molecular biology to the growth of the life sciences Technological advances rapidly are increasing the ability to sequence engineer DNA Know what recombinant DNA is and clone library 0 Know what restriction endonucleases are and how the length of the recognition site determines the frequency of cutting a given DNA fragment 0 Understand why enzymes with 8 bp recognition sites are more appropriate for the analysis of whole genomes while 6 bp cutters are less so Clicker what characteristic of a restriction endonuclease is most ideal to clone a 1000 bp gene 6 bp cutter because a 4 bp cutter will cut on average 4x4x4x4256 bp meaning the gene will be cut most often a 6 bp cutter will cut on average 46 or 4096 bp meaning it most likely won t cut in the 1000 bp gene but it could 8 bp cutters make too large of DNA fragements ca 60000 bp making it difficult to find and manipulate the gene Know the polymerase chain reaction powerful characteristics and limitations Function of template DNA primer Taq thermocycling 0 Basic characteristics of cloning vectors selectable marker origin of replication initiation blue white screening lacIlacZ genes polylinker or multiple cloning site 0 Know for cloning you want white colonies because they contain foreign DNA in lacZ Blue colonies are self ligated vectors containing no insert jMICROBIOLOGY Research Paper amp Presentation includes both guidelines useful websites and rubrics that will be used for grading material General Guidelines for Paper 1 Begin with a front page that includes title your name CRN date and citation style used AMA 7 American Medical Association isee 9 below 2 Must be typed or word processed in a typical 12 pt font doublespaced with a maximum margin space of 1 inch 3 Length of the body of the paper can vary Typically 3 7 5 pages are suf cient Graphs pictures and maps do not count toward the body of the paper 4 There must be an Outline preceding and a Bibliography following the body of the paper 5 The text should be well written in the student s own language with proper acknowledgement of resources used or quoted Use quotations sparingly and only when necessary or meaningful 6 Bibliography must be included with a complete list that includes a variety of resources such as books journals and internet URL addresses As a minimum there should be at least three peer reviewed scienti c journals one credible book and two credible websites Wikipedia or any wikies are not considered a credible research site although you can use them to identify certain relevant aspects of your report or a rst approach to the nature of your topic Examples of credible websites would be CDC BIOSIS WHO etc The journals used should be those that are credible peer reviewed scienti c journals A few examples would include Nature J AMA Journal of the American Medical Association Science JCM Journal of Clinical Microbiology and Journal of Bacteriology You can check online for whether a journal is peer reviewed or consult your instructor Journals can be accessed at online databases provided by the LCC Library but they will still be considered journal articles and not websites in your bibliography The majority of the research can be done at the LCC Library online at httpwwwlccedulibrary 7 Cite all references in the text of your paper unless it is information that you have gotten at several different sites AMA allows you to list and number the resources in your bibliography in the order of appearance in your paper which you can then cite by the number assigned So the rst citation would be 1 and then listed as the rst or 1 resource in your bibliography etc 8 LCC Library resources and recommendations for microbiology research report are available in a research guide titled BIOL 203 Microbiology at the resource desk in the library 9 A consistent style or format must be used for both bibliography and citations in the body of the paper You are to use the AMA American Medical Association citation style Details for AMA can be found at httpwww lin luttn quot quot wmhrnll edn mla html J li39urarvWm39 39 quot quot htm lO Outline and bibliography not body of paper can be emailed about two weeks prior to the due date for the report for teacher review and feedback Your instructor will either indicate that what you have is suf cient or that there are concerns These will not be graded but this as an opportunity to get meaningful feedback on the overall design of your paper prior to handing it in for a grade Handing it in ahead of time does not count 7 this is merely a sneak preview for your teacher to give you guidance without the loss of any points 11 There are many resources available to help you with your paper First of all there are many references in the articles that you read on Medscape that you can use to document or support your paper In addition to the URLs listed in 9 above you can visit the library in the TLC Building or you can go to the Writing Center on the second oor of the Arts amp Sciences Building Writing the Research Paper Title Should identify the purpose and scope N Outline Should give overview of the paper including a sequential list of information When you have nished writing the body of the paper then include page numbers for added clarity when a reader uses the outline Develop your outline as a way of determining the effectiveness of the organization Remember You can ask your instructor to review your outline and bibliography a couple of weeks before the paper is due and give you feedback You must however at least once during the process use the drop box in the folder for Paper and Virtual Presentation Materials to let your instructor know what title and thesis statement you are working with E Thesis A exible thesis that you can work on throughout the process of writing your paper is important However you are asked to let your instructor know about your thesis at some time during the process of writing your paper to get feedback A few sites that will help you formulate a thesis are httpwww indiana PflllNWtSI lthesis ltfafpmpnf hfml httpwwwcooradoedupwrwritingtipslOhtml httphomeworllfin ahmlf mmod I 39 ufusshtm gt Introduction It should be one paragraph An effective way to organize that introduction is to proceed from general to speci c You can begin with current understanding or knowledge in the area you have chosen to research U Body Organize your material logically The body of the paper should be at least 3 paragraphs and include a logical progression of the main issues or points relevant to the focus of the paper 0quot Conclusion This should be one paragraph that summarizes your main points and includes future applications predictions or the next questions to be answered in the eld 7 Bibliography Use AMA citation style and be consistent and accurate in the write up of the bibliography Use websites given above or just follow the format used by Medscape s website list of references Grade Grade of report will be based on both the text of the paper and following the guidelines given in rubric below Total points 50 points Late 1 pt each day Topics Selected for body of paper Conceptual Knowledge Mechanics Outline amp Thesis Statement Bibliography 10 points Main topics are presented Presents concepts or facts about the topic selected 1 point Cover page with name date CRN title There is an outline and a thesis statement Includes a variety of credible sources 12 points Main topics relevant and proceed in a logical order Shows some understanding of topics and own words used 2 points Paper is written with spelling and grammatical errors The outline is located before the text of paper amp after cover page The thesis is after title and before intro Includes at least 3 peer reviewed scienti c journals one book and two websites Rubric for Research Paper 14 points Topics supported by credible and accurate sources Indicates reasonable understanding amp ability to write clearly 4 points Paper is mostly well composed sentences with few spelling or grammatical errors The outline is an account of the body of the paper amp easy to use The thesis summarizes your paper Appropriate citations are made in body of paper with sources acknowledged 16 points successful demonstration of understanding issues involved Shows full understanding of topics with clarity and accuracy 6 points Paper is well written with no spelling or grammatical errors The outline is accurate logical and clear The thesis deals with a clear idea that asserts your conclusion AMA is used consistently and accurately both in citations and in the bibliography General Guidelines for Panel Discussion 1 Create a document of your paper for distribution to your colleagues during the discussion This should be a concise and meaningful summary or highlights of your paper a Be sure to include an introduction highlights of your library research and a conclusion Include enough background information to give your audience an understanding of your topic b Then make it visually appealing so that it is relatively easy to read understand by everyone You are free to get as creative as you would like while keeping it professional and informative 2 Your part of the panel discussion should give the audience some interesting aspects of your topic with a thesis statement that you have written It should not be an attempt to present your whole paper and it must not be the outline or table of contents that you have for your paper 3 Colleagues who are the audience need to be interactive with the panelist Each student is expected to ask a question or pose a dilemma for the discussion Panel Discussion Grading Rubric Points Possible Points Earned Introduction Thesis statements 4 points Body of Presentation 7 summarizes main points or 6 points highlights of research paper Information is provided in a clear concise and meaningful manner Demonstrates an understanding of topic 4 points Conclusion concisely summarizes highlights of the 3 points research paper Quality Presentation is neat and visually appealing It is 4 points spell checked and grammatically correct Questions or dilemma s posed for panelists in other 4 points Discussion Group SS11 MMG 301 Lecture 5 Bioenergetics metabonsm Ihe SUIT total OT all ChemlCal reactions In the CG has two components Catabolism Chemical reactions that are degradative and are regarded as carbon consuming and energy generating Anabolism Chemical reactions that are building in function and require an energy expenditure Anabolic reactions are also called biosynthetic reactions Three major energyyielding processes in biology Fermentation The growth substrate serves as electron donor A cellular organic compound so veg as slows accepto i tc wox reactcs mat provide energy for ATP production ATP is produced by substrate level phosphorylation does not consume oxygen Respiration Organic or inorganic com ounds serve as electron donors and exogenous organic or inorganic compounds serve as electron acceptors in the redox reactions that provide energy for ATP production ATP is produced from the proton motive force Photosynthesis Light energy is used to drive biosynthesis of carbon compounds from COZ may be oxygenic or anoxygenic light energy is used to generate a proton motive force to create ATP Review of carbon and enerqv sources for microbes METABOLIC PATHWAYS Carbon Source Inorganic 002 Note Mistake in slide 3 of lecture 3 Organic Most not all phototrophs Energy Source are autotrophs Inorganic Chemical Organic Light Comparison of Fermentation and Respiration Fermentation Respiration reduction e39 acceptors Purpose Oxuation of compounds gt energy ATP Substance Organic Organic or inorganic oxidized Oxidation No external terminal Requires external terminal e39 acceptors Method of ATP production Substratelevel phosphorylation Electron transport proton motive force Free Enerqv of Formation in Bioloqical Reactions Free energy of formation Free energy of a reaction energy released that is of a few compounds of available to do work biological interest Free energy of a particular reaction Gibb s Free Compound G r tnergy can be calculated trom the tree energy or Water 2372 formation of the reactants and products 002 3944 H2 0 02 0 Gibb s Free Energy AG In kJmole Acetate 3694 o o o Nitrous oxide 1042 AG ZG f of products ZG f of reactants Glucose 9173 Energyyielding reactions exergonic AGO is negative Energyrequiring reactions endergonic AGO is positive But AG is for standard conditions of 25 C 1 ATM 1 M reactants In nature this almost never occurs So we use AG which is the free energy for reactions under the actual conditions such that AG AG RTln K R constant T temperature K equilibrium constant Clicker question What is the Gibb s Free Energy AGO of the following reaction Compound Go in kJmol A B gt C D A 100 B 70 C 0 D 300 a We do not have enough information to calculate it b 470 kJmol C 470 kJmol d 130 kJmol e 1 30 kJmol Catalvsis and enzymes Activation energy energy needed to bring the molecules involved in a reaction to the reactive state Catalyst substance that lowers the activation energy of a reaction and increases the reaction rate The catalyst does not change the A60 of the reaction the reaction goes faster but the end point remains the same Enzm ilvi l tltrtin r RNA An enzyme can accelerate a reaction by 108 to 1020 times compared to the spontaneous reaction Free energy Activation energy Substrates A B with 1 enzyme AG G fc D 60f A B Pro ducts C D Progress of the reaction Reaction progress curve showing energy states during a non enzymatic and an enzyme catalyzed reaction Clicker question What does a catalyst do a IVIaKeS possmle OtherWISG impossmle reactions b Increases the amount of end products but does not change the rate 0 Changes the rate of the reaction but does not change the amount of end products d Changes both the rate of a reaction and the amount of end products e Changes the AGO39 of a reaction Exam Ie of enz mecatal zed reaction fructose1 6bis hos hate aldolase Iructose 16biP gt Glyceraldehyde BP Dihydroxyacetone P Substrate GcheraldehydeaP DihydroxyacetoneP Loinzc EHO CHZOH 0 0H 0H HfOH o CHz O P H C O P H C 0 P OH 2 2 Fructose 16bisphosphate F 0 W c a Produ 0H W 0 hrt Chgiovp Pal co 0 0H Enzyme substrate complex a Free aldolase a u Active site Free aldolase Capyrlgm e 2009 Pearson Educallnn Inc publishing as Pearson aemmm summmgs OxidationReduction Reactions Oxidationreduction redox reactions are the exergonic reactions that provide energy for all cells to grow The energy released from these reactions is conserved in the synthesis of energyrich compounds such as ATP The LOSS of an electron from an atom or compound is an oxidation The compound being oxidized is an electron donor The GAIN of an electron is a reduction The compound being reduced is an electron acceptor Because electrons cannot exist alone in solution an oxidation is always coupled with a reduction Thus electron transfer reactions can be thought of as two halfreactions an oxidation and a concomitant reduction Examples 2 H and H2 12 O2 1 and H20 and H2 2 O2 HZO Overall reaction NO3 and NOZ are H2 gt 2 e39 2 H Oxidative 12 reaction e39 loss from H2 called redox 12 02 2 e39 2 H gt H20 Reductive 12 reaction 2 O2 gains e39 H2 N05 gt N0339 HZO Overall reaction H2 gt 2 e39 2 H Oxidative 12 reaction e39 loss from H2 NO339 2 e39 2 H gt N0239 H20 couples often written as 2HH2 1202HZO and NO339NOZ39 oxidized on the Reductive 12 reaction NO339 gains eleft r9dUCed 0n the right Whether overall redox reactions are energetically favorable can be determined from the Electron Tower Energy of each 12 reaction is expressed in electron volts rather than Gof Reduced form Redox couple Eo V OXIdIzed form 503145 2 9V 4360 cogglucose 043 24 e 050 Examples of reactions 2 2 quotMm 9 4m H as e donor COJmethanoH O e 7 2 Electrons always go 1 s 7 03quot DOWN the tower 0st Ham39s 020 FADFADH 022 2 e 70m Pyruvatelactate D19 2 equot 1 H2 fumarate239 gt succinatez 50quotst 17 6 e o 0 sac6 752032 o024 2 e K 39 39 AGO 86 kJ EIeCtrons are Furnaratelsuccinate0032239 010 donated from redox Cytochrome ba md00351 e 020 couples above and Fe quotFezn2 1 2 pH 7 39 are accel ted bl 2 H2 N03 gt N02 H20 Ubiquinoneamed01122 e V o3u dox couples Cytochrome cawedho 5 1 e 0 40 re AGU39 163 kJ Cytochrome aw 0391 e 39 below gt NOfNOZ 042 2 e 050 060 1 3 2 3 02 H20 N03quotI N2074 5 e 070 quot AGO 237 kJ FemFe2 0761 2 pH 2 030 quot 024120 052 2 e 090 Clicker question In the following reaction compound is the electro uowr Glucose 6 H20 gt 6 002 24 e39 24 H COZIqucose 043 12 02 24 e39 24 H gt 12 H20 1202H20 090 Glucose 12 02 gt 6 002 6 H20 3 H20 b 02 c Glucose d 002 AGO ZGof of products ZGof of reactants IquotI2 N03 N02 HZO Table A11lt 39 0 kJmol 111 kJmol 37 kJmol 237 kJmol Appendix 1 in BBOM 2 7 products 6 311 reactants Overall reaction Oxidative 12 reaction e39 loss from H2 Reductive 12 reaction NO339 gains e39 163 kJmol How do electrons move in bioloqical systems NADH H m E H N H R F2 F i m l Oxidized Reduced H H Nicotinamide I H o II c NHZ H II 0 H07 P o CH2 Ribose OH OH NH2 N Ho Po lt I N I A 0 CH2 N N Ribose Adenine OH OH P0427 Phosphate added in NAEP Avariety of electron carriers are used they carry 1 or 2 electrons at a time no wires in cells These electron carriers transfer electrons by interacting with proteins or enzymes Two types of electron carriers Freely diffusible coenzymes Covalently attached to enzymes in the cytoplasmic membrane prosthetic groups NAD and NADP are common coenzymes that carry They can go from one enzyme to the next so they can increase the diversity of chemical reactions in the cell NADNADH and NADPNADPH have the same redox potential 032 V NADNADH couple involved in energygenerating catabolic reactions NADPNADPH couple involved in biosynthetic anabolic reactions NADNADH recycling Reaction 1 Enzyme 1 reacts with an electron donor and the oxidized form of the coenzyme NAD NAD blndlng Activesite Sit 7 c7 NAD39 Electron donor substrate complex NADH Electron donor oxidized Reaction 239 Enzyme 2 reacts with an electron acceptor and the reduced form of the coenzyme NADH NADH binding Active sit sue NADH Electron acceptor NAD39 Electron acceptor reduced Example of NADNADH recyclinq Fructose NADH gt Mannitol NAD Formate NAD gt 002 H2 Often represented as Fructose Mannitol NADH NAD 002 H2 Formate Recap Oxidationreduc ns edox terminoloqv Oxidation Loss of an electron Reduction Gain of an electron Electrons cannot be free in solution so oxidation reactions are coupled with reduction reactions reactants and products are called redox couples In some biological reactions a proton and electron hydrogen atom are transferred together Redox potential Eo a measure of the tendency to donate an electron High energy bonds and enerqvrich compounds e energy released from redox reactions is conserved in the form of energy rich compounds often phosphorylated compounds These compounds are then used by the cells to power energyconsuming functions Phosphate can be bonded to organic compounds in two ways Acid anhydride bond two acyl groups are bond to the same oxygen Hydrolysis of phosphate anhydride bonds releases between 31 kJ and 516 kJ Ester bond 3 R O Fgt 0H OH Hydrolysis of phosphoester bonds releases around 14 kJ 1 The cell uses mostly compounds whose AGO of hydrolysis is above 30 kJ as energy currency Hi h and low ener hos hatecontainin com ounds Hihenery CH2t coo 2 3907 eo o Phosphoenolpyruvate Anhydride bond zN Anhydride bonds Ester bond gt gm 9 9 9 oip o PoPao H H o o o NH2 Anhydride bond 0 0 O H CH H3CCOrquot039 6 0H 0H Acetyl phosphate Adenosine triphosphate ATP 4 Lowenery EHO quot0quot Ester bond OHCH Ht DOH HC OH 1427 of W 70 o Glucose 6phosphate ATP is the prime energy currency Compound GO kJmol 36039 gt 30kJ Phosphoenolpyruvate 51395 in all cells 13Blsphosphoglycerate 52o Acetyl phosphate 443 31 8 ADP 318 Acetyl 00A 31 AGO lt 30kJ AMP 142 Glucose 6phosphate 133 Clicker question AMP is a lowenergy phosphate containing compound because a It is a smaller molecule than ATP or ADP b The sole phosphate group is bonded through an ester bond 0 it is abundant in the cell cl It contains a single I hos hate Urou Coenzyme A derivatives are other highenergy compounds in the cell Coenzyme A Thioesterbond 7 Egg Example AcetylCoA oilmm H Hydrolysis of this bond releases enough energy 4160 31 kJ for ATP synthesis Coenzyme A derivatives are particularly important for the energetics of fermenting organisms Study guide lectures 2931 Le cture2 9 Clicker Dyeterminator cycle sequencing is different from PCR because C only 1 strand is used First lane is a marker with standard DNA fragments of known size The next lanes are the DNA that is being tested 4 bp cuts 1256 6 bp cuts 14096 and 8 bp cuts l65536 as template both use primers repeated cycles of DNA polymerization and thermostable DNA polymerases Microarray DNA can be synthesized and sequences of DNA of all the genes can be spotted onto a microarray Labeling the mRNA and then hybridizing base pairing or sticking it to the array will indicate which genes are expressed under a given set of conditions This can be used to identify genes differentially expressed in two bacteria strains two growth conditions or environment Commodities food additives fuel and solvents and fine chemicals Processes sewage treatment bioleaching extraction of metals biocatalysis production of chiral molecules and bioremediation degrading chemical pollutants Fermentation large scale growth usually in vats or tanks Primary metabolites metabolites produced during the primary phase of growth usually a byrpoduct of metabolism eg fermentation product Secondary metabolite metabolites of ten produced after cell growth and nonessential to growth and metabolism of the cells They are also unpredictable highly dependant on growth conditions Fermentation vessel 0 Mixing aeration and oxygen monitoring getting enough dissolved oxygen is a major problem 0 Temperature control 0 Sterilization 0 Sampling and harvest ports 0 pH control Detection of antibiotic producing microorganisms 0 Spread microbes from soil that they produce isolated colonies o Colonies are grown o Overaly with soft agar containing an indicator organism 0 Growth occurs 0 A zone of clearing of the overaly indicates the production of antibiotic by the colony below Antibiotic characterization Antibiotic producer is streaked and grown Antibiotic diffuses into the agar Test organisms are crossstreaked and grown 000 o The zone of inhibition indicates the antibiotic activity Some challenges about brining an antibiotic to the market antimicrobial probably not new antimicrobial compound may be toxic and antimicrobial compound may not be produced in enough quantities Products produced by mircroorganisms o Ethanol cephalosporin lactic acid xylitol Bioleaching is where microbes make soluble metals in ore The copper example mention involves bacteria that help make Cu2 but the more important process is oxidizing Fe to Fe3 which than reacts to form Cu2 This is the extent of detail I gave on this topic xenobiotic chemically synthesized molecular not thought to occur in nature Bioremediation degradation or removal of toxic pollutants by organisms usually microbes Mineralization the complete degradation of a compound to its elemental parts Lecture 30 Populations individuals of the same type multiply to form these Guilds metabolically related populations Communities mixtures of guilds conducting complementary physiological processes Ecosystem microbial communities interact with macroorganisms and environment Symbiosis the nontransient association between dissimilar organisms o Mutualism 2 organisms both bene ting from the association Commensalism one organism bene ts while the other is not effected Parasitism one organism is bene ted at the expense of the other Predation one organism engulfs and consumes other organism Amensalism one organism harmed without affecting the other Competition organisms competing for a common limiting resource OOOO POGIL The great plate anomaly Understand how serial dilution works to determine the number of bacteria cultured in a sample Understand the concept of enrichment 9 growth conditions select for microbes adapted to those growth conditions A bacterium may be enriched even if in low abundance in the microbial community This is why Azotobacteria grows on the plate from the top enrichment but not the bottom enrichment Know most living bacteria are uncultured 999 ie culture 104culturedout of 107totalbacteria Chemical gradients oxygen gradient around a soil particle Environmental heterogeneity Microbes live predominantly in structured bio lm communities with other microbes Lecture 31 C02 is xed by photoautotropths and chemoautotrophs C02 is generated by CH4 is formed by methanogens from C02 H2 and acetate Understand why methane is a concern to animals and global warming syntrophy where one species lives off the byproducts of another species The importance of nitrogen xation is it brings N from the atmosphere into living systems Nitrogen xation is catalyzed by nitrogenase that is oxygen sensitive in a reaction that consumes a lot of energy Heterocyst are specialized nitrogen xing cells formed by some lamentous cyanobacteria such as Nostocpunctiforme Cylindrospermumstagnale andAnabaena Sphaerica during nitrogen starvation They x nitrogen from dinitrogen N2 in the air using the enzyme nitrogenase in order to provide the cells in the lament with nitrogen for biosynthesis Nitrogenase is inactivated by oxygen so the heterocyst must create a microanaerobic environment Anammox another way nitrogen is lost to the atmosphere 0 NH4N02 9N22H20 o Contributes to 70 of the nitrogen cycling in the world s oceans 0 Being explored for a method to help remove ammonia and amines from the water The primary producers are cyanobacteria and algae in oxic environments Phytoplankton are oating or suspended photosynthetic microorganisms Benthic algae are attached to sides or bottoms of aquatic environments Organic matter can move up the food chain or be mineralized by phytoplankton in the microbial loop There is an inverse relationship between carbon and oxygen levels 0 Oligotrophic carbon is low then oxygen is high Little nutrient in ux o Eutrophic carbon nutrients high then oxygen low Signi cant inputs of nutrients Strati cation of freshwater lake 0 Oxygen is produced by phototrophs 0 Oxygen is consumed if lake is nutrient rich vs nutrient poor 0 Few eukaryotes live in hypolimnion o In the fall the epilimnian surface waters cool and sink to oxygenate the bottom waters This is called lake turnover Oligiotrophic lakes might never turn over Lakes that are very heterotrophic may turn over then become fully anoxic kills sh 0 Anoxic an area in the water that is depleted of oxygen Effect of sewage release into freshwater stream 0 Upstream high 02 but low algae cyanobacteria ammonia phosphate and nitrogen 0 Waste release causes drop in 02 high ammonia phosphate bacteria and nitrite o Algae and cyanobacteria bloom downstream o Stream eventually resembles upstream conditions Prochlorococcus most abundant oxygenic prototroph in the oceans It lacks phycobillin accessory pigment and appears green Estimated that oceans contain 13 x10A28 archea and 31 xlOA3l bacteria The ocean is the largest microbial biomass on earth s surface Barotolerant can grow under pressure Barophile grows best under pressure Extreme barophile growth under pressure Hydrothermal vents live off chemicals for primary production e g hydrogen sulfide oxidation by tubeworm symbionts Wastewater treatment is effective in limiting disease breaks down organic carbon to mostly C02 and CH4 the latter can be captured and used as fuel 0 Filtering and chlorinating water greatly reduced the incidence of disease 0 Goal is to remove organic and inorganic matter so not so support microbial growth Water quality is monitored by determining coliformcouts 0 Most probably number count 0 Colonies on membrane filter on media 0 Selective for coliforms and also may include a screen involving the conversions of IBDG to blue by E coli enzyme Beta glucoronidase
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