FIU Microbiology Lecture Notes (Exam1,2,3,Final)
FIU Microbiology Lecture Notes (Exam1,2,3,Final) MCB3020
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Date Created: 12/17/14
LECTURE 1 Unifying Concepts Robert Hooke l600s Built the first compound microscope Used it to observe mold Published lIicrographia the first manuscript that illustrated objects under the microscope Coined the term cell Antoni van Leeuwenhoek l600s Built singlelens magnifiers complete with sample holder and focus adjustment First to observe singlecelled microbes He called them wee animalcules 1600s to 1800s observations continued and questions arose where do microbes come from Spontaneous generation Louis Pasteur 1860s and his ask experiment demonstrated microorganisms are in the environment Showed that after boiling ask contents ask remains microbefree despite access to air led to pasteurization Robert Koch l880s Demonstrated the link between microbes and infectious diseases anthrax and tuberculosis Developed Koch s postulates Developed techniques solid media for obtaining pure cultures of microbes used today Since the 1880s much pressure to fulfill Koch s Postulates to prove a agent is the cause of a disease even now But Koch himself recognized that at times it was impossible to fulfill all of the postulates For example he believed cholera was caused by Vibrio cholerae but he also isolated this bug from healthy individuals which violated which postulate Since then many more examples Viruses can t be grown in pure culture Unculturable pathogens lt 1 of bacteria can be cultured Symbioses Polymicrobial diseases Subclinical infections Virulence vs nonvirulence switching genes on and off Induction by environmental factors Detection by molecular techniques Primary vs secondary pathogens Primary vs opportunistic pathogens Diseases vs syndromes Human diseases Still a big problem today Unifying concept Microbes are small technology is continuing optical tweezers confocal microscopy collaborations with physicists single cell genomics Microbes in uence the environment from the micro scale to the global scale Microbes are important in health and disease the human microbiome Many breakthroughs in basic sciences came from microbiology why LECTURE 2 Cell Structure and Function Prokaryote cell wall cytoplasmic membrane nucleoid cytoplasm plasmid ribosomes Eukaryotes cell wall cytoplasmic membrane mitochondrion nuclear membrane nucleus ribosomes endoplasmic reticulum cytoplasm golgi complex Diversity of shape coccus rod spirillum spirochete budding and appendage bacteria stalk and hypha filamentous bacteria Size range for prokaryotes 02 pm to gt700 pm in diameter Most cultured rodshaped bacteria are between 05 and 40 pm wide and lt 15 um long Examples of very large prokaryotes Epulopiscium shelsoni Thiomargarita namibiensis Size range for eukaryotic cells 10 to gt200 pm in diameter Cell Size and the Significance of Being Small Advantages to being small Small cells have more surface area relative to cell volume than large cells ie higher SV Support greater nutrient exchange per unit cell volume Tend to grow faster than larger cells The cytoplasmic membrane All cells in all organisms have a cytoplasmic cell membrane Basic structurefunction is the same All about 8 to 10 nm thick and all are highly selective permeability barriers only let certain things through Are definite differences between the three domains Bacterial Membranes Are phospholipid bilayers Two layers of phospholipids each with a hydrophilic end and hydrophobic end Hydrophobic two chains of fatty acids diagram Hydrophilic P04 bound to a glycerol Hydrophobic inside of the membrane acts like a uid fatty acids dissolve into each other Outside environment is usually aqueous charged and interacts with phosphoglycerol ends Result the membrane is held together by a combination of hydrophilic and hydrophobic interactions Membranes act as a selective barrier only allow certain molecules to pass these are small relatively uncharged H2O H2S N2 But need to get larger charged things across nutrients and energy sources in waste out done by specific proteins that move things across Carried out by the proteins in the phospholipid matrix Two main types of membrane proteins Integral membrane proteins firmly embedded in the membrane Peripheral membrane proteins one portion anchored in the membrane Also contain membranestrengthening molecules Eukarotes have sterols Rigid planar lipids strengthen and stabilize membranes cholesterol Some prokarvotes have hopanoids Structurally similar to sterols Archaeal Membranes Same overall structure hydrophobic interior hydrophilic exteriors But do not have fatty acids have chemically unique lipids called isoprenes Isoprenes are bound to glycerol with an ether linkage whereas Bacteria and Eukarya have ester linkages fatty acids to glycerol Archaeal Membranes Are two structural classes 1 glycerol diethers form membrane bilayers 2 glycerol tetraethers form membrane monolayers glycerolPO4 on each side of the membrane is covalently bonded to the other one are found in extremophiles why Membrane function Cells need to move molecules across the membrane Small relatively uncharged molecules move via diffusion depends on the relative concentration of the substance on each side of the membrane One biologically important example is water moves across membranes by osmosis osmosis movement of water from area of low solute to high solute Osmosis is very important Most bacteria have cytoplasmic solute concentrations of 10 mM amino acids salts sugars vitamins is much higher than the environment so water will diffuse in cells can swell and burst opposite is plasmolysis rigid cell walls counteract swelling Some cells have aquaporins membrane proteins to increase movement Charged molecules even small like H get bound to hydrophilic membrane surfaces Also can t pass through the hydrophobic membrane interior But cells need substances like sugars organic acids amino acids etc must be actively moved transported across such transport can result in gradients across membranes these gradients can be energy sources Why Transport systems allow cells to concentrate substances solutes against a concentration gradient These substances include nutrients Usually transport proteins are specific to what they transport they only transport one substance Requires membrane spanning transport proteins Often their synthesis is regulated by the cell Is faster than diffusion Are three categories of transport systems in prokaryotes based on energy source 1 Simple transport 2 Group translocation 3 ABC ATPbinding cassette system Why is energy required Energy for transport 1 Simple transport driven by a concentration gradient H Na 2 Group translocation substance transported is chemically modified why is this an energy source 3 ABC ATPbinding cassette system periplasmic binding proteins and ATP where is the energy Three types of energy source for transport of a substance across the cell membrane 1 Proton H or other ion gradient when the gradient is of protons is called the pmf proton motive force simple transport 1 Chemical modification with a high energy compound group translocation 2 ATP adenosine triphosphate ABC transport Are three categories of membrane transport proteins based on structure 1 Uniporters move 1 substance in 1 direction across the membrane 2 Symporters or cotransporters move 2 substances at the same time in the same direction across the membrane usually these are the desired substance and a second substance required to move the first often based on a concentration gradient of the second 3 Antiporters move 1 substance in one direction and a second substance in the other direction Simple transport Requires one membrane spanning protein Made of 12 alpha helices Energy from a gradient across the Example of simple transport Lac permease in E coli common enterobacterium Lactose disaccharide is an excellent carbonenergy source for E coli can t diffuse into the cell why transported in by the symporter Lac permease energy from the pmf pmf rebuilt by metabolism later Group translocation The A The substance transported is chemically modified Requires a set of proteins to transport the substance Common type of group translocation is the phosphotransferase system with PO4 from PEP transfered to the substance transported Phosphotransferase system is used to transport glucose mannose fructose more energycarbon sources for E coli into the cell Are phosphorylated chemically modi ed during transport this is the energy source PO4 is passed along to each protein phosphate cascade The final transfer is to the sugar Requires a set of five proteins for transport Four of the five are cytoplasmic Enz I and HPR are nonspecific Enz IIa IIb and IIc are specific to what why For glucose is metabolically efficient because phosphorylated glucose G6P is the first step of glucose metabolism When PO4 transferred to Enz IIc energy causes a conformational change that moves glucose across the membrane and phosphorylation to produce G6P BC System ATPbinding Cassette Requires a substratebinding protein a membrane transport protein an ATPhydrolyzing protein and ATP Used to transport sugars amino acids inorganic nutrients trace metals cofactors Are gt 200 identified Have high substrate specificity The substrate is first bound by a periplasmic binding protein Gram negative bacteria or a protein on the outer cytoplasmic membrane Gram positive bacteria binding proteins are specific for each substrate Substrate then transferred to the membrane spanning protein Energy in ATP used to move the substrate across the membrane unmodified and into the cytoplasm no PO4 transfer Above three classes of transporters are for relatively small molecules Also need to transport large molecules including proteins both into and out of the cell Done by specific translocases which are also used to insert proteins in to the membrane itself LECTURE 3 Cell Wall Structures Cell W alls Located outside the cytoplasmic membrane Function is to give rigidity to cells counteracts osmotic effects due to high solute in cells which leads to Wall structure is different for microbes in each domain Lysis of cell from osmosis 9 special attention to this area Cell W alls of Bacteria All bacteria divided into two groups Gram and Gram Based on major difference in bacterial cell walls Originally discovered based on staining procedure developed by Gram need to stain to see small cells one type retains the stain the other doesn t Difference is due to the amount of peptidoglycan Bacterial cell wall rigidity is due to peptidoglycan Found only in Bacteria used to tell if Archaea or Bacteria Composed of two sugar derivatives nacetylglucosamine and nacetylmuramic acid These alternate to form the peptidoglycan backbone 9 Know the 2 sugar derivatives names nacetylglucosamine nacetylmuramic acid The two sugars alternate to form chains The chains are covalently crosslinked Result is a three dimensional latticework that consists of one molecule surrounding the cell Each sugar has an amino acid side chain Amino acid side chains form links between peptidoglycan chains Are gt100 types of peptidoglycan based on differences in amino acids in the side chains Crosslinked differently in Gram vs Gram bacteria Gram have a glycine bridge 9 Amino acid side chain used as linkages Basic framework with sugars but alternate with cross linkage covalent bonds between amino acids 9 Figure 226 Peptidoglycan in Escherichia coli and Staphylococcus aureus Know the LAla DGlu DAP DAla chain order Linkage between DAP and DAla is important for drugs Know the Glycine bridge 5 residues in the linkage Gram negative vs Gram positive bacterial cell walls Gram negative are multilayered the most complex outside of cell membrane is a thin peptidoglycan layer inside a periplasmic space has a second outer membrane lipopolysaccharide layer Gram positive simpler and thicker outside of cell membrane is a thick layer of peptidoglycan Gram cells 90 of wall is peptidoglycan Gram cells only 520 usually l0 of wall is peptidoglycan Gram cells have a periplasmic space and LPS Gram cells have teichoic acids and different crosslinkages Gram negative bacteria LPS layer LPS lipopolysaccharide LPS layer is more permeable than the cytoplasmic membrane LPS layer has porins LPS spanning proteins with channels in center that allow low MW substances through even charged ones but not large ones From environment to periplastic space for porins LPS Basic properties inner hydrophobic outter hydrophilic Molecular model of porins in LPS Note Aquaporins last lecture are in the cytoplasmic membrane not the LPS layer outer membrane Porins in LPS allow large molecules to enter the periplasmic space Aquaporins for water to go faster than diffusion Porins will let fairly large larger sugars charged molecule but not huge ones Lipopolysaccharide LPS is made of two polysaccharides the 0 polysaccharides and core polysaccharides more physical integrity to the cell wall Lipids in LPS are also fatty acids like cytoplasmic membrane but are all lipid A with a glucosamine phosphate head not glycerol phosphate Also are proteins in the LPS that anchor LPS to peptidoglycan Often lipid A is toxic is a class of bacterial toxins endotoxins Lipid A toxic microbial endotoxin LPS structure in Gram negative bacteria 2 polysaccharides and organization of entire Lipid is specifically Lipid A and how it is different LPS in the cell wall Lipid A of the LPS replaces phospholipid in parts of the outer half of the outer membrane phospholipid bilayer how Peptiogycan layer in periplasmic space Lipid A inserted into hydrophobic part Held in place with hydrophobic and phillic interactions LPS in the cell wall Lipoprotein also in the LPSis present in the inner half of the outer membrane phospholipid bilayer Lipoprotein is covalently bonded to peptidoglycan so the LPS is attached to the rest of the wall properties Lipoproteins Covalently bonded to peptidoglycan Properties anchorage using covalently bonded to peptidoglycan and hydrophilicphobic interactions 9 EXAM Overall the LPS layer is functionally like the cell membrane hydrophobic interior hydrophilic exteriors but structurally it is slightly more complex than the cell membrane Lipid A and lipoproteins And it is relatively more permeable than the cell membrane porins Gram negative bacteria the periplasmic space Between the cytoplasmic membrane and LPS layer is a space Peptidoglycan is here but doesn t take up the whole space Protons of the pmf concentrated here 9 Functionally important for transport in terms of space actively transporting protons and harvesting energy 9 Gram negative profon motor force is organized here Periplasmic space Contains three types of enzymes hydrolytic degrade and lyse large organic matter that comes in through porins for uptake binding proteins first step of ABC transport chemoreceptors chemotaxis later 9 3 main groups of enzymes Photomicrograph of a Gram negative bacterial cell wall Gram negative bacterial cell wall 9 Understand organization 9 Know function of each layer Gram positive bacteria No periplasmic space and no LPS layer May have some binding proteins attached to the cell membrane ABC system Have teichoic acids on the outer part of the wall are negatively charged to maintain protons of pmf force how Lattice structure molecules diffiuse through peptidogycan to bond to binding proteins oating in periplasmic space 9 here these proteins are bound to cell wall Proton motor force 9since H are positive the neg acid particles stay in place through ionic bonding forces Also have lipoteichoic acids are covalently bonded to membrane fatty acids Photomicrograph of a Gram positive bacterial cell wall Gram positive bacterial cell wall THICK peptidoglycan layer 9 Know organization and functions On cell surgace Gram POS bumpy with polysacc sticking out The gram stain and bacterial walls Differences in bacterial cells walls are the basis of the Gram stain which in turn is the basis for the names Gram cells stain purple Gram cells stain pink Based on the thickness of the peptidoglycan layer The Gram Stain procedure Step 1 Result all cells purple 9 ood the heatfixed smear W crystal violet for 1 min Step 2 all cells remain purple 9 add iodine solution for 1 min Step 3 gram pos cells are purple gram neg cells are colorless 9 decolonize with alcohol brie y about 20 sec Step 4 gram pos G cells are purple and gram neg G cells are pink to red 9 counterstain with safranin for 12 min The alcohol step 3 washes away the stain crystal violet for Gram negative bacteria In Gram the alcohol dehydrates the thick peptidoglycan layer trapping the stain inside The last step is needed to stain Gram negative cells why Archaea walls Stain Gram or Gram but this is irrelevant because they don t have peptidoglycan Some Archaea have pseudomurein used to be called pseudopeptidog1ycan Found only in Archaea Function is rigidity Pseudomurein Composed of two sugar derivatives nacetylglucosamine and nacetylalosaminuronic acid Also has amino acid side chains that cross link like peptidoglycan Like peptidoglycan pseudomurein forms a lattice work of repeating structure Sugars alternate and parallel rows of sugars are linked by amino acid chains different than those of peptidoglycan Some Archaea with walls have no pseudomurein Can have pure protein walls differing glycoprotein walls etc Some are resistant to acids and high temperature Most common Archaeal cell wall is the Slayer Is a paracrystalline surface S layer Made of protein or glycoprotein interlocking Can act as a sieve Can keep proteins near the cell surface Some prokaryotes have no cell walls Examples Mycoplasmas bacterial pathogens that live in animal bodies osmotically protected environment Thermoplasma an Archaea that lives in hot springs how does it do this Concentration of solutes are comparable to animal cells 9 diffusion of water in balance 9 no lysis threat Hot springs survival 9 lipid monolayers covalently bonded tetraethers Cell walls of Eukarya Animal cells no walls Plants algae fungi all have walls Are highly variable range from cellulose chitin silica polysaccharides CaCO3 etc LECTURE 4 Cell Inclusions and Microbial Motility Cellular organization so far membranes walls and cytoplasm Cytoplasm an aqueous soup of solutes Also contains inclusions Many are for storage of carbon or nutrient compounds Some are functional ecological physiology Also are surface associated functional structures Cell capsules or slime layers surround some cells Are made of polysaccharide Different functions attachment to surfaces pathogens to hosts biofilms in many aquatic environments a strategy to avoid host immune response later Also often have fimbriae and pili Are made of protein extend from the cell surfaces Also have different functions attachment to surfaces specific receptors for viruses gene exchange conjugation motility twitching motility type IV pili extend from cell attach to surface then retract into cell powered buy ATP Cell Inclusions Intracellular or periplasmic inclusions are deposited materials Each type of inclusion is made of one substance Serves as storage for the cell Cytoplasmic inclusions are often surrounded by a single layer membrane Usually are made of a critical nutrient required for sustaining life such as carbon Carbon commonly stored as polyBhydroxybutyrate PHB or glycogen chains of glucose Only get storage in inclusions when carbon is in excess in the environment when carbon is limiting inclusions if present used for carbon source to the cell Also can have nutrients stored as inclusions Like carbon formed when that nutrient is in excess in the environment and used when nutrient becomes limiting 9 Nitrogen and phosphourus most abundant but competed for the most Sulfur can be stored and used for both a nutrient or as an energy source later are sulfur granules Some inclusions are common to many types of microbes PHB glycogen polyphosphate Some only found in specific rare types Sulfur granules usually in sulfide oxidizers later Other examples internal biomineralization magnetosomes Some cyanobacteria precipitate carbonate minerals made of benstonite composed of barium strontium and magnesium Function may be ballast to keep cells deep these cyanobacteria deep in lakes Magnetotactic bacteria Have magnetosomes Made of magnetite Fe3O4 or greigite Fe3S4 surrounded by a membrane Allow cells to detect orient to and move to a magnetic field is magnetotaxis Thought to be a way to move to low oxygen environments are microaerophiles movement to poles is actually down into the earth N vs S 9 Tiny magnets inside the cell required low levels of oxygen aerobic respiration Some inclusions common to different microbes in an ecological niche Example gas vesicles in aquatic bacteria and Archaea photosynthetic and nonphotosynthetic Allow cells to move up in the water column where the light is Gas vesicles are made of cone shaped structures of two proteins GVPA and GVPC hydrophobic Synthesized in the cytoplasm two ends first then hollow cylinder in between elongates Protein is impermeable to water but allows small gas molecules N2 O2 CO2 to pass how 9 Cones will expand into larger structure proteins impermeable Small gas does pass through 9 diffused gases inside the cell concentration gradient Causes cells to oat confers buoyancy As cells oat more light is available When accumulate photosynthate or DOC taken up in the cytoplasm increases turgor pressure gas vesicles collapse and cells sink synthesize more when cell needs more carbon 9 When they begin to synthesize begin to oat 9 cells cannot tolerate the UV lights at the area SO carbon molecules accumulate first prod of photosynth 9 increases solute level of cell cannot diffuse out are too big and charged accumulate and creates pressure and collapses the gas vesicles 9 sink back down away from the light 9 Populations move up and down in water column based on this cycle Can cause cyanobacterial blooms 9 Cyanobacteria that has oated up Endospore Produced by some Gram positive bacteria Are intracellular endo and allow cells to survive harsh periods heat chemicals dryness radiation etc Generate to form new vegetative cells when conditions get better are dormant Also good for dispersal Also inclusions When they are released the cell usually dies Microbial Motility Many microbes are motile both on a surface and suspended in liquid In liquid agella On a surface twitching or gliding motility Bacterial Flagella 9 are three arrangements Peritrichous Polar Lophotrichous Structure of bacterial agella Are long thin rigid and hollow 15 20 nm in diameter Are helices with constant and species specific distances between curves Composed of protein subunits agellin synthesized in the cytoplasm moved to agellum move along hollow center and added to growing tip via selfassembly grow until species specific maximum length is reached Flagella only thing made of protein agelin Attached to hook anchors it to cell wall moves cell by rotation This is a Gram Neg Bacteria 9 anchored on both outter and inner layer dw about ring names FOCUS Gram negative bacteria have an outer pair of rings in the LPS and peptidoglycan layers and an inner pair in the cell membrane Gram positive bacteria only have an inner pair in and above the cell membrane Bacterial agella impart movement by rotation The cell is propelled through the liquid Energy is from the pmf calculated that 1000 Hs move across the membrane for each rotation max rate 300 revsec 12000 rpm velocity 20 to 80 umsec The proton turbine model of pmf powering agella rotation Protons ow through channels in Mot proteins Charge interactions between protons and ring surfaces result in rotation Cell increase or decrease speed depending on the strength of the pmf what does this mean Flagella can move bacterial cells 60 cell lengths sec relatively faster than the fastest animal the cheetah 25 body lengthssec 9 Strength of concentration gradient Flagellum synthesis is sequential from rings in the cytoplasmic membrane to the tip 9 Growth of agella diagram Movement by peritrichously agellated bacteria Alternating runs and tumbles important for directionality later Counterclock wise direction line movement Changing rotation of rotation knocks each other around changes direction 9 Alternating runs Movement by polarly agellated bacteria Move by reversing direction Archaeal agella are different than bacterial agella Are 1013 nm wide Made of several types of protein not agellin Also cause movement by rotation but may be powered by ATP not pmf 9 Not made of agelin different proteins Gliding motility occurs on a surface benthos no agella For bacteria mainly filamentous microbes Is slower and smoother than with agella Are different mechanisms excretion of polysaccharide slime cyanobacteria twitching by extracting and retracting type IV pili some myxobacteria movement of proteins on the surface of cells Flavobacteriumjohnsoniae LECTURE 5 Metabolism Growth and OxidationReduction Reactions Cells carry out metabolism in order to grow For cells to grow they need both macro and micro nutrients Macronutrients needed in large amounts Are used to build cell constituents and for important molecules needed in quantity proteins RNAs etc Micronutrients needed in smaller amounts Usually are coenzymes cofactors etc Macronutrients C O H N P S K Mg Ca and Na C O H form most of the cellular material 7080 of the cell is water cytoplasm Other macronutrients are part of crucial cell components Micronutrients B Cr Co Cu Fe Mn Mo Ni Se W V Zn ex Fe in cytochromes and nonheme iron sulfur proteins Usually study microbes in culture in the lab need to provide them conditions that allow growth Macro and micronutrients are provided in media Also may contain growth factors such as vitamins or speci c amino acids that your bacterial strain can t synthesize KNOW Liebig s Law of the Minimum Whatever is present in the lowest amount will limit growth Barrel analogy the shortest slat determines how much water is in the barrel KNOW Two classes of culture media Defined everything is known in the medium including the precise amounts Complex or unde ned constituents are not specifically known ex Yeast extract Also use Enriched media selects for a specific type of microorganism Selective media has compounds which selectively inhibit growth of some microbes Differential media has an indicator usually a dye which tells when a diagnostic chemical rx has occurred Example EMB agar Eosin methylene blue Cultures that ferment lactose like E coli form colonies with a metallic sheen Besides macro and micronutrients and growth factors need to provide appropriate physical conditions pH oxygen Eh salinity temperature light etc Often media are made using agar special properties read in book Often work with pure cultures derived from a single organism called axenic Different techniques to obtain pure cultures enrichments streakingpicking colonies gliding technique sterile technique read 43 if not in lab Once a bacterium is obtained in axenic culture it must be maintained routine transfer into fresh media often stored frozen in glycerol Microorganisms are metabolically highly diverse Can carry out many different types of metabolism Metabolism is classified based on energy source and carbon source Are two main categories of each Energy source light or chemicals Carbon source CO2 or organic reduced Terminology to know 1 Energy source Growth with light as the energy source is phototrophy Growth with chemicals as the energy source is chemotrophy 2 Carbon source Growth with CO2 as the carbon source is autotrophy Growth with reduced organic carbon as the energy source is heterotrophy or organotrophy Reduced organic carbon can serve as both the carbon and energy source 9 Anything that its carbon dioxide is reduced The above terms can be combined to describe both the energy and carbon sources for a specific microbe Photoautotroph energy from Z carbon from Z Photoheterotroph Chemoautotroph Chemoheterotroph In addition a chemical energy source can be organic carbon organo or inorganic litho iron or sulfur Chemoorganotroph energy from organic carbon Chemolithotroph energy from an inorganic substance Can be further combined Chemolithoautotroph Chemoorganoheterotroph Metabolism Metabolism all chemical processes taking place in a cell Two basic categories of metabolic processes Anabolism cell constituents are built from chemicals obtained from the environment O2 H2 CO2 H2O N2 organic compounds etc also called biosynthesis Catabolism metabolic processes where chemicals are broken down to release energy for cell function Metabolic chemical reactions occur within a cell but chemical reactions are also very important in terms of the environment of the cell We need to consider environments within the cell on each side of the cell membrane and in the external environment around the cell 9 MUST consider environments within the cell as well as outside of the cell Also need to consider bioenergetics in microbiology energy measured in kiloj oules kJ free energy G is the energy available for cells to work Change in G during a bioenergetics rx is AGO Only reactions with a negative AGO exergonic will go without input of energy activation energy Much of the chemistry of cellular metabolism is based on electrochemical reactions Characterized by movement of electrons in a controlled process De ned by oxidation reduction reactions or redox reactions Definitions to know Oxidation the removal of an electron from a substrate Reduction the addition of an electron to a substrate When an electron is taken from a substance the substance has been oxidized When an electron is added to a substance the substance has been reduced An oxidant electron acceptor removes electrons ex O2 A reductant electron doner adds electrons ex H2 Electrons can t exist alone must be part of atoms or molecules Every time you have a reduction reaction you must have an oxidation reaction and vice versa Why Example 1 hydrogen H2 26 9 2H But can t have free electrons they have to go somewhere With oxygen around get 12 O2 2e 2H 39 9 H20 you mg have free H Both reactions are coupled H2 12 O2 9 H20 Overall this is the oxidation of hydrogen Example 2 water Can have the opposite last slide occur H20 9 12 O2 2e 2H Again electrons have to go somewhere Metabolic example in oxygenic photosynthesis the electrons go to a metabolic e acceptor O2 is a byproduct along with Hs where do you think they go Always have to have both an electron donor and an electron acceptor in a redox reaction why Chemical reactions in which an electron is transferred are very common in metabolic processes 9 When we consider these reactions think about electron transfer where the electron comes from the reductant or e donor and where it goes to the oxidant or e acceptor A very important aspect of electrochemistry is oxidationreduction potential Eh or E0 Definition Eh is the tendency of a substance to accept or give up electrons Eh is measured in volts and indicated as E0 reduction potential can be or A very strong reductant has a very negative Eo relative to a potential oxidant A very strong oxidant has a very positive E0 relative to a potential reductant Electron transfer can happen spontaneously depends on E0 of the potential e donor and the E0 of any available e acceptor The electron tower Often written on a vertical scale the electron tower Y axis is in volts values referenced to H2 at pH 70 most negative at the top positive at the bottom Top of the tower greatest amount of potential energy donors want to donate e shave the greatest tendency to donate e s Bottom of the tower greatest possible energy is released if the acceptor is here Why As es move from an acceptor to a donor the energy released available for metabolism is the difference in volts between the donor and acceptor Have to consider each substance as an electron couple the oxidized and reduced forms A substance can only be either oxidized or reduced at any one time ex cyt b oxred Example The amount of energy released when the donor is H2 and the acceptor is 02 the voltage between 082 and 042 volts 124 volts Calculate voltage for NADH and Fe H2 and NAD Pyruvate and Cyt a FAD and nitrate Note substances some can be partially oxidized or partially reduced if there is more than one e that can be donated sulfur carbon and others Many E yielding reactions of cells consist of a series of oxidation reduction steps electrons are passed along in a series chain of donors and acceptors later Why would this be efficient LECTURE 6 Fermentation and aerobic respiration Metabolic pathways The three most common energy yielding metabolic pathways Fermentation Aerobic respiration Oxygenic photosynthesis Carried out by eukaryotes and prokaryotes Respiration and photosynthesis involve electron transport fermentation does not Cells have two major sources of energy that are produced in energyyielding metabolism Reducing power NADH Hquot and ATP Both are present as pools in the cytoplasm NADH H 39 and NAD AMP ADP and ATP Will see how they are generated in fermentation and aerobic respiration Fermentation Fermentation is a common anaerobic energy yielding metabolic pathway Fermentation is not membrane associated it occurs in the cytoplasm The energy source is some form of reduced organic carbon that is partially oxidized no full to oxidation to CO2 Fermentation NO external e acceptor NO e transport chain NOT membrane associated Requires anaerobic conditions ATP is formed enzymatically by transfer of PO4 from phosphorylated compounds in the fermentation pathway to ADP in the cytoplasmic pool called substrate level phosphorylation In fermentation the partial oxidation of the energy source organic carbon is balanced by reduction reactions that lead to byproducts that are reduced organic compounds Overall fermentation is an internally balanced redox reaction Only a small amount of the potential E is released First NADH very important in fermentation When looked at as a redox couple what can you say about it Why is this an energy source The reduced form of NADH is correctly written as NADH H 39 because 2 e s are required in the redox reaction reduction of NAD 39 Overall reduction NAD 39 2H 9 NADH H 39 Example fermentation of glucose Occurs in three stages 1 ATP is used to produce 2 molecules of glyceraldehyde3PO4 from glucose uses 2 ATP 2 An oxidation occurs with NAD the oxidant produces NADH H to produce pyruvate get 4 ATP by substrate level phosphorylation 3 A reduction occurs to reoxidize NADH this is required for fermentation to continue Fermentation of glucose Steps 1 ATP is used to produce 2 molecules of glyceraldehyde3PO4 from glucose uses 2 ATP 2 An oxidation occurs with NAD the oxidant produces NADH H 39 to produce pyruvate Produces 4 ATP by substrate level phosphorylation 3 A reduction occurs to reoxidize NADH This is required for fermentation to continue In fermentation the fermented substance will serve as both an electron donor and an electron acceptor as it is metabolized along the fermentation pathway Where Additional points about fermentation Fermentation of one glucose to two pyruvate is called glycolysis Fermentation byproducts are produced as a result of the required oxidation of NADH Examples include EtOH lactic acid H2 CO2 and others There is no overall change in oxidized vs reduced NAD in the NADNADH H 39 pool after fermentation of a substrate Fermentation yields a small amount of energy compared to aerobic respiration Net E produced fermentation 2 ATP per glucose aerobic respiration 38 ATP glucose Why not only carry out aerobic respiration if glucose is available Aerobic Respiration Aerobic respiration is a membrane associated energy yielding metabolic pathway The energy source is an electron donor which is usually an organic carbon compound The electron is transferred to a terminal e acceptor which is 02 aerobic As opposed to fermentation in respiration all of the organic carbon energy source can be used all possible e s are donated and the energy source can be fully oxidized to CO2 In aerobic respiration pmf is generated the pmf is then used to produce ATP next lecture As electrons move along a respiratory e transport pathway protons are moved across the membrane The e transport pathway is organized in the membrane and carries out e transport The organic carbon energy source where the electrons come from is located in the cytoplasm Electrons are often donated to the respiratory electron transport chain via NADH H 39 or another cytoplasmic electron carrier such as FADH2 Electron transport and metabolism Consists of a series of oxidationreduction steps Electrons are passed along in a chain of donors and acceptors electron transport chain etc The donors and acceptors are held in place in the membrane by what Electron chains Electron chains consist of An initial or primary e donor A nal or terminal e acceptor Intermediate electron carriers in between the primary donor and the terminal electron acceptor Energy released difference in E0 between the primary e donor and the terminal e acceptor The most important e acceptor in biological processes is 02 Can calculate energy with different donors and acceptors how Electron carriers There are two general classes of intermediate electron carriers held in place in the cell membrane several types freely diffusible in the cytoplasm one type NADPH H 39 Are five types of membraneassociated electron carriers NADH dehydrogenases Flavoproteins Cytochromes Nonheme ironsulfur proteins Quinones All are held firmly in place except quinones these are freely diffusible in the membrane The one cytoplasmic electron carrier is NADPNADPH H 39 E0 032 V good electron donor reducing power Acts as a coenzyme facilitates chemical reactions in the cell that wouldn t normally happen ex movement of e39s between the cytoplasm and membrane bound e transport chains Are two categories of electron carriers Electron carriers only accept and donate electrons Hydrogen carriers have to take a proton along with an electron note are a type of electron carrier Hydrogen carriers NADH NADH dehydrogenase avoproteins quinones Electron carriers cytochromes nonheme ironsulfur proteins Example of an electron carrier Cytochrome electrons are associated with Fe present at either Fe or Fe Another electron carrier Nonheme iron sulfur protein Example of a hydrogen carrier Flavin mononucleotide FMN a avoprotein NADH is also a hydrogen carrier Protons are moved across membrane based on the type of e carrier the type of carrier it is passing the e to and where the e came from If a hydrogen carrier passes an electron to another hydrogen carrier hydrogen is passed if it passes to an electron carrier only the electron is passed and the proton is discarded usually to one side of the membrane to increase pmf If an electron carrier passes an electron to another electron carrier the electron is passed if it passes to a hydrogen carrier a proton must be picked up usually enhances pmf where does the proton come from Respiratory electron transport chain Substrate 9 NADH H 39 9 NADH dehydrogenase 9 avoprotein 9 nonheme iron FeS protein 9 quinone cycle 9 cytochromes 9 02 What are the relative Eo values of each of these What is the substrate Protons are moved across the membrane when an e is transferred from an electron carrier to a hydrogen carrier or from a hydrogen carrier to an e carrier organized so protons are picked up from the cytoplasm and released to the outside Pmf is also produced by protons being consumed in reactions in the cytoplasm Final step When the e reaches 02 Hs in the cytoplasm are consumed 4 are required 2 to form H20 2 needed to transfer the 2 e39s from cytag so is a proton consuming reaction in the cytoplasm contributes to pmf The energy used to produce pmf difference between the e donor and e acceptor Ex glucoseCO2 043 volts 12 O2H20 082 volts Difference 125 volts How does NADH H 39 get from the energy source to the etc Different paths For glucose can be same pathway to pyruvate as fermentation Are additional paths such as the citric acid cycle LECTURE 7 Energy production Summary metabolic redox reactions Metabolic oxidationreduction reactions proceed in three steps removal of an e from a primary e donor transfer of the e via intermediate e carriers addition of the e to a terminal e acceptor The energy released difference in volts Eo between the primary donor and terminal acceptor Often organized as a membrane associated electron transport pathway with protons moved across the membrane as the electrons are passed along Generates the proton motive force Energy and the pmf pmf a proton gradient across a membrane pmf composed of two forces a concentration gradient of protons the difference in electrochemical charges on each side of the membrane Hs are charged NADH H 39 is an important electron carrier in metabolism because it is cytoplasmic and can move between compounds associated with metabolic pathways in the cytoplasm and electron carriers in the membrane In this way pmf can be generated using reducing power electrons from the cytoplasmis pool Energyrich compounds and energy storage Chemical energy released in redox reactions is mainly stored in certain phosphorylated compounds ATP the most important Phosphoenolpyruvate PEP Glucose 6phosphate Can also be stored in coenzyme A energy released by hydrolysis ATP The most important source of biological energy is ATP which has two high energy PO4 bonds When the bonds are broken energy is available for the cell Longterm energy storage cellular inclusions made of substances that can be oxidized to generate ATP Examples in prokaryotes Glycogen Poly3hydroxybutyrate Elemental sulfur Examples in eukaryotes Starch Lipids simple fats Formation of ATP ATP adenosine triphosphate is produced from AMP adenosine monophosphate or ADP adenosine diphosphate The process is phosphorylation For ATP formation there are three types oxidative phosphorylation photo phosphorylation substrate level phosphorylation The first two are powered by pmf the second enzymatic ATP formation using the energy of the proton motive force is carried out by a membrane spanning protein Called ATP synthase or ATPase Is composed of two parts F0 and F1 ATPase Fo spans the membrane Serves as a channel that allows protons to ow along the gradient ATPase F1 is on the cytoplasmic side of the membrane sticks into cytoplasm ATPase F1 is catalytic carries out the phosphorylation reaction ADP Pi 9 ATP using energy released as protons move through Fo ATPase can run in reverse Occurs when cells need more pmf to power things like agellar rotation If there is a low level of pmf and a high level of ATP cells can use ATP to force protons to the other side of membrane and generate pmf Bacterial and Archaeal cells pmf is oriented such that protons are concentrated on the outside of the cell Eukaryotic cells pmf is associated with mitochondria with protons moved to the cytoplasm Catabolic Diversity Microorganisms demonstrate a wide range of mechanisms for generating energy Fermentation Aerobic respiration Anaerobic respiration etc with something other than 02 as the terminal electron acceptor Chemolithotrophy etc with electrons coming from a reduced inorganic compound not organic carbon or NADH Phototrophy etc with electrons entering using light energy LECTURE 8 Molecular microbiology and genomics Bacterial genetics Genome entire complement of genes in cell or virus Chromosome main genetic element in eukaryotes and prokaryotes not viruses are circular in prokaryotes but tightly packaged by supercoiling is only one chromosome in prokaryotes Also very important in prokaryotes are plasmids are small covalently closed circular pieces of DNA can carry important genes Since the prokaryotic chromosome is circular DNA replication is different than prokaryotes Is bidirectional two replication forks move in opposite directions around the chromosome 9 DNA replication is more efficient since its circular goes bidirectional as oppose to just one 9 2 replication forks moving at the same time Is carried out by a replisome protein complex that pulls the replicating DNA through the complex Are three natural methods of genetic recombination for bacteria Transformation Transduction Conjugation For eukaryotes main mechanism is sexual reproduction 1 Transformation Free DNA is taken up into bacterial cells Only some strains can be transformed are referred to as competent Are four steps in transformation a a small piece of DNA binds to the competent cell DNA comes from a lysed bacterial cell in the near environment binding occurs at a membranebound binding protein competent cell binding is at first reversible but becomes irreversible b One strand of the DNA passes into the competent cell nuclease degrades the other strand c The one strand in the cell is bound by specific proteins moves DNA to chromosome and recombines with the homologous region of the chromosome carried out by RecA d New DNA is incorporated and the cell is transformed Note if the new DNA is viral DS DNA this process is called transfection Transformation 2 Transduction DNA is transferred from one cell to another via a bacteriophage or phage a virus that infects bacteria Two types specialized and generalized Specialized a specific group of bacterial genes is packaged into the virus and carried to another bacterium happens only with temperate phages later Generalized any part of the host gene can be carried by the virus Transduction is important in nature many bacteria can do this Generalized transduction Transduction has occurred when the viral DNA is incorporated into the bacterial chromosome Specialized transduction moves part of the actual bacterial chromosome Happens when viral DNA has been inserted into the bacterial chromosome and when it comes out to form a new virus it takes some of the bacterial DNA with it Example lambda in E coli 2 Conjugation bacterial mating DNA transfer occurs via cell to cell contact Can happen two ways chromosome transfer or plasmid transfer Plasmid transfer plasmids extrachromosomal covalently closed circular DNA moves between cells If a conjugative plasmid integrates into the host chromosome part of the host chromosome can be transferred In both cases DNA passes between cells through a conjugation bridge Get membrane fusion of the donor and recipient Conjugation process When conjugation occurs a plasmid or part of the bacterial chromosome mobilized by a plasmid moves from a donor cell to a recipient cell The donor cell has a conjugative plasmid called F and the recipient does not F stands for fertility and the genome of F is known Contains genes for DNA replication genes to integrate into a bacterial chromosome and genes that code for transfer tra genes Tra genes include genes for sex pilus synthesis Transfer of the F plasmid by conjugation pilus retracts cell pairs stabilized F plasmid nicked in one stand transfer of one strand from F to F cell F plasmid simultaneously repliced in F cell synthesis of complementary strand begins in recipient cell completion of DNA transfer an synthesis cell separate During transfer as the DNA SS enters the recipient cell the complementary strand is replicated by rolling circle replication Same process occurs in the donor cell as the SS moves out Rolling circle replication HFR strains high frequency of recombination F is an episome a plasmid that can become integrated into the host chromosome Host cells with F present as a plasmid are F Host cells with F integrated into the chromosome are Hfr cells Hfr mobilizes a host cell s genome 9 A lot of genes transferred not just the plasmid including host chromosomes Transfer of host DNA in an Hfr cell F plasmid nicked in one strand Transfer of F followed by chromosomal DNA Synthesis of second strand in recipient Covalently bonded to host chromosome and pulling it into the new bacteria During conjugation with an Hfr strain SS DNA will keep moving through the conjugation tube until the tube membrane breaks The plasmid DNA is transferred and replicated first any chromosomal DNA that comes in after attached depends on how long conjugation occurs until the tube breaks Comparison of natural gene transfer in bacteria LECTURE 9 Growth and Environment Bacterial growth Most microbes grow by binary fission read Cell growth cell size increases and the cell divides into two new cells daughter cells requires cleavage of peptidoglycan between NAM and NAG Population growth increase in the number of cells in the population is exponential One generation of growth Cell elongation Septum formation Completion of septum formation of walls cell separation 2 cells result semi log scale used for measuring number of cells linear log plot parabolic arithmetic plot When measuring growth you typically consider two things 1 Growth rate change in cell concentration per unit time 2 Generation time doubling time for bacteria the range is about 10 min to days or even months both an intrinsic property and dependent on what is available to support growth in the environment Bacterial growth cycle Bacteria go through different stages when they grow in cultures Are 4 phases of growth lag log exponential stationary and death 9 explanation in textbook In the lab you normally grow bacteria in batch or continuous chemostat culture The growth cycle obtained is different for each Batch growth media is in a container with a fixed volume no new media is added Optical denisty absorbance Lag phase cells present but adapting to a new environment Log phase after adjusting grow exponentially Stationary something in medium limits their growth 9 stop growing Death if they don t produce endospores they start dying Continuous the growth container is an open system fresh media is continually added Chemostat continuously drips into ask w volume amount controlled Culture grows inside the over ow ef uent has microbial cells growth rate defined by dripping rate of esh medium Lag medium pump should not be dripping yet need to run as batch culture once there is growth then turn it on or else they will all come out Log media dripping in and making exponential growth Stationary not actually they are still growing exponentially NO DEATH PHASE Growth in a chemostat In a chemostat you need to start off growth of the culture as a batch culture why So you don t ush them out In continuous culture you can control the growth rate dilution rate growth rate Ex culture volume 1 L dilution rate 1 Lhr doubling growth rate 1 hr Examples of calculating growth rate in a chemostat 1 Culture volume l L dilution rate 500 mlhr doubling rate 9 2 hrs 2 Culture volume 500 mls dilution rate l Lhr doubling rate 9 12 hr 3 Culture volume 250 mls dilution rate l Lhr doubling rate 915 mins 4 Culture volume 250 mls dilution rate 50 mlshr doubling rate 9 5 hrs In addition to controlling the growth rate you can control cell density by the concentration of the limiting nutrient in your media Note if your dilution rate is too fast for cells to keep up what happens What does keep up mean 9 you end up ushing them completely out of the chemostat Whether growing cells in batch or continuous culture you often want to measure the number of cells to document growth Several methods microscopic counts viable counts optical density wet vs dry weight etc read in book Are equations to calculate growth Makemson handout on Blackboard Growth and the environment Microbes grow over a wide range of environmental parameters Temperature form psychrophiles coldloving to thermophiles heatloving Each has an optimal temperature for growth as well as a temperature minimum and maximum Figure 519 The cardinal temperatures minimum optimum and maximum Minimum membrane gelling transport processes so slow that growth cannot occur Enzymatic reactions occurring at increasingly rapid rates Optimum enzymatic reactions occurring at maximal possible rate most loving temp Maximum protein denaturation collapse of cytoplasmic membrane thermal lysis 9 psychrophile mesophille thermophile hyperthermophile hyperthermophile Psychrophiles Are many adaptations that support psychrophily enzymes that function optimally in the cold more polar and less hydrophobic amino acids fewer weak bonds modified cytoplasmic membranes with high unsaturated fatty acid content Only prokaryotes exist above 65 C Thermophiles organisms with growth temperature optima between 45 C and 80 C Hyperthermophiles organisms with optima greater than 80 C Live in boiling hot springs sea oor hydrothermal vents etc can live and growt at temperatures greater than 100 C Much work on hyperthermophiles in hot springs chemoorganotrophic and chemolithotrophic species High prokaryotic diversity both Archaea and Bacteria Adaptations to thermophily Enzymes and proteins function optimally at high temperatures Amino acid substitutions provide more heattolerant folds Increased number of ionic bonds between basic and acidic amino acids Modifications in cytoplasmic membranes Bacteria have lipids rich in saturated fatty acids Archaea have lipid monolayer rather than bilayer Hyperthermophiles produce enzymes widely used in industrial microbiology Example Taq polymerase used to automate the repetitive steps in the polymerase chain reaction PCR technique The pH of the environment greatly affects microbial growth Microbes are present across a wide range of pH values Acidophiles grow best at low pH lt6 some are obligate acidophiles membranes are destroyed at neutral pH stability of cytoplasmic membrane is critical Alkaliphiles grow best at high pH gt9 some have sodium motive force rather than proton motive force Neutrophones ph gt 55 and lt 8 Effects of pH on Microbial Growth The internal pH of a cell must stay relatively close to neutral even though the extemal pH is highly acidic or basic Internal pH has been found to be as low as 46 and as high as 95 in extreme acidophiles and alkaliphiles respectively Effects of pH on Microbial Growth Microbial culture media typically contain buffers to maintain constant pH Osmolarity and Microbial Growth Typically the cytoplasm has a higher solute concentration than the surrounding environment thus the tendency is for water to move into the cell positive water balance When a cell is in an environment with a higher external solute concentration water will ow out unless the cell has a mechanism to prevent this 9 plasmolysis prevention Halophiles organisms that grow best at reduced water potential have a specific requirement for NaCl Figure 526 Extreme halophiles organisms that require high levels l5 30 of NaCl for growth Halotolerant organisms that can tolerate some reduction in water activity of environment but generally grow best in the absence of the added solute Figure 526 Effect of NaCl concentration on growth of microorganisms of different salt tolerances or requirements as NaCl is increasing Nonhalophile Halotolerant Halophile Extreme Halophiles Oxygen and Microbial Growth Aerobes require oxygen to live Anaerobes do not require oxygen and may even be killed by exposure Facultative organisms can live with or without oxygen Aerotolerant anaerobes can tolerate oxygen and grow in its presence even though they cannot use it lIicroaerophiles can use oxygen only when it is present at levels reduced from that in air LECTURE 10 Metabolic regulation Chemotaxis and quorum sensing There are two major levels of metabolic regulation in the cell Control of the activity of preexisting enzymes Posttranslational regulation Very rapid process seconds Control of the amount of an enzyme Regulates level of transcription Regulates translation Slower process minutes Also are two categories of regulation Negative repression Positive activation Three examples of regulatory and regulated processes two component regulatory system chemotaxis and quorum sensing TwoComponent Regulatory Systems 9 very common in prokaryotes transmitted to a speci c target in the cell Prokaryotes regulate cellular metabolism in response to environmental uctuations Extemal signal is transmitted directly to a target in the cell so cells can respond External signals are detected by sensors and transmitted to regulatory machinery signal transduction Most signal transduction systems are twocomponent regulatory systems Twocomponent regulatory systems consist of two different proteins A Sensor kinase also called a histidine kinase located in the cytoplasmic membrane detects an environmental signal and autophosphorylates A Response regulator located in the cytoplasm Is a DNAbinding protein that regulates transcription when it is phosphorylated by the sensor kinase signal cascade Also involves a feedback loop 9 makes it no longer active either response regulator or something else in entire system Kinase an enzyme that adds a high energy phosphate group usually from ATP to another substance Sensor kinases involve histidine membrane When phosphorylated the response regulator moves to the chromosome and interacts with a gene represses or induces The feedback occurs because a phosphatase either the response regulator or another enzyme in the cytoplasm removes the phosphate group from the phosphorylated response regulator turning it off Two component regulatory system 9 with signal autophosphorylates phosphate group appears sensory kinase phosphorylates response regulator that turns on gene turns off gene on chromosome Are many almost 50 different twocomponent systems in E coli alone also found in many other species Control different metabolic processes phosphate assimilation nitrogen metabolism and osmotic pressure response etc Some Archaea also have twocomponent regulatory systems Chemotaxis Definition movement towards or away from a chemical taxis directional movement Chemotactic bacteria move toward an attractant and away from a repellant Can also have phototaxis movement toward a light source positive phototaxis or away from it negative phototaxis Gliding cells can glide toward an attractant or away from a repellant by simply reversing the direction of gliding For bacterial cells that move with agella it is more complicated and is determined by changing the ratio of runs agella moving what direction vs tumbles direction In the presence of an attractant chemotactic bacteria will run more in the presence of a repellant they will tumble more Also for an attractant the length of runs increases and the length of tumbles decreases For a repellant the length of tumbles increases and the length of runs decreases The result is a biased random walk that moves the cell towards the attractant or away from the repellant A biased random walk allows movement up a gradient of attractant The mechanism of bacterial chemotaxis CT was worked out using two model organisms Escherichia coli and Salmonella typhimurium both have peritrichous agella Chemotaxis was first seen with the Adler capillary assay Adler capillary assay Measuring chemotaxis using a capillary tube assay Bacterial cells are so small that they can t actually sense a spatial gradient they sense a temporal gradient Runs vs tumbles detected using a 3d tracking microscope and adding attractants etc to the medium The mechanism of bacterial chemotaxis at the cellular level involves proteins in the cell membrane are sensory proteins that combine with tactically active compounds attractants and repellants some are specific only combine with one compound others not ex groups of sugars 9 methyl accepting proteins bound to attractant or repellent bout to it 9 autophosphorylation 9 CheA and CheW associated with the MCPS attachement autopho of CheA 9 know how it works and how signals are transmitted Bacterial chemotaxis involves three overall steps 1 Response to a signal 2 Controlling direction of rotation of agella 3 Adaptation to the concentration of the signal in the environment 9 that one of the Che protein will do while the other does Overall model of chemotaxis Interactions of MCPs Che proteins and the agellar motor in bacterial chemotaxis First the attractant or repellent combines with the sensory protein a transmembrane protein called a transducer or methyl accepting chemotaxis protein MCP 9 chemoreceptors bind w signal and takes it over 9 Che W is coupling protein that assists CheA For Gram negative bacteria the attractant or repellant may first combine with a chemoreceptor in the periplasmic space in this case the complex moves to the MCP MCPs translate the signal from the chemoreceptor to the agellar motor controls direction of rotation There are five types of MCPs in E coli 9 Each responds to different attractants or repellants MCPs are methylated and demethylated methyl accepting with the methyl groups coming from S adenosylmethionine in cytoplasm Up to four methyl groups can be added to one MCP Mechanism of Chemotaxis MCPs bind attractant or repellant and for Gram negative may be a complex with periplasrnic chemoreceptors Attractants agella rotate CCW and cells run Repellants agella rotate CW and cells tumble Signal translation involve a class of cytoplasmic proteins the Che proteins are six Che A B R W Y Z CheA and CheW are membrane associated and the other Che proteins are in the cytoplasm MCPs are in contact with CheA and CheW CheA is a sensor kinase phosphorylates itself in response to an external signal and passes the PO4 to a response regulator CheW is a coupling protein assists CheA CheA is a sensor kinase When a MCP binds an attractant the rate of phosphorylation of CheA decreases When a MCP binds a repellant the rate of phosphorylation of CheA increases CheAP phosphorylates CheY a response regulator CheYP directly interacts with the agellum not CheY CheYP moves to the agellar motor and changes the direction of rotation to CW cell tumbles CheAP also phosphorylates CheB a second response regulator removes methyl groups but CheR continually adding at the same time 9 level of methylation adaptation of environment Phosphorylation rate is slower than phosphorylation of CheY CheBP removes methyl groups from MCPs and CheR continually adds methyl groups to MCPs The level of methylation of MCPs controls the ability of the cell to respond to attractants and repellants allows adaptation CheZ removes P04 groups from CheYP Responding to an attractant With a high level of attractant the rate of CheA 9 CheAP is low so low CheYP agella turn CCW and low CheB P The level of methylation of MCPs increases since CheBP is not there to demethylate CheR continues to methylate MCPs can get full methylation of the MCPs Adaptation to attractants MCPs no longer respond to attractants when fully methylated so the rate of CheA 9 CheAP increases CheYP is formed more rapidly and cells tumble only changes to clockwise CheBP formed more rapidly demethylates MCPs When the MCPs are no longer fully methylated they can again respond to attractants CheA 9 CheAP decreases 9 once fully methylated will not react to attracts 9 will increase rate and tumble 9 then demethylation of MCPs so they respond again because fully methylated don t respond Adaptation to repellants Fully methylated MCPs do not respond to attractants do respond to repellants CheA 9 CheAP rate increases CheYP is formed more rapidly and cells tumble CheBP formed more rapidly demethylates MCPs Once more cells respond best to attractants Note substances that are attractants are not necessarily usable for metabolism by chemotactic bacteria This system of cytoplasmic Che proteins also controls phototactic and aerotactic movement to oxygen responses of some bacteria sensors are different instead of MCPs phototactic bacteria have a light sensor protein and aerotactic bacteria an Eh sensor protein 9 they don t only respond to substances that they can metabolize 9 overall picture of how it works signals transduced and how its regulated Quorum sensing is another type of bacterial signaling system Chemotaxis detection and directed movement to or away from a chemical in the environment Quorum sensing up or down regulation of physiological or genetic processes based on detection of a signal produced by bacteria 9 not from environment like chemotaxis but from signals they product themselves 9 only works with mass where signal accumulates dense population of quorum sensing bacteria more outside that inside concentration gradient therefore move back into membrane 9 when they diffuse back in their produce a response Called quorum sensing QS because it only occurs when a population of bacteria is dense enough a quorum for the signal being produced to accumulate QS molecules are continually produced but diffuse out of the cell in a dense population as QS molecules accumulate in the environment they can diffuse back into the cell elicits a response QS is widespread in Gram negative bacteria also found in some Gram positive bacteria Each QS species produces a speci c QS signal autoinducer but different species can produce the same molecule the signal Are different types many Gram negative bacteria produce AHLs acyl homoserine lactones that are species specific and AI2 autoinducer2 that is produced and sensed by many Gram negatives universal AHL Species Specific AI2 universal Gram positive bacteria produce small peptides Acyl homoserine lactone structure 9 don t worry about structures General structure of AHL R can be 1 to 17 carbons AHLs are designated as C4 to C18 can have an additional 0 or OH group by R Examples C8HSL 3OHC4HSL 3oxoC12HSL Autoinducer 2 structure note boron 9 must have a lot of boron in the bacteria to use it General mechanism of quorum sensing 9 continually producing producing molecules and releasing them into the environment and turn on or off genes bind to activator that interacts w chromosomes 9 diffuse into lower conc Overall QS bacteria continually produce QS signals which diffuse out of the cell As the population of QS bacteria increases AHLs or AI2s accumulate diffuse back into the cell In the cell QS molecule will bind to an activator protein which interacts turns on or off specific genes Model of QS with AHLs Many properties are turned on or off via QS Biofilm formation production of different virulence factors toxins etc antibiotic synthesis bioluminescence sporulation etc 9 know examples of what can be used on and off First discovered in bioluminescent bacteria bacteria that produce light Mechanism worked out at the genetic and biochemical levels Very interesting symbioses between animals and bioluminescent bacteria Ex Hawaiian bobtail squid light organ contains dense populations of Vibrio scherii organ is evacuated every morning recolonized by cells in the environment don t need light during the day and then cells are slowly grow and accumulate by the time it gets dark autoinduction occurs to produce light when a quorum is reached due to population density increase in the organ throughout the day light is turned on by dusk and on all night MicroBiology Cumulative Topics 9 10 questions 1 Main source of Energy in cell ATP Reducing Powder PMF NADH H 2 E Source for microorganism Energy Growth with light as the energy source is phototrophy Growth with chemicals as the energy source is chemotrophy Carbon source Growth with CO2 as the carbon source is autotrophy Growth with reduced organic carbon as the energy source is heterotrophy or organotrophy Reduced organic carbon can serve as both the carbon and energy source 9 Anything that its carbon dioxide is reduced In addition a chemical energy source can be organic carbon organo or inorganic litho iron or sulfur Chemoorganotroph energy from organic carbon Chemolithotroph energy from an inorganic substance Can be further combined Chemolithoautotroph Chemoorganoheterotroph 3 Bacteria 9 species vs genes 16S rRNA 975 cannot only reply on the percent of similarity line blurred with horizontal gene transfer common basis to define species Human gut microbiota have identified 3500 species if use a 99 16S rRNA gene sequence homology to define specles 35000 species if use 97 homology the cut off for environmental species phenotypic lt genotypic 16sRNA databases not accurate no quantitative definition consensus based on data microheterogeneity in 16sRNA is common 0 ecological diversity 0 horizontal gene transfer 9 bacteria antibiotic resistance nonphylogenic interrupter 4 Metabolic Processes Determined by environment ability The three most common energy yielding metabolic pathways Fermentation Aerobic respiration Oxygenic photosynthesis Carried out by eukaryotes and prokaryotes Respiration and photosynthesis involve electron transport fermentation does not Cells have two major sources of energy that are produced in energyyielding metabolism Reducing power NADH H and ATP Both are present as pools in the cytoplasm NADH H and NAD AMP ADP and ATP Will see how they are generated in fermentation and aerobic respiration Fermentation Fermentation is a common anaerobic energy yielding metabolic pathway Fermentation is not membrane associated it occurs in the cytoplasm The energy source is some form of reduced organic carbon that is partially oxidized no full to oxidation to CO2 Fermentation NO external equot acceptor NO equot transport chain NOT membrane associated Requires anaerobic conditions ATP is formed enzymatically by transfer of PO4 from phosphorylated compounds in the fermentation pathway to ADP in the cytoplasmic pool called substrate level phosphorylation In fermentation the partial oxidation of the energy source organic carbon is balanced by reduction reactions that lead to byproducts that are reduced organic compounds Overall fermentation is an internally balanced redox reaction Only a small amount of the potential E is released First NADH very important in fermentation When looked at as a redox couple what can you say about it Why is this an energy source The reduced form of NADH is correctly written as NADH H because 2 e s are required in the redox reaction reduction of NAD Overall reduction NAD 2H 9 NADH H Fermentation of one glucose to two pyruvate is called glycolysis Fermentation byproducts are produced as a result of the required oxidation of NADH Examples include EtOH lactic acid H2 CO2 and others There is no overall change in oxidized vs reduced NAD in the NADNADH H pool after fermentation of a substrate Fermentation yields a small amount of energy compared to aerobic respiration Aerobic Respiration Aerobic respiration is a membrane associated energy yielding metabolic pathway The energy source is an electron donor which is usually an organic carbon compound The electron is transferred to a terminal equot acceptor which is 02 aerobic As opposed to fermentation in respiration all of the organic carbon energy source can be used all possible e39s are donated and the energy source can be fully oxidized to CO2 In aerobic respiration pmf is generated the pmf is then used to produce ATP next lecture As electrons move along a respiratory equot transport pathway protons are moved across the membrane The equot transport pathway is organized in the membrane and carries out equot transport The organic carbon energy source where the electrons come from is located in the cytoplasm Electrons are often donated to the respiratory electron transport chain via NADH H or another cytoplasmic electron carrier such as FADH2 Electron transport and metabolism Consists of a series of oxidationreduction steps Electrons are passed along in a chain of donors and acceptors electron transport chain etc The donors and acceptors are held in place in the membrane by what Electron chains Electron chains consist of An initial or primary equot donor A final or terminal equot acceptor Intermediate electron carriers in between the primary donor and the terminal electron acceptor Energy released difference in Eo between the primary equot donor and the terminal equot acceptor The most important e acceptor in biological processes is 02 Can calculate energy with different donors and acceptors how Electron carriers There are two general classes of intermediate electron carriers held in place in the cell membrane several types freely diffusible in the cytoplasm one type NADPH H Are ve types of membraneassociated electron carriers NADH dehydrogenases Flavoproteins Cytochromes Nonheme ironsulfur proteins Quinones All are held firmly in place except quinones these are freely diffusible in the membrane The one cytoplasmic electron carrier is NADPNADPH H Eo 032 V good electron donor reducing power Acts as a coenzyme facilitates chemical reactions in the cell that wouldn t normally happen ex movement of e39s between the cytoplasm and membrane bound equot transport chains Are two categories of electron carriers Electron carriers only accept and donate electrons Hydrogen carriers have to take a proton along with an electron note are a type of electron carrier Hydrogen carriers NADH NADH dehydrogenase avoproteins quinones Electron carriers cytochromes nonheme ironsulfur proteins Example of an electron carrier Cytochrome electrons are associated with Fe present at either FeH or FeH Another electron carrier Nonheme iron sulfur protein Example of a hydrogen carrier Flavin mononucleotide FMN a avoprotein NADH is also a hydrogen carrier Protons are moved across membrane based on the type of equot carrier the type of carrier it is passing the equot to and where the equot came from If a hydrogen carrier passes an electron to another hydrogen carrier hydrogen is passed if it passes to an electron carrier only the electron is passed and the proton is discarded usually to one side of the membrane to increase pmf If an electron carrier passes an electron to another electron carrier the electron is passed if it passes to a hydrogen carrier a proton must be picked up usually enhances pmf where does the proton come from Respiratory electron transport chain Substrate 9 NADH H 9 NADH dehydrogenase 9 avoprotein 9 nonheme iron FeS protein 9 quinone cycle 9 cytochromes 9 02 What are the relative Eo values of each of these What is the substrate Protons are moved across the membrane when an equot is transferred from an electron carrier to a hydrogen carrier or from a hydrogen carrier to an equot carrier organized so protons are picked up from the cytoplasm and released to the outside Pmf is also produced by protons being consumed in reactions in the cytoplasm Final step When the equot reaches 02 Hrs in the cytoplasm are consumed 4 are required 2 to form H20 2 needed to transfer the 2 es from cytag so is a proton consuming reaction in the cytoplasm contributes to pmf The energy used to produce pmf difference between the equot donor and equot acceptor Ex glucoseCO2 043 volts 12 O2H20 082 volts Difference 125 volts How does NADH H get from the energy source to the etc Different paths For glucose can be same pathway to pyruvate as fermentation Are additional paths such as the citric acid cycle Chemolithotrophy and anaerobic respiration Lithotrophy Energy is from the oxidation of inorganic electron donors Is another ETSassociated energy yielding metabolic pathway in which the energy source is chemical Different from anaerobic respiration for which the electron donor is an organic carbon compound In lithotrophy the terminal electron acceptor can be organic or inorganic Once again these metabolic modes are based on E0 and the difference between the electron donor and the terminal electron acceptor Which pair has the most potential energy available Electron donors that support lithotrophy Are many the most common are hydrogen various reduced sulfur and iron compounds and metals Each has a different amount of potential energy depending on its Eo Energy yield also depends on the of the Note that both the energy source and the terminal electron acceptor are shown to calculate energy yield 9 knowing the electron donors and acceptors For lithotrophs the carbon source is often CO2 but some can use organic carbon Each type of lithotrophy is associated with an ETS sometimes overlap with other ETS metabolic pathways The initial electronaccepting oxidoreductase is always Membraneintegrated specific for the electron donor can look for these genes in 39 Vd39 9 quot35 Emgron tra sport genomes to assess metabolic potential 2 Hydrogen oxidation Molecular hydrogen H2 is the electron donor E0 of the redox couple 2HH2 is 042 V This is low enough to donate an equot to nearly all biological electron acceptors including NAD This means many compounds can be used as the terminal electron acceptor Examples of hydrogen oxidation pathways NAD 2 I J co ATP ADP ETS There can be both membrane bound and cytoplasmic hydrogenases associated with hydrogen lithotrophy ATP is produced by the membrane bound type and NADH H by the cytoplasmic type 9 recognize H S Fe nitrate lithotropy 9 will have one of them Figure 1320 Bioenergetics and function of the two hydrogenases of aerobic hydrogen bacteria 9 Will ask which is the electron donor and acceptor check arrows 9 Ends with ace splitting something Most hydrogen bacteria are facultative chemolithoautotrophs They prefer to grow chemolithoheterotrophically but when no organic carbon is available they grow chemlithoautotrophically 6 H2 2 02 l C02 9 T 5 H20 Are microaerophilic growing best at 510 02 This is because their membrane bound hydrogenase is extremely sensitive to oxygen When considering compounds other than hydrogen that are electron donors for lithotrophy first look at the entire spectrum of oxidationreduction reactions for that compound most oxidized to most reduced or vice versa Sulfur lithotrophy Oxidationreduction pathway H2S gt s gt 12 S2O32 gt sof gt so4239 Hydrogen sulfide elemental sulfur thiosulfate sulfite sulfate The first three are normally used as an energy source for lithotrophy Usually O2 is the terminal electron acceptor but some can use nitrate NO339 As we ve seen before if sulfide is oxidized to S it is deposited intra or intercellularly Sulfur lithotrophy and production of reducing power The Eo ofNADNADH H is 032 V For S0HZS it is 029 V For s4o62 s2o32 it is 024 V This means sulfur lithotrophs need to use reverse electron transport powered by the pmf to reduce Hilt mm tun gtlt vt NAD A inno11prntim t 2 I I HM I39i gtlt y 9 9 9 9 v 9 i i 9 neither is negative enough to reduce NAD 39 39O39 39 therefore forces electron to go to something that s S S S S S S S S S S S S S S S S S S S S S S S S more electronegative pmf powered Out i 3 l 3 3 2 3 tzxzt W HS SO 239 S2032 0 0 Note that the three electron donors all of which are 2 common in the environment come in at two different 02 quot Nquot 504 points in the pathway quotADquotjLj Cequot Q material 11 izi Which is the best electron donor Most reduced sulfide elemental sulfur This is why elemental sulfur is deposited inon the cell Sulfur lithotrophy 9 NAD should not be in the membrane but outside of it illiiiiil it Iron oxidation Oxidationreduction pathway Fe2 gt Fe3 Ferrous iron ferric iron The ferrous form is favored by anoxia and low pH the ferric form by oxygen presence and high pH So iron oxidizers must live in very acidic environments with oxygen present Growth on iron occurs with this reaction 2 Fe 202 2 H 2 Fe3quot H20 Eo 12 O2H20 is 082 V and of Fe2Fe3 is 077 V So there is a very short ETS and reverse electron transport is needed to reduce NAD ETS includes a periplasmic enzyme called rusticyanin that removes the electron from Fe2 The pH of the environment of iron oxidizers is usually 12 pH units Since the cytoplasmic pH is 55 to 60 iron oxidizers can use this natural gradient for pmf Problem ATPase allows protons back into the cytoplasm making it acidic bad Problem solved by using these protons plus oxygen and the electrons from iron to make water Growth by iron oxidation does not yield much energy why 9 its very small So LOTS of iron has to be oxidized precipitates in the environment Nitrogen oxidizers xidation states of inorganic nitrogen Nnj gt NH2 H gt HN 2 gt N 2 gt HN 3 gt N 3 Ammoninm most redneed hydroxyiamine nitrons aeid nitrite nitrie aeid nitrate most oxidized Whieh is most redneed Whieh is most oxidized Whieh is hest for iithotrophy There are three main gronps of iithotrophie haeteria that nse nitrogen eomponnds Ammoniaoxidizing bacteria generates a proton 5 gmotivc force 4 Oxidation of 2 hydroxylamine I Electron transport N Oxidize ammonia The product is nitrite The nitrite produced is the electron donor energy source for the second group 2 Nitriteoxidizing bacteria Oxidize nitrite The product is nitrate In anaerobic environments nitrate is an important terminal electron acceptor for anaerobic respiration 0 39 O V 1 Pm 7 hl 8 Reverse e flow n 8 In ma a MADI Electron transport i generates a proton motive force Nitrite oxidation NXR nitrite oxidoreductase Figure 1327 Oxidation of NO2 to NO3 by nitrifying bacteria d The first two groups utilize oxygen and bacteria that can do K 3 this are obligate aerobes 0 Reverse e Mquot But there is also ammonia oxidation in anoxic environments the basis of the third group 3 Anoxic ammonia oxidizing bacteria Anoxic ammonia oxidization cmammox Ammonia is the electron donor Nitrite is the electron acceptor The product is N2 NH4 N02quot gt N2 2H2O39 The anammox reactions occur in cmammoxosomes which are membrane bound like organelles of Eukaryotes and take up about half of the cell Anammoxosomes do not have normal cytoplasm They contain no ribosomes The membranes are not phospholipids but are fatty acids with cyclobutane rings bound to glycerol with ester and ether bonds Are called laddercme lipids and are very dense to the point that nothing can diffuse across them This is necessary since one intermediate in the reaction is hydrazine NZH4 extremely toxic extremely strong reductant The three groups of nitrogen oxidizing bacteria live in close association with one another Why 9 the enviro has what is required to grow if you have one that produces one substrate then its enriching for the other What characteristics will this kind of environment have to have 9 have a gradient of oxygen and no oxygen Anaerobic Respiration Respiration with a terminal electron acceptor other than oxygen The electron acceptor is reduced and the end product is excreted to the environment a byproduct If the acceptor is nitrate or sulfate both are nutrients the process is called dissimilatory nitrate reduction or dissimilatory sulfate reduction If energy is used to reduce nitrate or sulfate to be used by the cell as a nutrient it is assimilatory nitrate or sulfate reduction Maj or forms of anaerobic respiration showing the terminal electron acceptor and the reduced byproduct Where is the electron donor The electron donor and electron acceptor used is based on 1 the availability in the environment 2 the energy yield The presence of a better terminal electron acceptor usually suppresses synthesis gene expression level of reductases for alternate electron acceptors ex oxygen represses genes for nitrate reductase nitrate represses genes for fumarate reductase Nitrate respiration Nitrate N03quot First consider the oxidized forms of nitrogen quotmt mducme quotmate reduction No gt N02quot gt NO gt 12 N20 gt 12 N2 Ef quot quotquot NItnte 39 N1trate n1tr1te n1tr1c oxrde n1trous oxrde nrtrogen gas Nitrite reductase Resprratolry reductron occurs successrvely 1n steps 1 39 beauti cation In genera any grven bacteria spec1es can carry out on y Nitric oxide NO pseudomonas one or two transformatrons 1n the ser1es lmmc oxide mductase smzen Each step is carried out by a specific enzyme Gases Nitrous oxide I ll J 1 A A Nitrate Reduction and Denitrification Biochemical pathway for dissimilative nitrate reduction has been well studied Enzymes of the pathway are repressed by oxygen Often facultative anaerobic respirers directly use part of the aerobic respiration pathway Figure 1341 Respiration and nitratebased anaerobic respiration Aerobic and anaerobic nitrate respiration Ec0lz39 Comparison of pathways These paths share which part of the ETS Where is the branch What determines which branch will be used Oxygen aerobic oxygen and nitrate aerobic nitrate anerobic Nitrate respiration this pathway involves one reduction nitrate to nitrite Some bacteria denitrifiers can transfer electrons to all of the oxidized forms of nitrogen resulting in N2 as the byproduct The additional steps generate more pmf Complete path of dissimilatory nitrate reduction 9 NOT IN THE EXAM Dissimilatory nitrate reduction is specialized among a few prokaryotes energy YIELDING form Assimilatory nitrate reduction is common carried out by prokaryotes and eukaryotes Is enzymatic and energy consuming Is the reduction of nitrate for use assimilation as a nutrient 9 pathways that overlap and the differences Sulfur respiration Sulfate is also reduced step by step in anaerobic respiration Oxidized to reduced forms of sulfur Most reduced to most oxidized lithotrophy Most oxidized to most reduced Anaerobic respiration SO42quot gt SO32quot gt 12 S2032 gt s gt HZS Sulfate sulfite thiosulfate elemental sulfur hydrogen sulfide ADP ATUPi 3 Sulfate respiration is very common in marine environments SO42 PAPS APS there is lots of sulfate in seawater ATP sumuyhse APS kmase Dissimilatory reduction of sulfate first requires activation by figsuctase ATP because SO42quot is too stable AMp pAp forms the compound adenosine phosphosulfate APS what does this remind you of 9 first step of 503239 503239 fermentation of glucose First step activation ATP Sulfite For assimilatory sulfate reduction a second phosphorylation reductase of APS occurs to form PAPS phosphoadenosine 1 1 phosphosulfate same activation step I I second phosphorylation occurs too 9 PAPS 325 325 Excietion Organic sullfur compounds cvsteine methionine Figure 1414 Biochemistry of sulfate reduction Activated sulfate The first part of the assimilatory and dissimilatory sulfate reduction pathways are shared Then the pathways branch These pathways are in the cytoplasm For dissimilatory sulfate reduction electron donors must be more electronegative than 017 volts sulfatesulfide 9 donor for sulfate S03 2 9 must be above it on the table more EN 5 Role of Microorganisms in biogeochemistrv 9 transforming Chemical to Energy 6 E tower 9 show energy ow of electrons E sources reducing power Often written on a vertical scale the electron tower Y axis is in volts values referenced to H2 at pH 70 most negative at the top positive at the bottom Top of the tower greatest amount of potential energy donors want to donate equot shave the greatest tendency to donate e39s Bottom of the tower greatest possible energy is released if the acceptor is here Why As es move from an acceptor to a donor the energy released available for metabolism is the difference in volts between the donor and acceptor Have to consider each substance as an electron couple the oxidized and reduced forms A substance can only be either oxidized or reduced at any one time ex cyt b med 7 Peptidoglycan Whatamp Where Cell walls of Bacteria All bacteria divided into two groups Gram and Gram Based on major difference in bacterial cell walls Originally discovered based on staining procedure developed by Gram need to stain to see small cells one type retains the stain the other doesn t Difference is due to the amount of peptidoglycan Bacterial cell wall rigidity is due to peptidoglycan Found only in Bacteria used to tell if Archaea or Bacteria Composed of two sugar derivatives nacetylglucosamine and nacetylmuramic acid These alternate to form the peptidoglycan backbone Gram negative are multilayered the most complex outside of cell membrane is a thin peptidoglycan layer inside a periplasmic space has a second outer membrane lipopolysaccharide layer Gram positive simpler and thicker outside of cell membrane is a thick layer of peptidoglycan Gram cells 90 of wall is peptidoglycan Gram cells only 520 usually l0 of wall is peptidoglycan Gram cells have a periplasmic space and LPS Gram cells have teichoic acids and different crosslinkages Gram negative bacteria the periplasmic space Between the cytoplasmic membrane and LPS layer is a space Peptidoglycan is here but doesn t take up the whole space Protons of the pmf concentrated here Gram positive bacterial cell wall THICK peptidoglycan layer 9 Know organization and functions On cell surgace Gram POS bumpy with polysacc sticking out The gram stain and bacterial walls Differences in bacterial cells walls are the basis of the Gram stain which in turn is the basis for the names Gram cells stain purple Gram cells stain pink Based on the thickness of the peptidoglycan layer 8 Horizontal gene transfer 9 importance transfer gene in pathogen In uences microbial populations The species concept in microbiology Big question what is a prokaryotic species prokaryotes are haploid no sexual reproduction but are lots of ways to get new DNA from other sources horizontal gene transfer 9 Why microbiology and microorganisms are good tools for biology Gene vectors plasmids amp phages Microbes are useful tools in research because of their rapid life cycle their simple growth requirements and their small size Due to this simplicity microbes have been essential in understanding core questions in biology Attempts to classify microorganisms have lead to a classification system that divides all organisms into three domains of life Archaea Bacteria and Eukarya Microbes provide tools for use in molecular biology These tools have allowed scientists to make rapid progress in investigating many types of microorganisms Plasmids as cloning vectors Plasmids are natural vectors useful properties as cloning vectors small size easy to isolate plasmid DNA independent origin of replication for control multiple copy number can get multiple copies of cloned gene per cell often have selectable markers can detect Insertion of the plasmid by chemical transformation or electroporation Plasmid pUC19 pUCl9 is a common cloning vector Is a modi ed naturally occuring plasmid Modifications ampicillin resistance and lacZ genes ampicillin used as a marker to tell your plasmid has been taken up lacZ a marker for DNA insertion has a polylinker multiple cloning site lots of restriction sites for gene insertion within the lacZ gene 9 you want to get the plasmid in BUT also make sure that gene is in there and be able to identify it To see if you have inserted the plasmid do antibiotic screening ampicillin resistance marker plate onto plates with ampicillin colonies that grow have the plasmid To see if you have inserted the new gene into LacZ do LacZ screening marker is blue vs white colonies LacZ gene codes for 3galactosidase presence intact LacZ detected if plated onto media with Xgal Xgal a colorless reagent that turns blue with 3galactosidase activity intact LacZ gene 9 We want to see if 1 Have plasmid in prokaryotic cell 9 antibiotic screening if colonies grow they have plasmid 2 Plasmid with new gene 9 LacZ screening using restriction sites in LacZ gene cleave and reanneal blue or white Blue colonies do not have new DNA inserted LacZ is intact White colonies do have new DNA inserted into LacZ so Bgalactosidase expressed Based on insertional inactivation of new DNA the M02 gene is inactivated by insertion of new DNA inactivated lacZ cannot target Xgal blue color does not develop Hosts for cloning vectors 9 host has to have speci c properties Properties of good hosts capable of rapid growth in inexpensive medium doubling time like 20 mins nonpathogenic capable of incorporating DNA when moving the genes genetically stable in culture nonmutating Can replicate the vector for plasmid to make more plasmid increasing copy of gene prod Methods in Microbial Ecology In microbial ecology usually want to study microorganisms from nature in the lab requires enrichment Enrichment is a process Goal is isolation separation of individual organisms from the mixed community Requires use of enrichment cultures that select for desired organisms manipulation of medium and incubation conditions The environmental sample you use to isolate microorganisms is the inoculum Enrichment example the Winogradsky column Create an artificial microbial ecosystem Named for Sergei Winogradsky used in late 19th century to study soil microorganisms Mostprobablenumber technique Serial 10x dilutions of inoculum in a liquid medium Used to estimate number of microorganisms in food wastewater and other samples Cultureindependent genetic analyses of microbial communities Several common approaches PCR community structure often targets the gene for 16S rRNA Microarrays phylogenetic and functional diversity 0 Environmental genomics and other omics Environmental Genomics Is called metagenomics DNA is cloned from microbial community and sequenced Detects as many genes as possible Yields picture of gene pool in environment Can detect genes that are not amplified by current PCR primers Can assessing phylogenetic diverstiy and metabolic potential Other omics Metatranscriptomics analyzes community RNA detects expressed genes in a community that are actually expressed reveals level of gene expression Metaproteomics measures diversity and abundance of different proteins in a community Measuring microbial activities in the environment Many approaches and techniques Chemical assays measure 02 for oxygenic photosynthesis sulfide for sulfate reduction etc Radioisotopes very sensitive expose population to the isotope kill sample measure radioactivity present incorporated with a scintillation counter Microsensors small electrodes that directly measure chemicals Microsensors Used to measure concentrations and activity pH oxygen CO2 and others can be measured are very small glass fragile electrodes electrodes are inserted into the habitat microbial mats biofilms etc can take measurements in 100 um increments can get profiles of the chemical within one mm scales for resolutions Microsensor tips not what they look like 9 O2 amp N02 Microsensor profiles be able to determine the graphs depths on left concentration below Microbial Ecosystems Read sections 191195 198 199 1912 Microbial ecology concerns two major factors that must be considered The environment habitat where the microbial population is living Microbial interactions within that habitat Environment already considered many times nutrients environmental factors such as salinity pH 02 etc Microbial interactions are several Can be positive negative neutral 00 or mixed eg 0 9 interested in specific terms how bacteria are interacting with each other Microbial interactions Positive interactions mutualism symbiosis both organisms require the other to live synergism both organisms enhance growth of the other but can live alont syntrophy both organisms need byproducts the other produces or consumes to grow Negative interactions competition both organisms compete for the same resource can be nutrients light etc parasitism one organism grows on or in at the expense of the other predation one organism preys on consumes the other Mixed and other interactions commensalism one organism benefits no effect on the other amensalism one organism is harmed no effect on the other neutral no effect of either one on the other Often are grey areas between types 9 often not completely clear cut Two case studies to illustrate the roles of environment and microbial interactions in microbial ecology 9 try to identify what kind of interactions are occurring between the organisms 9 will give 2 organisms and what they are doing and be able to identify their relationship 9 no specific biology of the case studies 0 Dawn to daylight arrows show Oscillatoria and Synechococcus populations o Dusk to darkness arrows show Oscillatoria and Synechococcus populations TEMs showing glycogen reserves o With 02 in darkness aerobic respiration but use up all stored glycogen in 810 hours can t survive the night o Ecological physiology switch to fermentation when anoxic conditions AND negative Eh Laboratory studies continually gliding filaments stop when they encounter sulfide negative chemokinesis Summary vertical migrations of Oscillatoria Migrate up at dawn due to positive phototaxis Clumps spreads out through the day based on light intensity combination of positive phototaxis and stepup photophobic response At night no longer a light cue Oscillatoria forms a layer below the oxygen sulfide interface In darkness switches to fermentation when in an environment with a negative oxidation reduction potential Potamocypris the ostracod can run up quickly to 48 C white dots highest temperature of an animal Feeds on filamentous cyanobacteria 0sciIIatoria Upper temperature limit of ostracods delineates arrow the population of microorganisms that can be eaten even though their lower temperature limit extends into ostracod territory Only thing present below this temperature threshold are PleurocapsaCalothrix nodules Two cyanobacteria Pleurocapsa and Calothrix are present in nodules filamentous Calothrix inside rubbery tough Pleurocapsa nodules Ostracod can t eat the nodules Black band disease BBD of corals The first reported coral disease 1970s Affects reefframework species of scleractinian corals Has spread since the 1970s to a worldwide distribution Does not open up new substrate for reef building corals Proposed BBD pathogens 19 70s Cyanobacterium Oscillatoria submembranaceae Antonius 1973 Sulfideoxider Beggiatoa or sulfate reducer Desulfovibrio Garrett and Ducklow 1975 Both of the above required Ducklow and Mitchell 1979 Marine fungus RamosFlores 1983 Renamed cyanobacterium Phormidium corallyticum Rutzler and Santavy 1983 May be no primary pathogen BBD may be a pathogenic microbial consortium Richardson et al 1997 Heterotrophic bacteria based on molecular analyses PCR based of the BBD community Cooney et al 2002 FriasLopez et al 2002 2004 Sekar et al 2006 found 9199 16S rRNA gene sequence homologies to Cytophaga sp and a uvenile Oyster Disease associated bacterium 2010s isolation of Roseo lum reptotaenium a cyanobacterium that causes BBD infection of corals in the lab but can t grow in axenic culture so what can t be Fulfilled Characteristic microorganisms make up the black band disease microbial consortium Beggiatoa spp cyanobacteria BBD functional groups Physiology Phototrophs cyanobacteria photoautotrohy photoheterotrophy mixotrophy Sulfate reducers Desulfovibrio and others Sulfide oxidizers Beggiatoa spp Heterotrophs various aerobic and anaerobic BBD Toxicity Sulfate reducers sulfidogenesis sulfide is toxic Cyanobacteria many produce cyanotoxins by cyanobacteria Heterotrophs various candidates and toxin producers Viruses 9 no one has researched BBD dynamics the entire community migrates horizontally across coral skeletons Use of microelectrodes to measure BBD chemical microenvironments The base of BBD is both anoxic and sulfide rich Exposure to sulfide anoxia kills coral tissue Thus BBD sulfate reducers Desulfovibrio spp and others contribute to toxicity Manipulataive studies of BBD using metabolic inhibitors of oxygenic photosynthesis DCMU and sulfate reduction Na molybate O2 electrodes used for confirmation of inhibition Summary Now we know that Cyanobacterial attachment is the first step of infection Leads to anoxic microenvironments that select for sulfate reducers Sulfide is required for attachment to progress to active infection The cyanotoxin microcystin is produced by BBD cyanobacteria and acts synergistically with sulfide BBD is a polymicrobial disease research in progress Toxins pathogens are acting together synergism Biogeochemical cycles Carbon cycle and nitrogen fixation Biogeochemical cycles or nutrient cycles consist of movement cycling of individual elements between biotic and abiotic chemical and geological reservoirs Microbes carry out many if not most of the transformations Will go over several of these important cycles Fermentation The byproducts are then fully metabolized to CO2 or CH4 Nitrogen Fixation 4 PVquotquotquot COA 4 A etyquotC A 4 co 0 Is an extremely important process E 2 X4 339c tquot39 1es quotate5 39 F o Nltrogen 1s major nutrlent often l1m1t1ng ectrofns e l avodoxin Reduced to ammon1a level reduc1ng or 4 Havodoxin 4 navodoxin nltrogenase o Ma1n reservo1r1s N2 atmosphere Ox i Red 0 Fixation is conversion of N2 to NH3 quot Flavodoxin reduces o Is very expensive metabolically and only a l dinitrogenase few prokaryotes can do this no eukaryotes X 39 d t35e39 4 Dinitrogenase 4 Dinitrogenase reductase reductase Very 1nterest1ng ecologlcally because the enzyme Red 4 ox complex that carries out N2 fixation nitrogenase is 16 ATP Electmns transferred ta irreversibly inactivated by oxygen dinitrogenase one at a A t t 39 t t t t se from D quotquot9 1 ATP 3399 re many s ra eg1es 0 pro ec n1 rogena mtmgenased 15ADp consumed per electron oxygen activity P 16 P g H pg Nitrogen ufixersquot I are varied and the process is Dinitmgenas quotDinitrogenase known to be carr1ed out at a temperature range of ox p Reg below 0 up to 92 C and a pH range of2 to 10 F I quot l Nitrogenase 2 quotH3 N2 8 9 don39t worry about table H2 9 know how man electrons 8 H and 16 ATP 2 e s 4 H 2 H 2 fl y Sum Nilljv HNNH H2N NH2 2Nl but given off as H2 in diagram 16 ATP D 16ADP 16 P Nitrogenase is composed of two proteins bl dinitrogenase splits triple bond dinitrogenase reductase reduces N2 Dinitrogenase reductase transfers donates the electrons and dinitrogenase splits the N2 molecule Electron transfer path Electron donor e dinitrogenase reductase e dinitrogenase a N2 Ammonia is the final product Nitrogen fixation is extremely expensive in terms of both types of cellular energy what are they The reduction has 3 steps with no free nonbound intermediates 8 electrons consumed only 6 reduce N2 Both proteins of nitrogenase contain iron and dinitrogenase also contains molybdenum The actual reduction occurs at a cofactor site shown here within the complex 9 reduction occurs at cofactor site micronutrient 9 don39t worry about structure Are different strategies to protect nitrogenase specifically dinitrogenase reductase from oxygen Anaerobes don39t have to protect it Freeliving aerobes do rapidly respire to remove oxygen when it comes into the cell production of slime layers impermeable to oxygen Azotobacter vinelandii grown under microaerobic 25 left and aerobic 21 right conditions arrow shows slime layer slows down diffusion of oxygen into the cell Strategies of freeliving aerobes cont production of anaerobic microenvironments diel nitrogen fixation fixation at night specialized cells with no oxygen conformational protection symbiotic relationships T richodesmium a marine cyanobacterium The center of these akes is anaerobic these filaments do not photosynthesize Laminated microbial mats can have vertically migrating oxicanoxic interfaces Nitrogen fixation can occur when that part of the mat is anoxic Some cyanobacteria produce specialized cells called heterocysts where nitrogen fixation occurs only found in SOME cyanobacteria Heterocysts Are produced only by some cyanobacteria NO photosystem II 9 no 02 production required Photosystem 1 kept cyclic photophosphorylation to make ATP for the process have thick walls to prevent oxygen from diffusing in have polar plugs between adjacent cells that allow fixed nitrogen incorporated into glutamine out and organic carbon from adjacent vegetatiVe cells in Image Heterocysts with polar plugs Visible 9 know WHY they allow Nitrogen fixation Heterocyst function 02 02 O2 02 02 02 9 Specialized cells for N fixation f T f f 9 why they are spread out over filament 0 h 9 quotquot N2 is reduced to NH3 which is incorporated 1 1 l 1 S into glutamine Iummine Passed to vegetative cells as glutamine 39 I I I Heterocyst I I I Vegetative cells Vegetative cells Often differentiate every 10 15 cells along a filament PatS is a protein synthesized in heterocysts Also moves to adjacent cells and inhibits expression of genes that code for heterocyst formation Conformational protection Some bacteria protect nitrogenase from oxygen by complexing it with a specialized protein when oxygen is present When oxygen is not present the protein is removed reversible Acetylene reduction assay Microbial ecologists often want to know if nitrogen fixation is occurring why Two methods detection of nif genes and acetylene reduction Nitrogenase also reduces acetylene to ethylene can be detected using a gas chromatograph Atmosphere 10 C H 2 in air aerobes or 10 C H 1 in N 1 or argon anaerobes Chart recorder for gas chromatograph 3939l H H H2 H2 39 C H C C 2 H C C Acetylene Ethylene C2 pl 2 2 can C2H2 I Nitrogenase C3 gt 9 Incubation Sample headspace time o A 1 h 2 h periodically and Genetics of nitrogen fixation Gene complex of nifgenes is organized into one regulon under common control Overall nif genes turned on with N starvation turned off when NH3 N03 some amino acids or 02 is present Model used is Klebsiella pneumoniae The nif regulon has 20 genes arranged in operons with genes for nitrogenase molecular structure the cofactor and regulators Biogeochemical Cycles Cont 9 Nitrogen and Sulfur The Nitrogen Cycle What are some energy yielding physiological processes that transform nitrogen 9 energy yielding process that transformed nitrogen dissimilatory nitrate reduction nitrate respiration nitrate lithotrophy 9 energy requiring processes and yielding identify FIGURE 207 know processes not organism know which are energy yielding and requiring and the energy transformations What are important energy requiring processes that transform nitrogen 9 assimilatory nitrate reduction nitrogen fixation Are additional processes involved Some of these processes we39ve seen under a different name Denitrification is also NITRATE RESPIRATION removing fixed nitrogen from system 9 back to N2 Nitrification is also NITROGEN LITHOTROPHY The anammox rx is also ANAEROBIC RESPIRATION But could be considered a form of New process ammonification Happens with decomposition of proteins etc amino group is released to the environment Note that this cycle is organized around an oxicanoxic interface Also note that this cycle is organized around the most oxidized NITRATE and the most reduced NH3 AMMONIA Nitrification N03quot 0 quotquot2 groups 39 Nitrogen 43 P9s a of protein quot 83903 No 1 9 xation 3 iTj 39 Oxic 3 T Anoxlc quotquot2 groups quot N02 of protein Nitrogen fixation Key Processes and Prokaryotes in the Nitrogen cycle Processes Example organisms Nitri cation NH gt N03 NHquot gt NO Nitrosomonas NO N03 Nitrobacter Denitrification NO gt N2 Bacillus Paracoccus Pseudomonas N2quotuxation Nn8Igt NH3 H2 Freeliving Aerobic Azotobacter Cyanobacteria Anaerobic Clostridlum purple and green phototrophic bacteria Methanobacterium Archaea symbiotic Rhizobium Bradyrhizobium Frankia Arnmonl cation organicN gt NIl Many organisms can do this Anammox NO NH3 2 N2 Brocadia 504 Know the processes and transformations Some nitrogen transformations are only aerobic some only anaerobic and some are found in both environments Examples good exam question 9 metabolic pathways depend on the presence of oxygen 9 Nitrification lithotrophy NH3 9 N2 anammox enzyme used to fix nitrogen nitrogenase irreversibly deactivated by oxygen BOTH ENVIROS nitrogen fixation ammonification assimilation Aerobic nitrification Anaerobic denitrification anammox Defences slime layer heterosis turn off PS2 thicker walls make protein complex to physically surround and protect nitrogenase complex Photosynthesis during the day 9 incompact communities diffusion cannot overcome the consumption of some substances The Sulfur Cycle What are some energy yielding physiological processes that transform sulfur What is an important energy reguiring process that transforms sulfur Again are more new for you processes involved We39ve seen the first 3 many of these already Desulfurylation is like what in the N cycle last 3 9 like deammonification Sulfur disproportionation This yields energy but a very low amount 219 k However it is very beneficial to be able to do this Why 9 product is strongly reduced sulfur and most oxidized form of sulfur most energy produced Organic sulfur metabolism An important source of sulfur as a nutrient why 9 catabolizing the organic carbon part of breakdown takes off sulfide group with byproduct 9 reduced sulfur compound released into cell and don39t have to use energy to reduce the sulfate Chemolithotrophic oxidation 50 SH groups of proteins Oxic Anoxic O SH groups 5 of proteins Key Processes and Prolcaryotes in the Sulfur cycle Process Organisms Sul delsulfur oxidation H28 s sof Aerobic Sulfur chemolithotrophs Thiobacillus Beggiatoa many others Anaerobic Purple and green phototrophic bacteria some chemolithotrophs sulfate reduction anaerobic SOquot gt Hzs Desulfovibria Desulfobacter Archaeoglobus Archaea Sulfur reduction anaerobic S gt H28 Desulfuromonas many hyperthennophilic Archaea Su ur d so3 gt H28 so Desulfovibrio and others Organic sulfur compound oxidation or reduction CH3SHgt C0 H28 DMSO gt DMS Many organisms can do this Deoulfurylltlon organicS gt H25 Many organisms can do this 9 energy yielding 9 energy producing Again some sulfur transformations are only aerobic some only anaerobic and some are found in both environments Examples more good exam questions 9 organized around oxicanoxic interface Chemolithotrophy oxygenated 02 terminal e acceptor Assilimations PAPS pathways assimilated as nutrient of sulfhydyl groups Desulfurilation deammonification releasedas product back to sulfide pool Disproportionation 9 missing thiosulfate split into sulfate and sulfide strongly reduced sulfide already available so oxygen not needed 9 is energy yielding just a small energy but get a great e reducer and acceptor 9 don39t worry about sulfate reduction Biogeochemical Cycles Fe Mn Hg and others The Iron Cycle Iron is one of the most abundant elements on Earth Already covered ferrous and ferric iron species ferrous iron most reduced can be an electron donor in LITHOTROPHY and ferric iron most oxidized can be a terminal electron acceptor in IRON RESPIRATION already a cycle There is another form F present as iron ore exposed during mining and enters the cycle The iron cycle is simpler than the S or N cycles but is similar in that oxicanoxic interfaces are extremely important 9 reduced iron favored by anoxic low pH FERROUS 9 oxidized iron favored by oxygenic high pH FERRIC Environments with oxicanoxic interfaces include those that are stratified which are common In these environments iron enters from ground water or leaching from the earth Stratified environments in microbial mats Also found in the surface layers of sediments 9 oxygen present in shallow and becomes anoxic creates an overlap 9 diffiusion inhibited by stratification read in book In the iron cycle chemical transformations in addition to biological transformations are extremely important since iron is so reactive Instantaneous oxidation of ferrous iron occurs under what conditions 9 aerobic and high pH super reactive precipitates with anything The newly oxidized ferric iron then precipitates with several other substances hydroxyl and sulfhydryl in particular to form FeOH3 and FeS2 if it precipitates to an anoxicacidic zone it will go back in solution become reduced 9 Can occur biologically microbially OR chemically 9 pH very important in iron BGC don39t worry about OH production don39t worry about metabolic process in second image 3 H20 FeOH3 3 H 9 ferric complexes as being very reactive Figure 209 igure 209 T Fe3 Ferrous iron F 39 Ferric iron ggtril a quot reduction 39 b ct i I h m39 339 lhaemlI r smelting Fe of ores Chemical oxidation Fez Ferrous Both oxiolatioh ahol reoloctioh of iroh occors hoth hiologically ahol cherhically Both cah he olirectly ihyolyeol Exarhple oxiolatiori of pyrite FeS2 Pyrite is yery corairhoh ih hattire fools golol Biologicalcherhical oxiolatioh of pyrite ihyolyes two electroh acceptors gt2 ahol Fettt oxidized iroh Wheri exposeol to air pyrite is cherhically oxiolizeol ih ah ihitiator reactioh which oxiolizes the 8 to Sm RX FCQS2 4 3 12 2 it lhl2 gt F it 2 S 4 it HT The pll is lowered hy this reactioh which is slow The Fett released is theh hacteriallr oxioliaeol lithotrophy to Fettt The hacterial hyprooloct Fettt reacts spohtaheotisly with more pyrite faster thah with Water gt stiper tihstahle therefore reacts spohtaheoosly The Fettt oxiolizes the pyrite to stiliiiric aciol the ehyirohirheht is hecorhihg aciolic which olissociates Rx FeS2 is 14 Fettt 8 lhl2 gtgt 15 Fett are 2 Sill i 16 hi it The olhl is lowered farther ahol agaih the Fett released is hacteriall oxiolizeol to Fettt 5 This processes repeats rhore ahol more rapidly ahol is called the propagatioh cycle Cherhicalrhicrohial pyrite oxiclatioh This cycle is associateol with aciol rhihe olraihage With rhtich of the aciol corhihg irorh iroh cyclihg lroh oxiolizihg rhicrohial that the Water is aciolic ahol the reololish color is precipitateol Fe lhl3 When bacteria grow by oxidizing iron the reaction occurs outside the cell and the oxidized iron precipitates arrows in figure This precipitate can form nanowiresquot that can move electrons between cells in a population can be 20 cm long are pili with cytochromies Nanowires can also be used as an energy source for other bacteria in the environment such as Geobacter Some microbes use living electrical cables to transfer electrons from sulfide to 02 at distances over 1 cm read page 631 The Mercury Cycle Mercury is in the environment mainly photochemical and due to use in industry other oxidations The Mercury Cycle lsrtr1x1c especially in the methylated quotgo I HQ2 c a gc a Elemental mercury Hg is volatile Al39 sphf39 o V oV goes into the atmosphere and is 31 V9 oxidized to Hg which dissolves in water Uptake by water vapor and can come down as rain Hg moves rapidly through the body t aquatic animals because it is excreted that is why it is 49 H92 c4lg cla gclla not very toxic l3ut methylated mercury accumulates V sediment I I in lipids and can b1omagn1fy increase in the food chain Hgwj Hg2quotj cHHgquotj CH3HgCl39l3 H25 J HgS CH 4 Hg Methylmercury can be a substrate for methanogens 9 methane and Hg Some sulfatereducing bacteria can precipitate Hg with sulfide to produce almost insoluble HgS which forms a reservoir in anoxic sediments When exposed to air it can be released oxidized to Hg and sulfate by S oxidizing bacteria The most toxic forms are methylmercury and dimethyl mercury CH3Hg and CH3HgCH3 Methylmercury is about 100 X as toxic as Hg or Hg and it is not excreted neurotoxin The methylation is carried out by bacteria in anaerobic environments Minimata disease and the Minimata story Industrial mercury was dumped in Minimata Bay Japan in the nontoxic form Bacteria in the anaerobic sediments methylated the mercury The methylmercury bioaccumulated in the food chain Most of the people in Minimata village ate fish Horrible neurotoxic damage to MANY children while in the womb hundreds born like this 9 Some bacteria are mercury resistant use organomercury Iyase to convert methylmercury to methane and Hg and then use mercuric reductase to reduce Hg to Hg again Hg is volatile and can move away as a gas These genes are often on plasmids which means what The Phosphorous Cycle 9 main limiting agent in freshwaters frees it up into soluble for cell to take it up Is the main limiting nutrient in freshwaters N is limiting in marine waters Are no redox transformations and no gaseous forms Present in inorganic rocks dissolved phosphate and organic forms Most easily used is SRP soluble reactive phosphate also called orthophosphate Many microbes produce and excrete phosphatases that release cleave bound phosphate from organic matter for uptake Ocean Acidification 9 top arrow dissociation acidification ca2 2 HC0339 CaCO3 co There is concern that increasing atmospheric CO2 not only increases temperature global warming but is leading to ocean acidification this will cause CaCO3 to dissolve including shells and skeletons and prevent growth precipitation perhaps resulting in ecological catastrophe Bioremediation Microbial Syntrophy and Symbiosis Symbiosis living together When the relationship is beneficial to all members it is mutualism remember if they can only live with the partner it is true symbiosis Microbes form symbiotic relationships with other microrganisms with plants and with animals First microbial symbioses Syntrophy when two or more organisms share their metabolic processes to make use of carbon or energy sources each organism cannot utilize the substance on its own cometabolize it All syntrophs are obligate anaerobes For microbes often involves byproducts of fermentation produced why are secondary fermentations The byproducts are substrates gun hpupnnay for other organisms in the HKIIOB nearby environment an p nor an r aoo KM KJ Very important in microbial M o anon0P o p gp pugg p syntrophy is interspecies H2 quotquot 39quot39quot quot quotquot39 39 quot39 quotquot quotquot 1 transfer H2 is produced by one n 1 I 2 39 prokaryote and used by another up v l Glytcvaldohydc 3 0 H2 is valuable metabolically l u quot M 1 Mo I why REDUCES OXIDIZED NAD 2 PIamphuqho yunIu P 1 cell does not use energy to do 2 O gfyutohuuw this It is used in a lot of general 1 p In 39 microbial metabolic pathways 1 3 llhcuuho gcuruu P Ifnolcw Syntrophy between an ethanol fermentor and a II Q T rvwvso methanogen IT If quotquot Pulling exergonic reaction pgg quot 39 7 Iynnoooo loannnu lyou and making it endergon1c Yo 1090 u H M I Iounuo Ian manna O Michel 050 dnhyorogonow NAD o lo Sloqol nd Hqun S Upon 1 Good Itoloqy of Muuoocguonmo II o Ethanol fermenter Methanogen co lnterspecies hydrogen transfer Hm quot Ethanol fermentation 2quot2 2quot A G0 194 kJIreaction Methanogenesis 4 H2 39939 CO2 gt CH4 399 2 H20 A G 39 1307 kJIreaction Coupled reaction 2quot quot 2 39 A 6 1113 kJIreaction 8 Reactions Ethanol fermenter Methanogen co lnterspecies hydrogen transfer Q pP quot Hydrogen consumption by the methanogen pulls the reaction of the partner AG0 won39t go Syntrophic bacteria are obligate anaerobes important in anoxic part of the carbon cycle These processes occur when there are no good electron acceptors for Are different types of microbemicrobe symbioses based on mutual benefits other than sharing metabolic pathways In freshwater lakes are motile phototrophic consortia composed of nonmotile photosynthesizers outside and motile heterotrophic bacteria inside The photosynthesizers are usually green sulfur bacteria Can make up 67 of bacterioplanktonic suspended bacteria biomass in the lake Figure 253 Drawings of some motile phototrophic consortia found in freshwater lakes Phototrophic consortia with different morphologies Figure 254 Phasecontrast micrograph of quotPelochromatium roseum from Lake Dagow Brandenburg Germany Arrow central rod shaped bacterium In this system the green sulfur bacteria are epibionts growing on the surface of a living organism Can have 1369 epibionts on one bacterial cell Some have gas vesicles but movement vertical migration is slower than with agellated partners Green sulfur bacteria need light and sulfide electron donor for photosynthesis The consortium is positively chemotactic to sulfide and negatively phototactic why is the latter important One consortium is called Chlorochromatium aggregatum The epibiont Chlorobium chlorochromatii can be grown in the lab in pure culture but has never been found in the natural environment without a bacterial associate 9 recognize both and what they do The central bacterium is a betaproteobacterium It grows on ocketoglutarate TCA cycle intermediate but only assimilates it in the presence of light and sulfide when the green sulfur bacterium is photosynthetically active SEM images suggest a shared periplasmic space How would this happen What would be the benefit Use of transcriptomics and proteomics to study Chlorobium chlorochromatii in the symbiosis vs in pure culture showed about 50 proteins unique to the symbiotic state about 350 genes are expressed in pure culture but repressed in the symbiotic state 19 are expressed more highly amino acid metabolism and regulation of N suggests the symbiosis is based on exchange of amino acids Metabolic role of the central bacterium not known Symbiosis with plants Plantbacterial symbiosis root nodules Root nodule bacteria Bacterial symbiosis with legumes plants with their seeds in pods soybeans clovers peas alfalfa beans Certain gram negative N2fixing bacteria Rhizobium Bradyrhizobium Sinorhyizobium and others all referred to as rhizobia symbiosis is specific Active in the rhizosphere area around roots where they infect plant roots and together form root nodules N2 fixation occurs inside the nodules Rhizobia can grow freeliving or in symbiotic association Don39t fix N2 in nature unless in symbiotic relationship with a legume Do fix N2 in culture but only when grown in microaerobic conditions Crops with and without root nodules don39t need to add fertilizer About 1 of all nitrogen fixation on Earth takes place in root nodules Steps in root nodule formation 1 Recognition and attachment of rhizobia to root hairs attachment by rhicadhesin 2 Excretion of Nod factors by the rhizobia 3 Rhizobial penetration of the root hair 4 Travel to the main root by an infection thread 5 Transformation to bacteroids inside plant cells 6 Increased plant cell and bacterial cell division forming the root nodule Rhizobial cell 39 39 V I Invaded plant cells Infection 39 2 ro oog mg and those nearby are thread 1 f I l stimulated todivide o Roothait 3 1 39 V 1 I 5 Formation ol bacteroid state within plant root cenls 4 Bacteria in T infection quot 3939 3 3 939 quot 6 Continued plant t 939 3900 A and bacterial ce v 2 Bactetium 3 Invasion Rtnizobia V Ce d39V39539 quot secretes Nod factors PEquot U316 3900 5393quot ads O d quot 539 V Nodules cauisung root hair and multnply within curlling an quotinfection tlnnadquot 1 Recognition and sttactunc nt Ijrlhicsdhe5in mediated 9 know this process After attachment rhizobia penetrate root hairs which curl in response to substances secreted by the rhizobia Then induce plant to form an infection thread is a hollow tube made of cellulose Rhizobia move through the root hair stimulating plant cell division forming nodules around the bacteria Bacteria growing in nodules are transformed into bacteroids swollen misshapen cells can do N2 fixation Figure 2211 The infection thread and formation of root nodules Bacteroids red color is leghemoglobin Bacteroids are contained in structures called symbiosomes N2 fixation occurs only after these have formed Surrounded by a membrane obtained from the plant cytoplasmic cell membrane Symbiosome membrane has special transport proteins that exchange nutrients between the plant host and the bacteroid N2 fixation in nodules Remember in culture these microbes only fix N2 under microaerobic conditions Need 02 but too much poisons nitrogenase In nodules 02 levels controlled by leghemoglobin produced by the plant Leghemoglobin An 02 binding protein in root nodules ratio of leghemoglobinbound O2 to free 02 is 100001 is induced by interaction between the legume plant and the bacteria contains Fe and acts as an oxygen buffer cycles between oxidized Fe and reduced Fe forms The inside of each bacteroid inside the symbiosome is anaerobic Leghemoglobin delivers 02 to the bacteroid to support aerobic respiration of the bacterium 02 does not come in contact with nitrogenase Aerobic respiratory electron transport path in membrane N2 fixation in cytoplasm Han cytoplasm Photosynthesis Symbiosome l Sugars membrane Bacteroid 03993quot acids membrane V Iactoroltl swam Malato Fumlarate Pyruvate Proton 99 399 motive force Nntrogenase Electron transport 9 397 quot iquot 024 Lb Glutaminc 02 pQ 39 0239lb Asparagine Lb Leghemoglobin 9 don39t worry about where the electrons are coming from 9 from inside the bacteroidssymbiosomes 9 leghimoglobins are delivering oxygen Whole process of root nodule formation is carried out by rhizobial nod genes Code for nod factors that induce root hair curling stimulate plant and rhizobial cell division etc Biochemistry is known Water fern cyanobacterium symbiosis Azolla water fern has a symbiosis with a heterocyst forming cyanobacterium Anabaena Used by rice farmers The cyanobacteria live in small pores in the leaves of Azolla Photosynthesis by the leaves occurs how is nitrogenase protected from oxygen MISSING SLIDE AGROBACTERIUM AND CROWN CALL DISEASE 9 Ti plamid tumor induction Crown gall Tumor formation process 1 A tumefaciens cells attach to a wound site on the plant 2 Attached cells synthesize cellulose microfibrils to hold cell to plant 3 Transfer a portion TDNA of the Ti plasmid to plant cells like conjugation 4 DNA transfer mediated by Vir genes on the plasmid 5 Vir gene transcript induced by compounds produced by wounded plant tissue Ti plasmid of A tumefaciens Z1DNAS oncogenes Opine Transmissibility genes vi 4 synthesis jgengs Opine encode O catabolism virulence Qe es factors 9 l 9 I 4 Transfer of TDNA from the Ti plasmid to the plant 9 phenolics from plant wound causes cascade and expresses Vir genes frees up TDNA kicks Piplasmid and forms a pore 9 don39t need to know specifics of intermediaes Phonetics from pun gonna Yranscriptuon of olive am gonna l Nichln A060 ww gt39 J VirA autophosphorylates transfers P04 to VirG VirG is the response regulator and does what VirD nicks the Ti plasmid niext to TDNA VirB moves to plant cell membrane and acts as a conjugation bridge VirE is a single strand binding protein that binds carries TDNA to and through the bridge Plant DNA polymerase synthesizes complementary strand which is inserted in to plant chromosome In the plant chromosome TDNA genes expressed oncogenes and genes for opine synthesis Oncogenes cause uncontrolled growth of plant cells forms a tumor the gall Opines now produced by the plant are weird amino acids that serve as a C N and P source for A tumefaciens but not other bacteria Ti plasmid used in genetic engineering of plants Animal Microbe Symbioses Microbiome all microbes in habitat like body The Human Microbiome All the microorganisms that live on or in a human skin mouth throat stomack intestines etc Your microbiome has 10 x as many cells as you have human cells Is unique to each individual in terms of bacterial species Human gut microbiome is important in human health probiotics The human gastrointestinal tract note pH changes as food goes through Colon 1 W9 to9 T 7 10quot1o39g I PH 4 PH 5 H ls PH 7 J magi lamaInxnxlnnjj Human gut microbiota determined by 16S rRNA gene sequencing of whole community DNA from fecal samples Lachnosplnccaa 1 Uncertain al llatlon 2 Copmcoccus 3 Dora 4 Lachnospln 5 Rosaburia 5 mm 900 Snntoooccacoao quot Actinobacleria ms L I I W Unclassmodandothor minor bacmrlal groups Vnmrcomlcmbla omquot Fmmc Prntaabactona 8 Rmninococcacaae 1 Uncertain af liation 2 Faocanbactonum 3 Papmdbactcr I Ruminoooccus 5 Subdoligranulum 5 Minot groups Other Clostridiales Vamanoltacoao Human gut microorganisms produce enzymes and amino acids humans can only synthesize 10 of the 20 amino acids This microbial community always dominated by one of four major phyla Firmicutes Bacteroidetes Proteobacteria or Actinobacteria Most dominant usually Firmicutes or Bacteriodetes in each case can be gt90 of the population Human gut microbiota have identified 3500 species if use a 99 16S rRNA gene sequence homology to define species 35000 species if use 97 homology the cut off for environmental species 9 take fecal matter and examine diversity also used for patients with weak immune systems to reestablish the bacteria community in their intestines Microbial pathogenicity and toxins Have talked a lot in here about the positive roles of microorganisms symbiosis nitrogen fixation BGC cycling microbiota etc Now pathogenic microbes First overview of more beneficial humanmicrobe associations 231 Beneficial Human Microbial Interactions Most microorganisms are benign 0 Few contribute to health and fewer pose direct threats to health Normal microbial ora Microorganisms usually found associated with human body tissue Humans are colonized by microorganisms at birth Animals provide a favorable environment for the growth of many microorganisms Infections frequently begin at sites in the animal39s mucous membranes Figure 231 232 Micro ora of the Skin The skin surface varies greatly in chemical composition and moisture content Three microenvironments Dry skin Moist skin Sebaceous skin Skin micro ora examined by molecular ecology methods 19 phyla detected Each microenvironment shows a unique microbiota Figure 232 0 The skin micro ora Composition is in uenced by Environmental factors e g weather 0 Host factors e g age personal hygiene Metagenomic analysis of human micro ora shows a complex microbial community Most microorganisms are facultatively aerobic Some are obligately anaerobic 0 Some are obligately aerobic 234 Micro ora of the Gastrointestinal Tract 0 The human gastrointestinal GI tract Figure 235 Consists of stomach small intestine and large intestine Responsible for digestion of food absorption of nutrients and production of nutrients by the indigenous microbial ora Contains 1013 to 1014 microbial cells Microbial populations in different areas of the GI tract are in uenced by diet and the physical conditions in the area The acidity of the stomach and the duodenum of the small intestine pH 2 prevents many organisms from colonizing the GI tract Functions and products of intestinal ora Intestinal microorganisms carry out a variety of essential metabolic reactions that produce various compounds 0 The type and amount produced are in uenced by the composition of the intestinal ora and the diet Compounds produced include 0 Vitamins Gas organic acids and odor 0 Enzymes Microbes as pathogens Much is known about pathogenicity our focus virulence and bacterial toxins Virulence properties of a pathogen associated with pathogenicity Are many virulence factors enzymes that allow pathogen to invade host enzymes that allow pathogen to bind to host first step of infection toxins etc read in book 9 recognize examples of Vir Facts Virulence can be measured Common method LD50 dose amount of toxin number of cells or number of virus particles that will kill 50 of a test population Virulence is highly variable bacteria usually 1005 to 1000s of cells viruses can be one 9 LD LETHAL DOSE will give LD 50 amount and know which one is more virulent 5 vs 50 9 5 more virulent Figure 2714 Microbial virulence Mechanisms of pathogenesis following exposure 9 know these steps Further exposure at local sltes I TOXICITY co N39 AT39 N toxin effects are TISSUE 3quot H quotlocal or systemic EXPOSURE ADHERENCE INVASION Row139 DAMAGE to pathogens to skin or mucosa through epithelium production of DISEASE 399 9 1 virulence factors 0 quotquot quot quotquotquotquotquotquoto 0 INVASIVENESS 0 N go 0 0 further growth at 39 L 0 original and distant sites 0 0 0 Further exposure 9 each mechanism allowing each step virulence factor Adherence of pathogens to tissues Adherence facilitated by slime layers capsules fimbriae pili 9 glycopolysaccharides in mucus Once in a host body the pathogen can t grow everywhere limited by the immediate environment and availability of materials necessary to grow nutrients trace elements etc speci c for different pathogens chronic wound speci c habitat After successful invasion and once growing some pathogens can spread through the body Examples release of enzymes like hyaluronidase breaks down polysaccharide hyaluronic acid that binds tissues occurs in gas gangrene 9 enzyme virulence factor release of collagenase breaks down collagen 9 collagenase also virulence factor Some bacterial diseases are characterized by intracellular growth of the pathogen in host cells problem intracellular immune system doesn t know it s a foreign cell 9 the pathogen usually stays alive for a long time Some grow in the cytoplasm some in the nucleus In each case the pathogen is protected from the host immune system when growing intracellularly Some can grow intra or extracellularly Some people are disease carriers of these pathogens no symptoms but can infect others Each of the different mechanisms involved in every step of microbial pathogenesis is a virulence factor Enterotoxin Virulence factors in Salmonella 9 recognize the dz vzr facts diarrhea siderophofes lnjectisome iron uptake inv and prg products form Bacterial Toxins quot39P39 Endotoxin in Type I mbriae adherence Virulence LPSlayer 9 virulence factor carried on plasmid Ti plasmid quot quot quot 39 39quot39 Many pathogenic bacteria produce toxins these damage host cells Are three 333 quot V main classes gggzfgg c byoxyR inhibits host cell protein synthesis caquot eftlux Exotoxins are released extracellularly are proteins can travel to other parts gnmggquot quot quot39 39T quot39 quot39 39 quot VI capsule antigen of host body 9 most toxic i39l397ilg3Y Pamogmcity inhibits complement binding islands on Flagellum motility I I chromosome 0 antigen adherence Enterotoxins subclass of exotoxins also proteins act on the small so mhbts Pquot3939 Y1 Kquotquot9 intestine Endotoxins toxic LPS of some Gram negative bacteria not as toxic as exotoxins Exotoxins Most in are in one of three categories Enterotoxins subclass of Exotoxins l cytotoxins also cytolytic toxins attack cytoplasmic membranes cause cell lysis activity is enzymatic 2 AB toxins have two covalently bonded subunits A and B B binds to a host cell surface receptor A goes across cell membrane and causes damage 9 mechanistic level 3 Superantigens stimulate large amounts of immune response cells 9 in ammatory reactions Chapter 24 9 anaphylactic shock Cytolytic toxins cytotoxins Lyse cells in the lab can easily detect on plates Ex Hemolysin assay with red blood cells positive result is hemolysis clearing completely lyses rbcs Streptococcus pyogenes growing on blood agar plate Staphylococcal oitoxin oi toxin subunits bind to the host phospholipid bilayer oligomerize into heptamers seven units in the membrane change conformation to form a membranespanning pore cell contents are released and cell dies Mechanism of toxicity of Staphylococcal oitoxin Forms a pore in the host cell membrane and contents leak out 0 co 0 5 Efflux of o o cYt l3935m39 0 9 3 9 cytoplasmic quot39939quot quot19 9 components A Out IAJJIJJJJJJJJkJJJ39J39JJ amp 9zti2i Lat33lliiiiitttalllitiallitaf i quotquotquotquotquotJ39Ji T Ji39R if1i39 T Jy f il l JAIJJJJJJJJJJJ J oiToxin pore In In ux of extracellular 9 0 components 0 0 o 0 00 AB toxins 9 know MECHANISM Many inhibit protein synthesis Example diptheria toxin 1 exotoxin discovered produced by Clostridium diptheriae AB subunits together MW 62000 A MW 21000 Subunit B binds to a cell receptor Subunit A enters the cell cytoplasm Disrupts protein synthesis by blocking transfer of amino acids from tRNA to growing polypeptides at ribosomes Very powerful one molecule of toxin can kill a cell 9 one toxin cell can spread and kill Mechanism of diptheria toxin Specifically inactivates elongation factor 2 a protein involved in growth of the polypeptide chain by catalyzing attachment of ADP ribose from NAD to elongation factor 2 9 have to have specific toxic gene that has to be lysogenized with phase B phage has infected it and inserted its gene into chromosome on which is a toxin Only strains of C diptheriae lysogenized with phage B with a toxin gene produce this AB toxin Figure 2320 Diphtheria toxin activity EF2 has ADP ribose attached can t bind tRNA with amino acid for growing peptide Botulinum toxins are seven related AB toxins produced by Clostridium botulinum 9 2 Exotoxins to know Enterotoxins A subclass of exotoxins also proteins act on the small intestines Example cholera toxin Produced by Vibrio cholerae is an AB toxin MW of A subunit 27200 B has five B subunits each with a MW of 11600 Mechanism of cholera toxin B binds with ganglioside GM1 complex glycolipid icytoplmasic membrane in intestine in the epithelial cytoplasmic membrane A moves into the cell and activates adenyl cyclase converts ATP to cAMP 9 Active part of AB toxin into the cell then needs to be activated 9 activated Adeny Cylase A must be activated by a host cellular enzyme requires NAD and ATP when cAMP increases results in active secretion of Clquot and HCO339 from mucosal cells into the intestinal lumen In ux of Clquot and HCO339 causes a change in ion balance which results in large amounts of H20 moving into the lumen why 9 osmosis conc gradient created with relatively less water in lumen Massive net uid loss due to diarrheae can lead to death Here toxic activity mimics normal mammalian hormones form of cAMP Figure 2323 The activity of cholera enterotoxin 9 don t worry about Na Endotoxins Are LPS toxins lipid A part of the LPS are not proteins are released when cells pathogens lyse Stimulate host cells to release endogenous pyrogens 9 causes a FEVER are proteins cytokines that affect the temperature control center of the brain cause fever large doses can cause death due to hemorrhagic shock Are less toxic than exotoxins for many enolotoxins LD5 2 4l pig mierograms E12 for botulinum toxin an exotoxiin LD5 25 piieograms ll vvlriiielri is lit million times more tome Comparison of toxins know this table 9 exam qu Propefll Exotoxins E ndotoxins Chemical properties Proteins excreted by certain grampositive LipopolysacchandltLlipoprotein complexes released or gramnegative Bacteria generally on cell tysls as part of the outer membrane of heatlabile gramnegative Bacteria extremely heatstable Mode of action symptoms Specific usually bonds to specific cell General fever diarrhea V0mlllng receptors or structures either cytotoxin enterotoxin or neurotoxin with detined specilic action on cells or tissues Toxicity Often highly toxic sometimes fatal Weakly toxic rarely tatal Immunogeniciry response Highly immunogenic stimulate the Relatrvely poor imrnunogen immune response not production 01 neutralizing antibody sufficient to neutralize toxin antitoxin Toxoid potential Treatment of toxin with lormaldehyde will None destroy lOX7CllY but treated toxin toxoad remains lrnmunogenic Fever potential Does not produce lever I host Pyrogenic olten induoes lever in host Immunity and immune mechanisms Immunology the study of immunity Immunity the ability of an organism to resist infection Two general types 1 Innate or nonspecific immunity 2 Adaptive or specific immunity Innate immunity Anatomical barriers skin etc General ability to resist pathogenic viruses bacteria and fungi after they have broken through anatomical barriers Involve nonspecific responses that develop immediately against any invading microbe includes phagocytes cells that recognize ingest and degrade pathogens Pathogens with and pathogen products quotAquotquot 3 Responses can break pathogens down to immunogens if so are acting as 1 antigen presenting cells APCs which interact with the adaptive immune E139 39fy t39 f1 p t o3939r39 quotquotquot 5 system Phagocytes include macrophages monocytes neutrophils and dendritic cells most have lysosomes with degradative enzymes proteases phosphatases lipases nucleases etc and H202 Phagocytes respond to PAMPs pathogen associated molecular patterns by pattern recognition receptors PRRs examples of PAMPs LPS agellin teichoic acids are speci c molecules but common to many bacteria Adaptive immunity 5 LI ta i Amiggn Phagocytosis Involves mechanrsms to develop rmmunrty to 1nd1v1dual pathogens presenting 9 Pathogen destruction A tquot A new pathogen m1crobe must rnteract w1th the 1mmune system quot V i n 3998quot processmg before the response can occur usually several days required Spec1f1c 1mmune responses are el1c1ted by rmmunogens Wm MHC II I U include surface proteins of the pathogen agella pili breakdown Pathogen antigen Antigen Pathogen antigen product of phagocytes etc Tc 39 39 S quot 39 quot f 7 quot When immunogens are recognized the immune system are antigens Cells defend against antigens by making antigen specific antibodies p Tc Cequot immunoglobulins 391 quot t cg are soluble proteins in serum that react with the antigen and destroy quot t 0 Cvwkines 0 zTne or neutralize it p 33 2 Cytokme Perform and release 1 1 granzyme release 9 relationship between innate and adaptive immunity via 3939 39 2 PH Inflammation Target cell Iysis j innane bind to PAMPs circulatory system adaptive immunity respond to these phagocytes that have interacted with PAMPS Microbes counteract host immune defenses can neutralize phagocytes degrade phagocytes via leukocidins such cells are pyogenic pusproducing pus is dead cells Can avoid phagocytosis by changing cell surface evolutionary interactions by the presence of capsules or by invading cells intracellular pathogens Antibodies Antibodies immunoglobulins are soluble proteins made by B cells in response to exposure to nonself antigens B cells display antibodies on their cell surfaces the antibodies directly interact with antigens to cause B cells to ingest pathogen phagocytosis B cells then produce many pathogenderived peptide antigens these are presented to antigenspecific Th2 cells Clinical Microbiology CLINICAL AND DIAGNOSTIC MICROBIOLOGY Aimed at isolation and identification of pathogenic microbes Needed to precisely ID a pathogen to treat infectious disease Labs can usually isolate ID and determine antibiotic susceptibility using standard techniques in 48 hours genetic techniques or detection of surface proteins can ID in a few hours Often just prescribe broad spectrum antibiotics and don t assess antibiotic susceptibility To isolate a pathogen need to sample directly from site of infection and culture or assay the sample blood urine sputum cerebrospinal uid etc use of sterile techniques is important Once isolated need to ID Traditional methods include a suite of growth dependent assays Growth from patient sample often based on two types of media Selective has compounds which selectively inhibit growth of some microbes Differential has an indicator usually a dye which tells when a diagnostic chemical rx has occurred Example EMB agar Eosin methylene blue Cultures that ferment lactoselike E coli form colonies with agreen metallic sheen Table 313 Important clinical diagnostic tests for bacteria 9 identify whats differential and what selective and whats both Yes Principle 9 will pick one straight forward one for exam Procedure Most common use Carbohydrate fermentation C atalase Citrate utilization Coagulase Decarboxyiases lysine ornilliine arginine BGalactosidase ONPG test Gelatin lrquelaction Hydrogen Sulfide H33 production Acid andor gas is produced during lermentative growth with sugars or sugar alcohols Enzyme decomposes hydrogen peroxide HO Utilization of citrate as sole carbon source results in alkalinization ol medium Enzyme causes clotting of blood plasma Decarboxylation of amino acid releases CO and amine OrthonitrophenyiBgalactoside ONPG is an artilicial substrate for the enzyme Hydrolysis of ONPG lorms nitrophenol yellow Many protease3 hydrolyze gelatin and destroy the gel H38 is produced by breakdown of sulfur amino acids or reduction of thiosullate Broth medium with carbohydrate and phenol red as pH indicator inverted tube for gas Add a drop ot H O2 to dense culture and look lor bubbles Oti Citrate medium with bromthymol blue as pH indicator Look tor intense blue color alkaline pH Mix dense liquid suspension ol lizicteria with plasma incutnale and look for fibrin clot Medium enriched with amino acids Brorncresol purple pH indicator becomes purple alkaline pH if there is enzyme acton Incubate heavy suspension of lysed culture with ONPG Look tor yellow color Incubate in broth with 12 gelatin Cool to check for gel formation It gelatin is hydrolyzed tube remains liquid on cooling H35 detected in ironrim medium from formation of black lerrous sulfide many variants KIigler39s iron agar and triple sugi I iron agar also detect carbohydrate fermentation Gelatin liquefaction differential shows result not selective Selective or differential Eosin a dye and a pH indicator when acidic changes from no color to black Methylene blue inhibits the growth of Gram bacteria SELEC amp DIFF A question about this will be taken from table 313 previous two slides on the next exam Enteric bacteria difterentiation Bacillus lrorn Clostridrurn Streptococcus from Micrococcus Staphylococcus l r l Klebsiellafnrerooacrer from Escherichia Edivardsaella from Salrnone a Staphylococcus aureus from Slaprrytococcus epiderrriidis Aid in determining bacterial group among the enteric bacteria Crrrobacter from Salmonella t Identifying some Shigella and Pseudornonas species Aid in identification of S4rralia Pseudornonas FiaiOCaC39C VIt 7l Clostridurn Among enteric bacteria to aid in identifying Sa39mone3939ai Edivardsietla and Proteus After ID the pathogen test for antibiotic sensitivity often not done these days unless a problem KirbyBauer assay prepare spread plate lawn of cultured isolated pathogen put filter discs with known concentration of antibiotics on plate incubate measure zone of inhibition around each disc diameter of no growth in mm 33 66 look at table to see if isolate is resistant intermediate or sensitive 3 3 now know if can use each antibiotic at what concentration KirbyBauer assay Example Zone of Inhibition mm Antibiotic Concentration Resistant Intermediate Sensitive Gentamycin 10 ug 12 or less 1314 15 or more Methicillin 5 ug 9 or less 1013 14 or more Choose one to which the pathogen is sensitive Don t choose intermediate can select for antibiotic resistant strains during treatment Sensitive bigger zone of inhibition When choosing an antibiotic Select sensitive lowest concentration 9 then doctor narrows it down Intermediate selects for antibiotic resistance Resistant no way Second technique antibiotic dilution assay Prepare series of 2X dilution of each antibiotic in tubes with growth media Inoculate each tube Look for MIC minimum inhibitory concentration of antibiotic lowest concentration where no growth Want to use minimum concentration highest dilution that completely inhibits growth many antibiotics have toxic side effects Figure 318 Antibiotic susceptibility testing Antibiotic dilution series with P aeruginosa Each row a different anitibitic with the highest concentration on the left Look for MIC Growth yellow wells No growth clear wells Where is MIC in lane row 1 Lowest cone Row 9 Lowest cone What is happening here around 7 Resistant to Etest not based on diffusion of the antibiotic on a plate or need to perform dilution series E for easy Is a kit preformed and predefined gradients of antimicrobial compounds bound to a plastic strip results show the exact concentration of antibiotic for the MIC result Figure 278g Antibiotic susceptibility testing Antibody Titer Sometimes you can t isolate the pathogen so can t ID or treat 9 guessing Can use the Antibody Titer approach First need to hypothesize as to ID of the pathogen Then look for antibodies the patient is producing to con rm presence of the pathogen Do this by titering Set up 2X dilution series of serum from the patient where antibodies located determine the highest dilution lowest concentration where antibodyantigen reaction is detected antigen a substance that elicits an antibody calculate the concentration of antibodies Are different techniques to detect antibodyantigen interactions can target either the antibody or the antigen Fluorescent labeled antibodies will react with the antigen if in the serum microscope based Monoclonal antibodies more specific selective for a single antigen ex membrane protein ELISA enzyme linked immunosorbent assay direct detects the antigen indirect detects the antibody Agglutination based on physical binding of antigen to antibody clump and precipitate can see You need to do an antibody titer more than once to show a rise in antibody concentration this indicates an active infection Antibody presence may be due to previous infection by the same pathogen exposure to the pathogen successful antibody response or immunization Antibody titer doesn t work if the infection is highly localized ex gonorrhea Antibiotics often called antimicrobials Mechanisms of antibiotic activity Targets include Cell wall synthesis Cytoplasmic membrane integrity Protein synthesis Nucleic acid replication synthesis packaging Metabolic pathways inhibition of Are different specific mechanisms for each target Cell wall synthesis inhibitors 9 slight modification ensures getting rid of the resistance 9 all have B lactam ring and variable R groups N acyl groups determines the differences in the class 1 3lactam antibiotics Are two main types penicillins and cephalosporins are naturally produced by fungi Are subgroups in these including more natural compounds as well as semisynthetic chemical modification of a natural compound All Blactam antibiotics contain a Blactam ring Inhibit the last step in peptidoglycan synthesis transpeptidation Mechanism the Blactam antibiotic binds to a transpeptidase that normally cleaves a DAla located at star from the peptide chain on one NAM and forms a bond with a DAP on neighboring NAM The transpeptidase was discovered during research on penicillin activity named the penicillin binding protein but actually binds all B lactam antibiotics 1Rmisumon iniiiaiion Covalent bond formed between the a serine site on the Q transpeptidase and the 3 lactam ring a V Initiator Large 508 The enzyme transpeptidase is inactivated GT9 E Em quotbquotquotquot 3lactams act on growing peptidoglycan but also induce synthesis xguliirtge of bacterial autolysins 3939quotcI 39 quot squot i i s 39 39 3 Initiation complex subunit Autolys1ns degrade peptidoglycan I I I TRANSLATION Elongation are usually active during growthassociated cell division mm Why am quot Codon recognition 3lactams disrupt control of the process Protein synthesis inhibitors Target bacterial ribosomes Overall size is 70S Svedberg units Two subunits small 30S and large SOS 39 quot L Small subunit l6S has 21 proteins plus RNA 9 phylogenetic analysis P site A SR9 AI eptida bond formation Large subunit also has two subunits SS and 23 S plus RNA Antibiotics that target bacterial ribosomes usually form H bonds to P04 backbones of 16S or 23S rRNA subunits Examples of protein synthesis inhibitors l Aininoglyeosioles are trisaeeliarioles 2 have aniino sugars with glyeosiolie linllts target 3 S subunit of loS rRNA Function by binding to the l6SrRNA on 30S subunit and preventing it from binding to the SOS subunit eanit niallte a full ribosome so not functional Many types llt lllll Il lllS9 Slltpl l Ily lll9 gentaniyeina ll lOIly lll9 toloraniyein DDDD M These are T XlC hearing loss and oleereaseol lltiolney funetion 2 Tettaeyelines have four eyelie rings also loinol to l6SrRNA on 3 S sulounit are loaeteriostatie change conformation of the site that interacts with tRNA can t get amino acid to the growing protein 3 Maeirolioles have laetone rings loonoleol to sugars loinols to 23 SrRNA on 5 S sulounit blocks exit tunnel where growing peptide chain leaves the ribosome Antibiotics that target DNA synthesis Antivirals and antibiotic resistance TRANSLATION Initiation Initiator Large 508 subun Add large subunh Small 305 subun Ribosome binding site RBS TRANSLATION Elongation Small 308 Initiation complex subunit Incoming tRNA Codon recognition Cycle continues three times u o I eptide bond formation I P quot Large sos GTP subunit 3 E E Eslte t E Add large subunit binding site small 305 R38 TRANSLATION Elongation subunn Initiation complex Incoming tRNA Codon recognition g p 8quot A site Cycle continues three times Peptide bond formation Antibiotic resistance Pathogens have evolved to counteract antibiotics become resistant Based on mutations and gene exchange Five main categories Resistance Mechanisms l Impermeable to antibiotic 2 Can inactivate the antibiotic 3 May modify the target of the antibiotic 4 May develop a resistant biochemical pathway 5 May be able to pump out the antibiotic ef ux Table 268 Baetetiai t SiSimC to antibiotics gt alon t memotJize Different mechanisms in each category Many of the mechanisms involve housekeeping proteins why adapt the target of the antibiotic to counteractattack the antibiotic can have multiple mechanisms in one bacterium acting against one antibiotic can have multiple drug resistant bacteria with different speci c mechanisms Mechanisms of antibiotic resistance Limit access of the antibiotic 1 Change outer membrane porins make them smaller so antibiotics can t pass through 2 Reduce uptake across the cell membrane some make use of transporters can mutate so transporter no longer can carry the antibiotic 3 Evolve ef ux pumps antiporters and ABC transporters that pump the antibiotic out 9 Proton gradients Enzymatic inactivation of the antibiotic 3lactamase very common especially for Gram negative bacteria has an active serine site that forms a covalent bond with the Blactam ring at the same site penicillin binding site or PBP recognized by the transpeptidase the 3lactamase hydrolyzes and opens the ring releasing the Blactamase to inactivate other molecules of B lactam antibiotics New approach when treating pathogens that have 3lactamase can add mechanismbased Blactamase inhibitors Ex Add clavulonic acid Augmentin is amoxycillin and clavulonic acid or sublactam are not antibiotics but prevent 3lactamase from inactivating Blactam BUT bacteria have developed resistance e g by increasing the rate of synthesis of 3lactamase so the inhibitor is overwhelmed bound up by excess the increase is due to gene duplication multiple times Modi cation of the target Also seen for resistance to Blactams Penicillin binding proteins PBPs can be altered so they no longer bind to Blactam antibiotics Ex Staphylococcus aureus has a gene mecA that codes for PBP2 which replaces normal PBP confers resistance to methicillin MRSA Resistance to aminoglycosides Bacteria inactivate by adding OH acetyl NHg and other groups to the antibiotic 9 wont function anymore Results in interference of Hbinding to target 16S rRNA so translation is not inhibited Figure 2733 Sites at which antibiotics are modified by enzymes encoded by R plasmid genes Resistance Mechanisms and Spread of Resistance Most drugresistant bacteria have drugresistance genes located on R plasmids and raising livestock selects for the spread of R plasmids Are many examples of overuse of antibiotics Figure 2734 Patterns of drug resistance in pathogens Antibiotic use and presence of drug resistance Almost all pathogenic microbes are resistant to some antimicrobial agents A few pathogens have developed resistance to all known antimicrobial agents Resistance can be minimized by using antibiotics correctly and only when needed Resistance can be lost if the antibiotic is not used for several years Search for new antimicrobial drugs Is ongoing development of and search for new antimicrobial compounds Can be modi cation of current antimicrobial compounds or use of automated chemistry methods combinatorial chemistry for drug discovery 7 million compounds must be screened to nd a single useful clinical drug Search in new environments coral reefs etc Computers now used to design molecules to interact with specific microbial structures New methods of screening natural products are being used combinations of drugs e g ampicillin and sublactam bacteriophage therapy drug delivery Resistance Mechanisms and Spread of Resistance Most drugresistant bacteria have drugresistance genes located on R plasmids The use of antibiotics in medicine veterinary medicineand raising livestock selects for the spread of R plasmids Are many examples of overuse of antibiotics Figure 2734 Patterns of drug resistance in pathogens Antibiotic use and presence of drug resistance Almost all pathogenic microbes are resistant to some antimicrobial agents A few pathogens have developed resistance to all known antimicrobial agents MRSA methicillinresistant S aureus and VRSA vancomycin resistant S aureus Resistance can be minimized by using antibiotics correctly and only when needed Resistance can be lost if the antibiotic is not used for several years 9 use antibiotics when required and sparingly Search for new antimicrobial drugs Is ongoing development of and search for new antimicrobial compounds Can be modification of current antimicrobial compounds or use of automated chemistry methods combinatorial chemistry for drug discovery antimicrobials from fungi 7 million compounds must be screened to find a single useful clinical drug Search in new environments coral reefs etc Computers now used to design molecules to interact with specific microbial structures New methods of screening natural products are being used combinations of drugs e g ampicillin and sublactam bacteriophage therapy drug delivery In uenza and resistance Section 298 In uenza an RNA orthomyxovirus group with a segmented genome Are three different types of in uenza viruses A B C this year the u shot is effective for strains within all three types In uenza A is the most important human pathogen highly infectious airborne many deaths Figure 2925 In uenza A virus Each in uenza A strain has two unique surface glycoproteins antigenic Hemagglutinin HA or H antigen attaches the virion to the host cell during infection Neuroaminidase NA or N antigen releases the new virion from the host cell In uenza outbreaks occur yearly and new immunizations are required because of the plasticity of the in uenza genome Antigenic drift minor change in in uenza virus antigens due to gene including point mutation BUT important since RNA viruses have no nucleic acid repair mechanisms and point mutations are common Antigenic shift major change in in uenza virus antigen due to gene reassortment of very different virus types lots of new DNA can lead to major changes in virulence and surface properties In uenza genome changes due to antigenic drift predictable and the basis for new shots each year based on the prevalence of that in uenza type the previous year Genome changes due to antigenic shift are not predictable so no immunization available Epidemiology amp Epizootiology 9 Read all Chapter 28 Epidemiology the study of the occurrence distribution and control of infectious diseases in human populations Epizootiology the study of the occurrence distribution and control of infectious diseases in animal populations Also called medical ecology Prevalence proportion of diseased individuals in a population Incidence number of newly diseased individuals in a population over a speci c period of time Epidemic unusually high numbers of diseased individuals in a community at one time Pandemic worldwide epidemic Endemic disease continually present usually at low levels in a population Sporadic individual cases of disease cases not related Outbreak sudden increase in the number of cases of a disease cases are related Subclinical infection mild or no symptoms carrier Mortality incidence of death in a population Morbidity incidence of disease in a population Zoonoses disease that occurs in humans and animals Nosocomial hospital acquired infection Disease reservoir a site where a pathogen remains alive and can infect a susceptible host varies soil contaminated food for botulism the Anopheles mosquito for malaria rodents for plague etc Hostpathogen relations I Coevolution of a host and its pathogens is common quotquot 39 quotquotquot 39quot39quotquot39quot39 39quotquotquot quot39quotquotquot 739quot quot739339 A pathogen that kills its host before it can infect v another host may become extinct 4 39 wv 0 For a pathogen to remain present in populations it W 0 must reach equilibrium with the host population based on coevolution p 1 g Herd immunity when a high proportion of n 39v I individuals in a population are immune to 3 an infection then the whole population will be protected A 3 39 i The immune hosts protect nonimmune hosts because w 39 the pathogen cannot be transmitted 39 3 3 39a Epidemiologists study disease transmission by S correlating geographic climatic social and population data These data are then used to identify possible modes of transmission Often patterns will give clues to the basis of disease dynamics Disease Transmission Direct hosttohost transmission Infected individual transmits a disease directly to a susceptible host in uenza common cold STDs ringworm Indirect hosttohost transmission Transmission involves a living or nonliving agent Living agents are called vectors Nonliving agents are called fomites Major epidemics are usually commonsource or hosttohost epidemics Commonsource epidemic usually from contamination of water or food sudden outbreak cholera Hosttohost epidemic usually see a slow progressive rise in incidence then a gradual decline in uenza Figure 286 Types of epidemics Zoonosis a disease that primarily infects animals but is occasionally transmitted to humans Control of a zoonotic disease in the human population may not eliminate the disease as a potential public health problem Some infectious diseases have complex life cycles involving an obligate transfer from a nonhuman host to humans followed by transfer back to the nonhuman host Emerging and Reemerging Infectious Diseases Worldwide distributions of diseases change rapidly Diseases that suddenly become highly prevalent are called emerging diseases Re emerging diseases again become highly prevalent after having been under control White Plague Type II on Caribbean Coral Reefs A reemerging disease First described in 1995 in the Florida Keys Spread to affect 41 Caribbean species in 21 genera gt75 Maj or coral disease Distinctive etiology Tissue lysis initiates at the base of colonies Disease line migrates at rates up to 2 cmday May recrudesce or halt Affects nonacroporid scleractinian Caribbean corals The plague type II pathogen Aurantimonas coralicida gen nov sp nov the golden colored coral killer Disease line no distinct band mat or obvious presence of microorganisms 1995 outbreak in the Florida Keys gone the next year Same pattern throughout the Caribbean outbreak then disappears THIS MONTH new outbreak on reefs off of Miami Is a reemerging disease the same pathogen FINAL 30 from last subjects 10 big concepts 16S question energy ATP and reducing power 16 S area species concept based on 16S Viruses Virology the study of viruses A whole area of microbiology Viruses are extremely small 002 to 03 umm 20 to 300 nm Much smaller than bacteria some infect bacteria bacteriophages or phages Also infect animals plants etc Outnumber all living cells on planet by at least 10 to 1 but are they living Viruses are genetic elements that alternate between two stages Extracellular how transmitted between hosts is inactive consists of pure DNA or RNA surrounded by protein and sometimes an envelope in this stagec called a virion or virus particle 2 Intracellular active inside a host cell purpose is viral reproduction the virus takes over the host cell machinery to replicate new viruses genetic material proteins etc Many viruses cause diseases when present in host cells can kill cells or not if not usually alter the host genome which may be beneficial to the host new genes or have no effect on the host Viral structure extracellular The entire structure in the extracellular stage the virion or virus particle is a nucleocapsid Is highly variable in size shape and chemical composition Viral nucleic acid NA is present inside a protein capsid Viral NA can be SS or DS DNA SS or DS RNA or have DNA and RNA at different stages of reproductive cycle Genome DNA viruses 9 ssDNA OR dsDNA RNA viruses 9 ssRNA OR dsRNA RNA to DNA viruses 9 ssRNA Nuc39e aP5id Envelope retroviruses OR dsDNA hepadnaviruses Capsid Viral NA is surrounded by the capsid a ggigleic Nucleic shell made of capsomeres amd are protein subunits arranged in an Capsid repet1t1ve pattern composed of forms by selfassembly while in the capsomeres iI1fCCtCd hOSt C611 Naked virus Enveloped virus Some viruses are enveloped 9 0 surrounded by membrane extra to others but nonfunctioning present only in extracellular form When inactive 9 aids in infection as virulence factor and sticks to your mucus to infect the host 0 surrounded by a membrane consisting of lipid bilayer and some proteins 0 lipid bilayer acquired from the host later but proteins are viral virulence factors 0 If no envelope are naked Proteins in envelope can be enzymes often help to get virion into the host cell 9 hydrophobic interactions There can also be enzymes inside the capsid often required for viral NA replication later Many surface viral proteins are antigenic 9 illicit an immune response cause antibodies to be formed 9 and used for treatment Nucleocapsids are highly symmetrical Two basic forms With two kinds of symmetry Forms rods helices or rough spheres isocahedral Symmetry helical or icosahedral Helical symmetry capsomeres are arranged in a helix characteristic number of capsomeres per each tum of the helix for different viruses also characteristic width and length of the overall helix Icosohedral symmetry like a soccor ball Characteristic number of planar surfaces 20 60 180 240 Can also have complex viruses often have icosahedral head and helical tail may have tail fibers and endplates Tail fibers attach to host cell attached and contract so that base of virion endplate touches host cell enyzmes on endplate dissolve through host cell wall 9 attack bacteria 7 Tlnjf 8 3 1 W Tall plns Endplato f39 391 r V Tall bers D The amount of viral NA overall percentage of the Virion varies between viruses Enveloped viruses can be 12 Naked viruses 2550 Viral NA can be a single molecule or many molecules Viral NA is the basis for the Baltimore classi cation of viruses Table 92 The Baltimore classi cation system of viruses 9 be able to recognize know bases Viral replication Viruses use living host cells to synthesize everything to Make new virions Assemble new virions Allow virions to escape host cell and reinfect M Wurtz The whole process occurs in an orderly manner With five steps attaches to cell host adsorption penetration injection viral RNA synthesis of nucleic acid and protein taking over mechanisms assembly and packaging release lysis 9 virions 1 Attachment adsorption the virion attaches to the outside of a host cell is host specific proteins on the outside of the virion interact with receptors on the host cell surface receptors can be proteins polysaccharides lipoproteins often are part of host agellum or pilus If the receptor is not present the virus can t infect 2 Penetration entry or injection injection of viral NA into the host cell for some viruses the entire virion enters process varies depending on host cell Wall or lack of Wall Phages have to get through bacterial cell Wall often are complex Viruses that infect animals just have to go through the cell membrane many are enveloped often the Whole virion is taken up can be by fusion or by endocytosis 3 Synthesis of viral NA and viral protein 9 when they are active This is the active intracellular stage The host cell is taken over and host metabolism may be altered End result is production of new virions Virus enzymes may be synthesized depends on the virus later To synthesize viral proteins viral mRNA is required may be brought in or synthesized Is much variation with different strategies If DS DNA can be read directly be host cell machinery transcribed directly to viral mRNA using host RNA polymerase IF SS DNA can proceed as SS DNA 9 DS DNA 9 mRNA again using host machinery For RNA viruses may require viral enzymes brought in during penetration Examples virus speci c RNAdependent RNA polymerase to make mRNA from viral RNA reverse transcriptase carries out SS RNA 9 DS DNA 9 mRNA Some SS RNA Viruses are plus sense RNA reads directly as mRNA by host cell Some are minus sense RNA need Viral RNAdependent RNA polymerase to synthesize complementary strand which is RNA now read as mRNA Viral mRNA read by host ribosomes using host pool of tRNA ribosomes amino acids etc 9 don t memorize table 39 dsDNA virus ssDNA dsRNA 2 8RNA ti 88RNA H ssRNA Class I virus virus Vquotus virus retrovirus Class VII Class II Class III Class IV Class V Class VI S nthesie of I r strand Used directly Reverse dsDNA intennadiam Transcription as mRNA transcription mmscrlptlon of minus strand of minus strand Transcription jot minus strand T 39 t quot fn J39squots39 39nd dsDNA intermediate Genome Genome 39 replication 0383 l classical semiconservatlve 399PquotC3 l0D1 Class Iquot make ssRNA 1 and transcribe from this to give ssRNA partner Class II classical semiconservaiive Class N make ssRNA H and transcribe iiom this to give ssRNA oi genome discard ii strand Class V make ssRNA M and transcribe irom this to gve seRNA genome Class VII mmscnpuon fonowgd by Class VI make ssRNA ii genome by transcription of H strand oi dsDNA reverse transcription DNA viruses RNA viruses In hi 9 understand this know diff classes and how they Work Eclipse Maturation but don t memorize the whole thing l 227 Once all necessary enzymes are present Viral NA is Era lyymes ggiglelc zggt ln replicated in 2 39 U 3E 0 3911 o M Now need to package everything E Virus 3 added Assembly ii J 3quot Now capsomeres are synthesized in large amounts 5 Latent period release Also may synthesize lytic enzymes depends on Virus and proteins on outer surface of Virions Time to complete one cycle infection to release Very Time different for phages Vs animal Viruses Phage life cycle 3060 minutes Animal Viruses 1248 hours 9 spike up is when new viruses are being produced understand stages 4 Assembly and packaging Once everything is replicated capsomeres selfassemble to form new virions 5 Release lysis May require viral lytic enzymes to exit host cell May involve budding through host membrane envelope aquired Are different processes depending on the type of virus Overall can diagram a virus life cycle one step growth curve Temperate lysogenic phages Temperate virus 7 Host DNA Have two potential life cycles 39339 DNA Anachmem L tic virions re licated host cell 1 ses and virions H 39 y P y 1 CeI host released Lysogenic most of the viral genome is not Injection expressed repl1cates along with the host a Lytic pathway 1 Lysogenic pathway Latter case host is a lysogen and has been i L Viral DNA lysogen1zed by the temperate phage replicates 9 integrated in the chromosome F Induction Example Lambda Very well known temperate phage Infects E coli Efgghgg gigs Bgged Is DS DNA genome known 48502 bp quot 39quotd 39 5 quotquot ms 939 Lysogenized cell C 1 3 Q Pro ha e 1 Lysis Cell pdlvision Lambda circularizes almost immediately after i A infection PD E p Carried out by lambda integrase Lytic vs lysogenic state For lysogeny to occur lambda must be integrated into the host chromosome and production of late proteins must be prevented But early protein production is required integrase Since lambda is DS DNA host transcribes lambda DNA once it is in the host cell Lytic vs lysogenic state of lambda is controlled by competition between two gene products coded by lambda c1 and Cro both are repressors 9 competition between 2 stages Are two sets of genes which control lytic or lysogenic growth With a new infection both sets of genes are expressed Are two promoters PL and PR near each other Tum on genes to the left PL and right PR of the gene for cl lambda repressor Winner of the race is determined by the relative accumulation of gene products When cl lambda repressor accumulates it turns off all lambda genes except the one that codes for itself stays in chromosome and replicates along with host cl is turned on by cII but cII is unstable and degraded by host proteases cII is stabilized by cIII So cII and cIII allow cI to be expressed but once expressed genes for cII and c111 and all other lambda genes are repressed Cro proteins can accumulate before cl has repressed all other genes Cro represses genes for cII and cIII so cI is not not turned on Result is lytic cycle all genes are expressed Cells lysogenized with lambda can undergo induction to the lytic state Host produced RecA normally involved in genetic recombination later can convert to a protease and degrade cI Can artifically induce with UV radiation or chemical mutagens Lambda widely used in genetic engineering later Viral diversity Both Bacteria and Archaea can be infected by phages Usually but not always have complex struture Most are virulent and lyse kill host cells some infect without killing Size can vary Replication strategies of viruses are highly diverse Just like evolution based on bacterial pathogenhost interact have viralhost evolution Seen for viruses and eukaryotic bacterial and archaeal hosts Example Phage MS2 Infects E coli Genome is sequenced 3569 nucleotides Is SS RNA meaning it can be read as mRNA by the host cell Codes for four proteins RNA replicase coat protein lysis protein maturation protein 9 FIGURE 212 plus sense is used in both plus and minus sense for protein synth and replication RNA replicase two functions synthesizes minus RNA from viral plus RNA synthesizes more plus RNA from minus RNA Plus RNA read as mRNA to produce more viral proteins Animal viruses Are highly diverse and many are enveloped Four classes in terms of effect on host cell Virulent infection host cell lyses when released 9 u when bacteria giving out cytokines and feel terrible Latent infection viral NA not replicating a delay between infection and reproduction Persistent infection host cell remains alive virions produced slowly over a long period Transformation transform normal cells to cancer cells Retroviruses Are RNA Viruses that replicate with a DNA intermediate AIDS in this group Used for gene therapy integrate in the host genome Have reverse transcriptase called this because it makes DNA from an RNA template Have two identical SS RNAs Both are plus sense but are Hbonded together so are not read as mRNA 9 RNA Virus with DNA intermediate 9 lifecycle is What is different lalll U U JG RNA Enzymes reverse r transcriptase integrase 39 protease quot Lipid membrane b ayer Core shell protein Core protein a Retrovirus replication Formation of proviral state and transformation Into tumor cell quot lltY9r Virus 6 T I Virus multiplication 4 virus without Shiv release of 0 causing cell death Tumor con Transformation 49 Surface envelope protein Transmembrane envelope protein quot i39 cell and release Ly ofthevirus A T quot 0 4 I g I I O 0 39 2 I Persistent 39 infection I I Virus present 39 but not m39 39Jquot greplicating 1 quot T r 39 May revert to lytic infection 1 Virion enters the cell by endocytosis fuses with the host cell membrane at a specific host cell receptor 2 Reverse transcriptase brought in copies one of the two RNAs into DS DNA by carrying out three functions 0 synthesizes complementary DNA from RNA template 0 degrades the RNA of the RNADNA hybrid acts as a 0 ribonuclease 0 during degradation of the RNA strand synthesizes 0 complementary DNA strand to produce Viral DS DNA 3 DNA integrates into the host genome using Viral integrase 4 Transcription of Viral DNA to both Viral mRNA and more Viral genomic RNA 5 Production of Viral nucleocapsid in the host cytoplasm 6 Release by budding through the host cytoplasmic clel membrane envelope acquired 9 brings in own enzyme RT 9 overall function by carrying out 3 specific enzymatic functions 9 don t worry about WHERE things are happening 2 single strands plus sense H bonded synth complementary DNA 9 degrades RNA part synth new DNA double stranded DNA DNA is now made from single stranded RNA 9 integrated into host genome 9 AIDS so hard to treat because the body does not treat it as a foreign body and doesn t attack it Herpesviruses Large group of DS DNA Viruses viral genome 1 Entry and uncoating ol the mtrnvlrus sol NA 2 Reverse transcnptasa Q activity two steps d3DNA 3 Vlral DNA ontors nucleus and H0 DNA Wm DNA 1 lnugratu into the host genome polymerase lorrns viral mRNA 4 Transcription by host RNA and gonomo copies BQRNA 5 Translation of mRNA forms viral proteins new nucleocapsid assembled and released by budding Host cytoplasmic membrane Many cause diseases cold sores herpes simplex venereal herpes chicken pox shingles infectious mononucleosis cancer Epstein Barr virus Some are latent with symptoms caused by stress Varicellazoster virus chicken pox 9 shingles Are structurally complex consist of four units Core is DS DNA linear Core surrounded by an icosahedral nucleocapsid Nucleocapsid is surrounded by a tegument brous structure unique to herpesviruses Tegument surrounded by an envelope with spikes Herpesvirus genome is large codes for at least 84 proteins Infect cell by adsorbing to specific host cell receptors followed by fusion of viral envelope with the host cell membrane Nucleocapsid enters the cell and migrates to the nucleus where viral DNA is uncoated All macromolecular synthesis in the host is shut down by proteins brought in by the virus Host machinery is taken over to produce three classes of viral mRNA Code for immediate early delayed early and late proteins Immediate early five regulatory proteins Delayed early DNA replication proteins Late structural proteins capsomeres tegument and spikes 9 integument In uenza virus 9 u airbom Viral nucleic acid is negativestrand minus sense RNA SS 9 cannot be read as mRNA only RNA can be read as mRNA Is an orthomyxovirus myxo interacts with mucus or slime of a host cell surface ex mucus of respiratory tract ortho distinguishes this group from paramyxoviruses another group of negative strand RNA viruses Viral RNA is transcribed to RNA by RNAdependent RNA polymerase RNA replicase brought in by the virus synth RNA from RNA Is an enveloped virus Is polymorphic no de ned shape 9 not symmetrical like others Acquires envelope by budding through the host cell membrane 9 hiding from host cell Has protein spikes on the surface that interact with the host cell Hemagglutinin binds to sialic acid in mucus cell membrane adsorption site 9 specific receptor called this since it agglutinates red blood cells also have sialic acid antibodies made against this Neuramindase breaks down sialic acid For infection virion is taken up by endocytosis Replication occurs in both the nucleus and the cytoplasm once inside the nucleocapsid leaves the envelope and migrates to the nucleus Viral RNA is replicated in the nucleus using viral replicase and viral endonuclease also brought in In uenza RNA is present in 8 separate pieces Is a segmented genome Codes for 10 proteins When virions assemble in the host cytoplasm different segments from different u viruses can be packaged together 9 leads to antigenic shift spiral nucleic acid that code for 10 proteins different packaging W shit the antibodies made from injection will no longer Work Antigenic shift changes virion surface properties no longer recognized by host antibodies when released Also have antigenic drift accumulation of mutations that also change virion surface properties Have to guess at vaccine development each year Viroids Prions and Prokaryotic Genetics Viroids Are the smallest known pathogens 246 to 399 nucleotides infect plants ONLY Are small circular SS RNA molecules with no capsid exist extracellularly as naked RNA Do not code for any proteins only more viral RNA Are completely dependent on host machinery since don t code for their own proteins Virions are circular SS RNA but they form base pairs 9 complementary binding not like plasmid Along their length so are not plasmidlike and no protein coat Enters a plant host cell through a wound Move from cell to cell through the plasmodesmata like plant RNA viruses or through the plant vascular system prions move along W water mov in cells through plasmodesmata Viroid replication is in the plant nucleus or chloroplast Done by plant RNA polymerase Since the viroid NA is circular result is one long RNA molecule with repeating viral RNA sequences Part of the viroid RNA has ribozyme activity can cleave the long molecule into individual viroids Many plant diseases are caused by viroids No viroids are known to infect animals or prokaryotes Infections in plants can be symptomless mild or range to lethal Mechanisms of pathogenesis are not known Prions NO DNA or RNA but infective proteins Are infective proteins they have no nucleic acids Have an extracellular stage pure protein which is infectious Are not killed by radiation Cause diseases in animals Mad cow disease scrapie in sheep CreutzfeldtJakob disease in humans chronic wasting disease in deer and others none are known to infect plants Prion diseases are transmissible spongiform enchephalopathies Infections produce holes in the brain tums it spongelike 9 don t worry about mechanism Normal hosts that codes for R prionli lte protein p is Prnp lt66p1 i 111fD1 lii1199gt 2 j pH product is prion protein cellular Neuronal cell is normally in healthy neurons p in the Infectious prions LP1 PS prion protein scrapie which 5 but conformation a 0c lot of ozelielices but of LP1 PS is LB 5 do not perform riormal function 5 5 5 5 insoluble p in p rieural tissue ano forming holes V39 C1 A A A On O A A mOnf W V7lf 1E1Li 1i Pgt the host cell it interacts Wltli i 9 1 On A A C1 A O L normal it to the LP1 P form this J is irifectious prion not just on its own but changirig the riormal proteins opmo 6 O on O 6 mm m D po M n mm 7AAfA m mp o f f G also S gporadio prion in Wliiclri r1orrnalPrP is imsfolded to produce the PJJQQ form which can 7 theii convert other molecules that produce once iii 0 million people is also iriheriteol priori Prokaryotic Genetics and Genetic Engineering Bacterial genetics another entire field of study Most basic research on genetics has used microbes Why Much involved mutant strains Mutation inherited change in the sequence of genomic nucleic acid Genetic recombination genetic elements in two separate genomes brought together to form a new genome Any characteristic of a bacterium can be changed by mutation pigmentation motility nutrition antibiotic resistance etc Many mutants can be detected Visually motility pigments Some can t but can often detect using replica plating Replica Plating come up with 2 identical plates with the same colonies Velveteen bunch of inoculating needles and stamp it on fresh media same colonies and same orientation used to try and identify a mutant you cannot see manipulating the properties and genetics of the bacteria Master plate growth on complete medium Pm plate onto Transfer imprint Nam of colonies to 3 39 1 fresh media Replica plating works when you put the mutant s fequotr39ifff 1 1quot on a plate that it now can t grow on for i la sFtiG if39 tquot V 39 0 jzmphge medium example antibiotic presence ico n go mies 7 1 A Nutritional mutants need a new growth factor rm 39 Mutant is an auxotroph for the newly needed quotquotquotiquot quot 39quot quot 39 growth factor wild type is the prototroph quot quotquot 3 P P P 39 gg 9 How to form your mutant and then use it to grow 1 P h JrJ 7 iquotHf 39 C39J 39 1 comma medium 2 wrutarr u3 to run mcclu cuiquotu39 putt that specific colony MullI plau owth Replica plating to detect an auxotroph ofoomplvtrmv um Master plate growth on complete medium Transfer imprint of colonies to fresh media Press plate onto Minimal medium does not i Wm contain the growth factor y0u f6 I F tarii zacwi x M v 5 p 39 Vel ete n quot39 8 intafastad in w o3 0 gfitalinimg nt W Complete medium All colonies grow quotquotw d quot colonies v 39 39 b39 quot i U 3 I 9 only prototrophs can grow Minimal medium Mutants do not grow when you compare the plates you compare the colonies that do not grow on the minimal media 9 work out their metabolism Can also detect mutants using penicillin selection or other bacteriostatic antibiotic that inhibits growth Inoculate the culture with hopefully a mutant on the appropriate minimal medium and add penicillin or other antibiotic incubate cells that can grow will die Wash away the penicillin and incubate now cells mutants can grow and be isolated 9 the ones that grow have the mutagen with penicillin To make a mutant expose your bacteria to a mutagen Assigned reading section 102 know molecular basis of mutation base pair substitution frameshifts insertions deletions The Ames Test testing for mutagents but mostly carcinogen Many mutagens are also carcinogens not all but a high proportion 9 a lot of mutagens are carcinogens Basis of the Ames Test a mutagenicity test for carcinogens 9 uses engineered bacterial strain with easily detectable mutagen can back mutate to the Wild type Makes use of bacteria with a mutation that s easily detected and that can back mutate to the wild type usually also has poor DNA repair enzymes Often use an auxotroph that will backmutate to the prototroph the mutation is usually a point mutation Colonies that grew on the left had naturally occuring back mutations frequency of spontaneous mutations is 10396 to lO 7 9 More colonies on the right indicates mutagenesis Subtances testing positive in the Ames Test are suspect carcinogens need to further prove Often the test includes rat liver enzymes since many carcinogens only become carcinogenic and mutagenic when exposed to enzymes mixed function oxygenases in the liver that normally detoxify substances 9 if it back mutates to prototroph 9 positive result accumulation of back mutated substance because it diffuses through the agar 9 high probability that this is a carcinogen increase rate of back mutation suspect carcinogen The Prokaryotic Immune System CRISPR Prokaryotes have a system CRISPR that recognizes both RNA and DNA viruses and some foreign DNA brought in by conjugation CRISPR Clustered Regularly Interspaced Short Palindromic Repeats are areas on the chromosome of the prokaryote are present in 90 of Archaea 70 of Bacteria Consist of sequences corresponding to viruses the strain has encountered which alternate with identical repeated sequences palindromes CRISPR system also has CRISPR associated proteins CAS proteins coded by CAS genes on chromosome upstream of CRISPR Can recognize the viral sequences on CRISPR Cuts transcribed CRISPR RNA at in palindromic areas so Viral sequence RNA is released CRISPR RNA can then associate with corresponding Viral sequence on Virus CAS proteins Mummy Bactenal CHIOCVIOSOME ggqucncgg recognize l CRISPRVirus complex on 39 1 i F and destroy the Viral 8W R0para PP V Ir1r rI v 1v 391 NA C pmuins ram Ipn1 and V trarvnrv r crusrn RN A 39 K J uv I L CI1 n c om 3939 fa 3939c1391 R Many prokaryotes destroy foreign DS DNA including i A L i i v D Viral Viral DNA with restriction 39quot quot quot 39 395 COOJUIIUOO Vlfll or plasmid DNA enzymes I 39 39 ffquot 39 quot 39 Cut foreign DS DNA I ampv3 3 M r n43ltMmi i s not own DNA own DNA is modified at site where enzymes cut restriction sites Some DS DNA Viruses have modified those sites in their own DNA resistant to this mechanism 9 understand 1028 Genetic Engineering the isolation manipulation and expression of genetic material Used in both pure genetic processes and applied antibiotic production microbiology Makes use of natural and unnatural bacterial genetic recombination mechanisms Unnatural artificially induced competence if its competent it takes up DNA automatically electroporation mainly splasmids Arti cially induced competence Only some bacteria are naturally competent Can treat E coli lab rat with high Ca and cold and they cells can be transformed Electroporation allows cells to be transformed and to take up plasmids Method expose cells to short high Voltage electric pulses opens up small pores in cell membrane and DNA or plasmids added to media can pass through Both natural and unnatural genetic recombination of bacteria can be used to make genetic maps create new strains and study biological processes at the genetic level Also important are in Vitro techniques often used in combination with in Vivo experiments In Vitro does not inVolVe living cells Restriction enzymes restriction endonucleases Are prokaryotic enzymes that recognize specific sequences of DNA cut DNA at that site Evolved to protect cells from foreign DNA Usually recognize sequences that are 4 to 6 base pairs long Bacteria with restriction enzymes most don t cut up their own DNA because shortly after replication DNA is modified at restriction sites by methylation carried out by modification enzymes 9 3D structure Wont let it bind and therefore cannot cut Necessary because with only 4 nucleotides a specific sequence of 4 has a probability of l in 256 4x4x4x4 of 6 has a probability of 4096 4X4x4X4x4x4 not rare 9 look to see Where the sites are going to cut Sometimes R for restriction Since DNA has two complementary strands restriction enzymes recognize sequences on each side of the complementary doublestranded sequence The enzyme cuts the single strand at each recognized site cuts both single strand Can cut both strands opposite each other and cleave the DNA Ex BsuRI Can also out both strands at different points Ex EcoRI Result is two single stranded sticky ends Figure 111 Restriction and modi cation of DNA Sticky ends are good for cloning genes Example of modification of EcoRI restriction site No longer out here by EcoRI 9 this is how you do clone genes that have compliments 1 9 example of modification 9 presence of methyl means it cannot be cut the substrate is 5 G AATIc 3 the DNA and the enzyme cannot reach it due to the methyl EcoRl Ecollv I 1 I 1 1 3 539 9 eAsA rre 339 s39 ecAIArc 4 339 Singlestranded Doublestranded quotstickyquot ends quotbuntquot ends Singlestranded stickyquot ends a 8 Use in genetic engineering can cut DNA and insert new genes usually with complement to sticky ends Available in catalogues are several thousand available Molecular gene cloning Isolation and movement of DNA using a Vector Three main steps of gene cloning 1 Isolation and fragmentation of source DNA 2 Insertion of DNA fragment into cloning vector 3 Introduction of cloned DNA into target organism 1 Isolation and fragmentation of source DNA o source DNA can be genomic DNA RNA or PCRamplified fragments must first be restriction digested 2 Insertion of DNA fragment into cloning vector o most vectors are plasmids or viruses o DNA is inserted using DNA ligase an enzyme that o joins two DNA pieces 3 Introduction of cloned DNA into target organism 0 usually by transformation Plasmids as cloning vectors Plasmids are natural vectors useful properties as cloning vectors small size easy to isolate plasmid DNA independent origin of replication for control multiple copy number can get multiple copies of cloned gene per cell often have selectable markers can detect Insertion of the plasmid by chemical transformation or electroporation Plasmid pUCl9 pUCl9 is a common cloning vector Is a modified naturally occuring plasmid Modifications ampicillin resistance and lacZ genes ampicillin used as a marker to tell your plasmid has been taken up lacZ a marker for DNA insertion has a polylinker multiple cloning site lots of restriction sites for gene insertion within the lacZ gene 9 you Want to get the plasmid in BUT also make sure that gene is in there and be able to identify it To see if you have inserted the plasmid do antibiotic screening ampicillin resistance marker plate onto plates with ampicillin colonies that grow have the plasmid To see if you have inserted the new gene into LacZ do LacZ screening marker is blue vs white colonies LacZ gene codes for 3galactosidase presence intact LacZ detected if plated onto media with Xgal Xgal a colorless reagent that turns blue with Bgalactosidase activity intact LacZ gene 9 We want to see if 1 Have plasmid in prokaryotic cell 9 antibiotic screening if colonies grow they have plasmid 2 Plasmid with new gene 9 LacZ screening using restriction sites in LacZ gene cleave and reanneal blue or white Blue colonies do not have new DNA inserted LacZ is intact White colonies do have new DNA inserted into LacZ so 3galactosidase expressed Based on insertional inactivation of new DNA the lacZ gene is inactivated by insertion of new DNA inactivated lacZ cannot target Xgal blue color does not develop act AmpR t 9 Hosts for cloning vectors vector F 39 399quot DNA 9 host has to have specific properties 1 Digestion with restriction enzyme 1 Properties of good hosts D T 5 capable of rapid growth in inexpensive medium doubling time like 20 mins 3 nonpathogenic Joquot Wm capable of incorporating DNA when moving opened cm 0quot quot u make more plasmid increasing copy of the genes genetically stable in culture nonmutating Can replicate the vector for plasmid to gene prod Recyclized vector without insert Vector plus foreign Examples Escherichia coli Bacillus subtilis DNA insert irenstorm IHIO Escherichia Saccharomyces cerevisiae con and met on ampicamn plates containing Xgal rransformants blue Egalactosidase active Transformants white Bgalactosidase inactive Chang QQA4g Aul CIA1 I A A Phage lambda as a cloning vector Cloning with lambda multiple copies of the gene and move it 1 Isolate DNA from lambda Virion open capsid and cut it with restriction enzyme 2 Connect the lambda fragments to foreign DNA using DNA ligase lacZ and multiple cloning sites foreign gene isolated gene with sticky ends 3 Package the new DNA adding cell extracts containing the head and tail proteins Infect E coli cells and isolate cells with lambda clones by picking plaques 5 Check for the presence of foreign DNA Baeteriephage M13 as a elehihg yeeter Carl elerie up te 5 kileleases ef DNA Alse eeritairis laeZ fer hleeWhite sereeeirig lll all ef these DNA eari lee iriserted irite the yeeter Whether it is a plasrhid er a phage arid esed te rheye DNA irite the leaeterial hest their haye rheltiple hestsrrmiltiple plasmids Firial teel Pelyrrierase Chairi Reaetiee PCR fer arriplifieatierl Eleela 2 laterlt yires Microbial evolution and metabolic diversity l2l Ferrriatierl arid Early Histery ef Earth 0 The Earth is 45 Dllll il years eld 0 First eyideriee fer rhiereleial life eari lee feerid ill reeks 386 Dllll il years eld Figere l22 0 Plariet Earth is aleeet 45 Dllll il years eld 0 The first liyirig ergariisrres err Earth Were rhierelees 39Evenmquot pd b aym took the aim at rnlnera compartments allowing the e EVidm 39 rst cells to dispense to new habitats j O Bierharkers er eherhieal sigriatdres ef life iri arieierit reeks these eerresperld te eerrerit leielegieal preeess marry erily rreiereleial 0 The fessil reeerd the earliest fessilized reeegriizalele D D D D D D icomputmentsl life ferrhs are strematelites er fessilized mierehial gallow coupling E l of ecrgngettsc ma S Ok OTIS 0 molecular I grepltcalion I V139L T k E Dldest lltiil Wil strerhatelite is 35 leillierl years eld 39 feerid ill Western Aiistralia 1 Mmetal pores alse iridiyidelal eells reek reeerd lll Seiith Afriea igt39quot 3939 I compartmenui Dee leillierl year eld rhierefessils Dldest fessils leek like rhederri day Chlere exes arid eyarleleaeteria The three domains of life are considered to have evolved from a common ancestor the last universal common ancestor or LUCA based on the presence of similar structures and functions in all living organisms examples 9 NDA and RNA first individual cells formed populations populations interacted competition etc and got ongoing evolution shaped by extemal environment Earliest life 386 billion years age before fossil record based on biomarkers and isotope ratios 0 Major events included oxygenation of Earth due to cyanobacterial evolution of oxygenic photosynthesis which also involved formation of the ozone layer protocyanobacteria 0 Before this event the atmosphere was mainly N2 and CO2 plus some others 0 First organisms believed to be methanogens and other microbes that could carry out anaerobic energy yielding processes including anoxygenic photosynthesis 0 Earliest bacteria methnophotosynthesis hypothesized Accumulation of oxygen the great oxidation event was followed by the appearance of multicelled life forms eukaryotes and aerobic respiration 9 multicellular forms of life Also 02 in the atmosphere iswas converted to ozone 03 which absorbs light up to 300 nm ultraviolet light Formed an ozone shield which protects life from lethal DNA damage from UV light were concems that the current ozone shield was being depleted due to release of gases like freon that destroyed it Molecular Phylogeny Pioneered by Carl Woese in the 1970s Showed the three domains of life based on analysis of ribosomal RNA rRNA Worked with small subunit RNAs l6S rRNA for prokaryotes 18S rRNA for eukaryotes Properties for use of these are conserved change very slowly over time are functionally constant basis of being conserved are long enough to allow comparisons chains of only 4 nucleotides 9 highly conserved function very important in DNA translation crucial role makes it stable mitochondria from Proteobacteria chloroplasts arose from Cyanobacteria the endosymbiotic hypothesis Molecular phylogeny is based on comparative rRNA sequencing routine procedure amplification of the gene encoding SSU rRNA single subunit RNA sequencing of the amplified gene sequencing DNA that codes for the RNA analysis of sequence in reference to other sequences are online databases GenBank 16S rRNA or 18S rRNA for eukaryotes Phylogenetic trees are graphs of the relationships among sequences of different organisms contain nodes divergence in sequence and branches number of changes along lineage branches define the order of descent and ancestry of the nodes branch length represents the number of changes that have occurred along that branch evolution and relatedness The species concept in microbiology Big question what is a prokaryotic species prokaryotes are haploid no sexual reproduction but are lots of Ways to get new DNA from other sources horizontal gene transfer 66 Today consider prokaryotic species as phylogenetic species a group of strains that based on DNA sequences of multiple genes cluster closely with others phylogenetically and are distinct from other groups of strains There is no universally accepted concept of species for prokaryotes just philo relatedness defined Current definition of prokaryotic species Collection of strains sharing a high degree of similarity in several independent traits Most important traits include 70 or greater DNA DNA hybridization and 97 or greater 16S rRNA gene sequence identity BLAST search in GenBank but some strains with identical 16S rRNA sequences are very different Why other traits are considered Naming a new prokaryotic species is based on the International Code of Nomenclature of Bacteria using two main references Bergey39s Manual of Systematic Bacteriology The Prokaryotes Oxygenic and anoxygenic photosynthesis Oxygenic photosynthesis Is a major metabolic process found in all higher plants eukaryotic microalgae and some Bacteria cyanobacteria Will be presented in three Ways 1 An overview of What happens in this metabolic process 2 How oxygenic photosynthesis is organized in terms of e ow using the e tower 3 How electron transport is organized in the cell membrane Steps in oxygenic photosynthesis 1 Light energy is absorbed by pigments photosynthetic pigments Vary for oxygenic photosynthesis the photoreactive pigment Which is involved in transfer of light energy to chemical energy is chlorophyll a also needed are lightharvesting pigments Three major groups of pigments for ETS associated photosynthesis Chlorophylls eukaryotes cyanobacteria Green sulfur bacteria and bacteriocholorophylls bacteria Can be photoreactive or lightharvesting Carotenoids all three domains mainly photoprotective but some are lightharvesting Phycobiliproteins cyanobacteria and some eukaryotes ex red algae lightharvesting Each pigment has a unique absorbance spectrum Each organism has more than on type of pigment Absorbance spectra are ecologically relevant Bloom of purple bacteriatop with a green microalga Reaction center photosynthetic pigments Generalized view Figure 134 Arrangement of lightharvesting chlorophyllsbacteriochlorophylls and reaction centers Within a photosynthetic membrane Are specialized structures in different phyla For each group photosynthetic pigments and ETS are associated with membranes Eukaryotic microalgae as Well as higher plants Pigments are in chloroplasts membrane bound 9 KNOW THE ELECTRON DONORS FOR EACH and how they make ATM cyclic or noncyclic if goes to NAD Z scheme Steps in oxygenic photosynthesis 1 Light energy is absorbed by pigments photosynthetic pigments vary for oxygenic photosynthesis the photoreactive pigment which is involved in transfer of light energy to chemical energy is chlorophyll a also needed are lightharvesting pigments 2 Energy from light boosts an e to a higher 0 energy level more electronegative o the e donor is H20 o 02 is produced as a byproduct oxygenic 3 As the e moves to a lower E level in a controlled process a chain of relatively more positive e carriers E is converted to chemical energy by moving protons across the membrane pmf 4 Both ATP and reducing power are produced ATP from the pmf produced ATP the electrons end up reducing NADP 5 NADPH H and ATP are then used to reduce CO2 to organic carbon CO2 fixation Oxygenic photosynthesis presented on the electron tower is called the Z scheme of photosynthesis since the path of electrons follows the shape of a Z In this scheme each compound the electrons are associated with including the donor are positioned on the tower at the level of their The two main types of cellular energy what are they are produced in the two paths one in one path one in the other Figure 1318 Electron flow in oxygenic photosynthesis the Z scheme The two main types of cellular energy what are they are produced in the two paths one in one path one in the other If the electron keeps getting boosted at PSI it is called cyclic photophosphorylation If the electron goes all the way to NADP it is called noncyclic photophosphorylation Why is it called this since reducing power is formed Energy for photosynthesis is from the photoexcitation of a lightabsorbing pigment Photoexcitation leads to photolysis the lightdriven separation of an electron from an electron donor For anoxygenic photosynthesis electron donors include H2 Fe2 H2S S2032 and S but not H20 why ETS associated photosynthesis Each electron is transferred to a membrane associated ETS as the electron is passed along the ETS pmf is produced how the electron may 1 end up reducing NADP not NAD or 2 be boosted over and over to produce pmf the above two are called 1 noncyclic and 2cyclic photophosphorylation Four types of ETSassociated anoxygenic photosynthesis carried out by 0 Cyanobacteria only some use only PSI 0 Purple bacteria 0 Green sulfur bacteria 0 Heliobacteria Each type of psn is supported by a different taxonomically distinct set of photoreactive photoprotective and lightharvesting pigments Oxygenic photosynthesis review Anoxygenic photosynthesis carried out by some cyano bacteria Electron ow from PSII is blocked often by H2S Electrons come in at PSI Donor can by H2S or H2 Some cyanobacteria can have PSI and PSII operating at the same time more metabolic exibility Purple Bacteria Reaction center is P870 Electron donors H25 S2032 So and Fe2 Need reverse electron ow to reduce NADP Reverse electron ow energy is used to force an electron to a more electronegative carrier NAD powered by pmf Figure 1315 Arrangement of protein complexes in the purple bacterium reaction center Photosynthetic electron transport is organized in the thylakoid membrane in the same Way as in other energy yielding membrane associated electron transport pathways respiration lithotrophy Overall comparison of photosynthesis of Purple bacteria Green sulfur bacteria and Heliobacteria Photosynthesis of Purple Green sulfur and Heliobacteria Electron donors H2S S2O3239 H2S H2 Fe2 Organic S Fe2 carbon Note the different Eo levels of each photosystem where the electron is boosted Which types need reverse electron transport to reduce NAD What do you need to know Cyanobacterium using sulfide as electron donor What would you see if it was using water Photoheterotrophy Many photosynthetic bacteria and cyanobacteria can grow by photoheterotrophy Some are facultative photoheterotrophs cyanobacteria some obligate Chloro exus Chemolithotrophy and anaerobic respiration Lithotrophy Energy is from the oxidation of inorganic electron donors Is another ETSassociated energy yielding metabolic pathway in which the energy source is chemical Different from anaerobic respiration for which the electron donor is an organic carbon compound In lithotrophy the terminal electron acceptor can be organic or inorganic Once again these metabolic modes are based on E0 and the difference between the electron donor and the terminal electron acceptor Which pair has the most potential energy available Electron donors that support lithotrophy Are many the most common are hydrogen various reduced sulfur and iron compounds and metals Each has a different amount of potential energy depending on its Eo Energy yield also depends on the of the Note that both the energy source and the terminal electron acceptor are shown to calculate energy yield 9 knowing the electron donors and acceptors For lithotrophs the carbon source is often CO2 but some can use organic carbon Each type of lithotrophy is associated with an ETS sometimes overlap with other ETS metabolic pathways The initial electron accepting oxidoreductase is always specific for the electron donor can look for these genes in genomes to assess metabolic Membraneinteg rated potentlal hydrogenase i E clttron transport generates 3 proton 2 Hydrogen oxidation quot2 motquot quot quot39e39 2 Out 1 p I A Molecular hydrogen H2 is the electron g 39 39 3 39f 0 s C O 1 C I E0 of the redox couple 2HH2 is 042 V This is low enough to donate an equot to R L nearly all biological electron acceptors 0 o o o v r including NAD In This means many compounds can be used as the terminal electron acceptor NADH Examples of hydrogen ox1dat1on cytomasmic A1 pathways hydrogenase 0 ETS There can be both membrane bound and cytoplasmic hydrogenases associated with hydrogen lithotrophy 0 ATP is produced by the membrane bound type and NADH H by the cytoplasmic type 9 recognize H S Fe nitrate lithotropy 9 will have one of them Figure 1320 Bioenergetics and function of the two hydrogenases of aerobic hydrogen bacteria 9 Will ask which is the electron donor and acceptor check arrows 9 Ends with ace splitting something Most hydrogen bacteria are facultative chemolithoautotrophs They prefer to grow chemolithoheterotrophically but when no organic carbon is available they grow chemlithoautotrophically 6 H2 2 02 C02 9 CH2 5 H20 Are microaerophilic growing best at 5 10 02 This is because their membrane bound hydrogenase is extremely sensitive to oxygen When considering compounds other than hydrogen that are electron donors for lithotrophy first look at the entire spectrum of oxidationreduction reactions for that compound most oxidized to most reduced or vice versa C8 material FT Sulfur lithotrophy Oxidationreduction pathway H2S gt So gt l2 S2O32 gt SO32 gt SO42quot Hydrogen sulfide elemental sulfur thiosulfate sulfite sulfate The first three are normally used as an energy source for lithotrophy Usually O2 is the terminal electron acceptor but some can use nitrate NO3 As we ve seen before if sul de is oxidized to S it is deposited intra or intercellularly Sulfur lithotrophy and production of reducing power The E0 ofNADNADH H quot is 032 V For S0H2S it is 029 V For S4O62 s2o32 it is 024 V This means sulfur lithotrophs need to use reverse electron transport powered by the pmf to reduce NAD 9 neither is negative enough to reduce NAD therefore forces electron to go to something that s more 919CtT011 gatiV PmfP0W f d Hm tum hmspnt tv39H39YU 39 prT um HHIIV fotco Out OV Q ooossso oooosoeoco Sulfur lithotrophy 9 NAD should not be in the membrane but outside of it Reverse e o 0 cc NAD I 1 1 I I I Note that the three electron J H I 11 5 I U L 1 donors all of which are oooocououoouo skaos u O I common in the environment In S1x 2032 or 02 4 39 come in at two different points 3 in the pathway E Y 39 o quot20 Cell quot quot 39 material ADP ATP Which is the best electron 1 donor Most reduced sulfide elemental sulfur This is why elemental sulfur is deposited inon the cell Iron oxidation Oxidationreduction pathway Fe2 gt Fe3 Ferrous iron ferric iron The ferrous form is favored by anoxia and low pH the ferric form by oxygen presence and high pH So iron oxidizers must live in very acidic environments with oxygen present Growth on iron occurs with this reaction 2Fe2 20242H 2Fe3H2O E0 2 O2H20 is 082 V and of Fe2Fe3 is 077 V So there is a very short ETS and reverse electron transport is needed to reduce NAD ETS includes a periplasmic enzyme called rusticyanin that removes the electron from Fe2 The pH of the environment of iron oxidizers is usually 12 pH units Since the cytoplasmic pH is 55 to 60 iron oxidizers can use this natural gradient for pmf Problem ATPase allows protons back into the cytoplasm making it acidic bad Problem solved by using these protons plus oxygen and the electrons from iron to make water Growth by iron oxidation does not yield much energy why 9 its very small So LOTS of iron has to be oxidized precipitates in the environment 39 Electron transport gnnorzitos a proton I N 5 motive force Oxsdation of 239 hydroxyammo 49 20 Q stcscoio 5 oi Ammoniaoxidizing bacteria Oxidize ammonia The product is nitrite The nitrite produced is the electron donor energy source for the second group NH39H NH 239 e ADP Oxadahon at S Roduchon P A11 ammomn of oxygen 2 Nitriteoxidizing bacteria 0 Oxidize nitrite 0 The product is nitrate 0 In anaerobic environments nitrate is an important terminal electron acceptor for anaerobic respiration Nitrite oxidation Electron transport T NXR n1tr1te oxidoreductase genumtz 9 a proton motive fotce x Figure 1327 Oxidation of NO2 to NO3 3 quotT g V y D by nitrifying bacteria ii i 39 i V The first two groups utilize oxygen and bacteria that can do this are obligate aerobes K K Reverse e ow But there is also ammonia oxidation in anoxic 1 k 0 make NADH environments the basis of the third group 3 Anoxic ammonia oxidizing bacteria 0 Anoxic ammonia oxidization an A P R ductoon D T Oxidation iof mtritrr I anammox of oxygen P 0 Ammonia is the electron donor 0 Nitrite is the electron acceptor 0 The product is N2 NH4 N02 gt N2 2H2039 The anammox reactions occur in anammoxosomes which are membrane bound like organelles of Eukaryotes and take up about half of the cell Anammoxosomes do not have normal cytoplasm They contain no ribosomes The membranes are not phospholipids but are fatty acids with cyclobutane rings bound to glycerol with ester and ether bonds Are called ladderane lipids and are very dense to the point that nothing can diffuse across them This is necessary since one intermediate in the reaction is hydrazine N2H4 extremely toxic extremely strong reductant The three groups of nitrogen oxidizing bacteria live in close association with one another Why 9 the enviro has what is required to grow if you have one that produces one substrate then its enriching for the other What characteristics will this kind of environment have to have 9 have a gradient of oxygen and no oxygen Anaerobic Respiration Respiration with a terminal electron acceptor other than oxygen The electron acceptor is reduced and the end product is excreted to the environment a byproduct If the acceptor is nitrate or sulfate both are nutrients the process is called dissimilatory nitrate reduction or dissimilatory sulfate reduction If energy is used to reduce nitrate or sulfate to be used by the cell as a nutrient it is assimilatory nitrate or sulfate reduction Major forms of anaerobic respiration showing the terminal electron acceptor and the reduced byproduct Where is the electron donor The electron donor and electron acceptor used is based on 1 the availability in the environment 2 the energy yield The presence of a better terminal electron acceptor usually suppresses synthesis gene expression level of reductases for alternate electron acceptors ex oxygen represses genes for nitrate reductase nitrate represses genes for fumarate reductase 1 j Nitrate respiration Nnrate N03 Nitrate First consider the oxidized forms quot 39 d reduction of nitrogen Escherichia name cow NO3 NO2 N20 Nitrite TBUUCYBSG gt l2 N2 Denitri cation N1trate n1tr1te nitric oxide n1trous Nlt c oxide D pseudomonas oxide nitrogen gas Nitric oxide reductase 5 031971 Respiratory reduction occurs Gases itous gxide Successively in Steps Nitrous oxide reductase In general any given bacterial species can carry out only one or Dinitrogen M two transformations in the series Each step is carried out by a specific enzyme Nitrate Reduction and Denitrification Biochemical pathway for dissimilative nitrate reduction has been well studied Enzymes of the pathway are repressed by oxygen Often facultative anaerobic respirers directly use part of the aerobic respiration pathway Figure 1341 Respiration and nitratebased anaerobic respiration Aerobic and anaerobic nitrate respiration Ecoli Comparison of pathways These paths share which part of the ETS Where is the branch What determines which branch will be used Oxygen aerobic oxygen and nitrate aerobic nitrate anerobic Nitrate respiration this pathway involves one reduction nitrate to nitrite Some bacteria denitrifiers can transfer electrons to all of the oxidized forms of nitrogen resulting in N2 as the byproduct The additional steps generate more pmf Complete path of dissimilatory nitrate reduction 9 NOT IN THE EXAM Dissimilatory nitrate reduction is specialized among a few prokaryotes energy YIELDING form Assimilatory nitrate reduction is common carried out by prokaryotes and eukaryotes Is enzymatic and energy consuming Is the reduction of nitrate for use assimilation as a nutrient 9 pathways that overlap and the differences Sulfur respiration Sulfate is also reduced step by step in anaerobic respiration Oxidized to reduced forms of sulfur Most reduced to most oxidized lithotrophy Most oxidized to most reduced Anaerobic respiration so42 gt so32 gt 12 S2O32 gt s0 gt H2S Sulfate sulfite thiosulfate elemental sulfur hydrogen sulfide Sulfate respiration is very common in marine environments there is lots of sulfate in seawater Dissimilatory reduction of sulfate first requires activation by ATP because S042 is too stable forms the compound adenosine phosphosulfate APS what does this remind you of 9 first step of fermentation of glucose First step activation ATP For assimilatory sulfate reduction a second phosphorylation of APS occurs to form PAPS phosphoadenosine phosphosulfate same activation step second phosphorylation occurs too 9 PAPS ATP ADP Figure 1414 Biochemistry of sulfate ATP PP V reduction Activated sulfate 5042 ATP sulfmyhs PS APS k39inase PAPS The first part of the assimilatory and dissimilatory sulfate reduction pathways W APS are shared Kfductase NADP Then the pathways branch 4 AMP PAP These pathways are in the cytoplasm 5032 5032 For dissimilatory sulfate reduction m electron donors must be more Smme 1 reductase lgt electronegative than 0 17 volts sulfate sulfide 9 donor for sulfate S03 2 9 must be 0 above it on the table more EN quot2 quot25 Excretion Organic sulfur compounds cysteine methionine and so on Dissimilative Assimilafive sulfate reduction sulfate reduction Oxidation of H W generates a proton motive force 401 8 8e V Q 39v O I 0 0 Q I 0 9 recognize the pathway i Aps I 6 9 Electron donors Hydrogen Lactate pyWage e ADP ATP ANDOR lactate Ferredoxi I 803239 quot2 ANAEROBIC RESPIRATION 9 Acemte CO2 A1P quot25 Nitrate amp Sulfate Dissimilatory product will be used and gotten rid of Can also have anaerobic respiration of metals Most common are iron and manganese oxidizers These reactions are also considered to be dissimilatory energy yielding with byproducts precipated as iron or manganese oxides that accumulate in the environment 9 image table not in exam Anaerobic environments such as sediments in aquatic environments or stratified layers in lakes usually have a series of different electron acceptors As each successive acceptor is depleted its reduced form appears and the next best electron acceptor is used generally by a different species Organisms at the surface will be aerobic 9 closer to the atmosphere Fermentations Review of fermentation NO extemal e acceptor NO e transport chain NOT membrane associated ATP is formed enzymatically by transfer of PO4 from phosphorylated compounds in the fermentation pathway to ADP NAD musts be regenerated 2nd state is energy yielding and requires NAD So far one example three stages of glucose fermentation in terms of overall processes Are different fermentation pathways Fermentations are classified by either the substrate fermented or the products formed Many organic compounds substrates can be fermented 9 oxidization of energy yielding source substrate Uptake Excretion 39 Redox cyclin M lIIIIm llg IImIIIIquot Substratelevel phosphorylation BrIch b mu jg Lactic Acid Fermentation 9 named by byproduct lactatelactic acid Carried out by lactic acid bacteria are Gram positive nonsporulating Product is lactic acid lactate Two pathways homofermentative product is only lactate Heterofermentative products are lactate EtOH and CO2 Homofermentative pathway Net 2 ATP glucose Note the first part glucose to pyruvate or glycolysis we have seen before 9 glucose to pyruVate it is pyruVate going to lactic acid that makes it different to glucose an ADP an ADP g I i I Fructose 16 Ammo 2Glycoroldohydo Dihydroxyocotom T quot quot bisphospmuo 3prmprmo 039 pnoaptum I o a n u n n n u u c o n c u n n n u on u n n u Q n n u Q Q a n o o o an 2 13BiphOIphO 2 03934 glycedc acid a Homotormentatlva 9 don t need to know the diagram Heterofermentative pathway Net 1 ATP glucose Glucose to pyruvate is different from glycolysis 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 MAD I I 0 I 39 39 39 39 39 39 quot 0 1 Acetaldehydo 1 I L t it 1 I NAo 39 9 Pvruvate 63P 39 39 Ace I ATP ADP y n n quot0quot Pt phoyphate Glucose 6Phosphogluconlc Ribulose Xylulose G39 L 6phosphate acid 5phosphate coquot 5phosphate 9 kotolau quotquotquotquotquot II39IIII39I39Z III39IIIIIIIIIIIIIIIISIIIIIIIIIquotquotquot 39V quot 39 39 aldehyde 39 O I 33939 0 O I P anon App ATP App ATP NAo 138isphospho 0 9 t Lncuu glycarlc acid Pym W Hotcrofnrmantativa 9 know that after you activate glucose it then goes to pyruvate and lactate 9 know 2 pathways and what they are called and the biproducts MixedAcid Fermentations Carried out by enteric bacteria facultatively aerobic Gram negative rods Pathway generates a mix of three different acids acetic lactic acid and succinic acids also can generate EtOH CO2 and H2 as well as neutral products common one is butanediol substrate is glucose or sugars that can be converted to glucose Where is ATP produced How much per glucose 9 2nd state of glycolysis is Where energy is yielded Net 2 ATP P HC C 0 Pyruvalo 9 pyrophosphato H Glucose cooquot 3 799 co OH I 39quotUquot quot 9 Put nldwl 39It 39 I 39 lquot Lacfago Kquotquot7391739739h39 3l3939I 3939rquot3v i H 39 C07 Succinate 39I I rmh ryorogmvpaoo Ethanol Clostridial Fermentation Clostridium species are obligately fermentative anaerobes ferment sugars amino acids purines pyrimidines and others product is commonly butyric acid butanol and acetone can also be produced Note more ATP produced Secondary fermentation Further fermentation of fermentation products Example Propionic acid fermentation cheese made Propionibacterium propionic acid is the major fermentation product produced by further fermentation of lactate responsible for the taste of Swiss cheese hole are from CO2 another fermentation product 2 39 noon 0 C O l Hgc c coo Lrgc C H H30 OH OH TPP uAcotoloctaio Acotom cu cat on cm on 23Butnnodlol p V r vrunwouanu gt2co ohutnnodol Acetate A7 ADP Glucose womb 2 Pyruvate 2 NQDH P 1 N Acetalde hyda 2 D Ethanol AcetoacetylCoA O no CH3 CH 4 I AcetylAPv2 AcetylCoA 0 tan ButyrylCoA quotcm c cH c can 3HydroxybutyrylCoA CrotonylCoA Phosphorochmc mlctbon co m n K AcetylCoA Acetoacetate 00 Acetone 39 CH 0 c cu 3 6 lsopropanol ADP ATP ran Butanol cu Butyraldehyde CF13 cupro 2610000by f3990 mucous 0value 3939IlilI 1 H l3 FIII 0il39 obuIn6lo 8 Oo4l4 jf l ound Butyrate CO0 3 Lactate o quot rogonau I I13 3 Pyruvate IAN ACO ADP Acetate 003 2 Oxaloacetate ATP r 2 quotn Further Works on lactate CoA transfct 2 Malato zfggzgitsazld from ptopionyl b cycle working O main 2 H30 in reverse 2 Fumarato Am 2 Prop1on1c ac1d fermentatlon can also start from 2 r I glucose 9 lactate 0quot I quot L 2 SuccinylCoA 1 539 aian ma1o7syucoA 9 3 ATP per lactate Bacterial functional diversity Many but not all Archaea live in extreme environments Are both chemolithotrophs and chemoorganotrophs Here will just highlight a few of the unusual physiological processes Extremely halophilic Archaea Are called haloarchaea Live in environments with extremely high salt hypersaline Are extreme halophiles which means they require extremely high salt require at lit 15 M NaCl 9 Wtvol Most require 2 to 4 M 1223 All can grow at 55 M 32 limit for saturation above this NaCl crystals form but they can tolerate this too Hypersaline environments Halobacterial physiology most are obligate aerobes and can grow on organic carbon as the energy source Need high amounts of M Na and K The high Na is needed to stabilize the cell Wall Cell walls are glycoprotein have a very high content of negatively charged acidic amino acids These charges repel each other and without being stabilized the Wall would fall apart The wall is stabilized because the Na ions interact bind to the negatively charged carboxyl groups The high K 39 is needed to maintain a balance of cytoplasmic osmolarity in the hypersaline environment With such high salt in the environment What would happen to a normal cell or a nonextreme halophile The high cytoplasmic K is maintained by actively pumping in K J from the environment to a concentration inside the cell that is greater than Na outside the cell High intracellular K is also required because K ions are required for protein activity enzymatic etc other organisms is Na Also K and Cl is specifically required to stabilize ribosomes in this group Photosynthetic halobacteria another type of anoxygenic photosynthesis This is NOT associated with electron transport It does NOT involve chlorophylls or bacteriochlorophylls involves the pigment bacteriorhodopsin Is called bacteriorhodopsin dependent anoxygenic photosynthesis put in your table Bacteriorhodopsin is synthesized when the oxygen concentration in the environment is low is inserted into the cell membrane transmembrane This pigment is very closely related to rhodopsin eyes Absorbs green light 570 nm so appears red When light energy is absorbed part of the pigment retinal changes configuration from trans to cis In the trans relaxed state a proton is associated with the cytoplasmic side of the retinal Light absorption energy causes the molecule to rotate to cis as it relaxes back to the trans state the proton is moved across the membrane and another proton associates with the now relaxed pigment on the cytoplasmic side This results in What Halobacterial bacteriorhodopsion z xx Mutnbtmto Bacwrimhodnpulr I ATP 39 2 v Aw ADP P 121 Methanogens Produce methane from mostly Cl compounds Carried out Q by E obligately anaerobic archaea Is energy yielding and important in the carbon BGC as Well as global Warming methane is a gas see text page 517 Are present in diverse anaerobic habitats 9 table 192 CO2type substrates including CO2 are reduced using hydrogen as the electron donor Methylated substrates can also be reduced with hydrogen as electron donor or if no hydrogen is available some of the substrate can be oxidized to CO2 yielding hydrogen that can then be used for methanogenesis Acetate is the least common substrate Is metabolized by cleaving to produce CO2 and CH4 Both of these are green house gases Methanogens are a very ancient lineage genomics has revealed housekeeping genes that are Eukaryotic Bacterial and Archaeal Thermophilicextremely acidophilic Archaea Are three genera Thermoplasma Picrophilus and Ferroplasma All grow at pH values around even below zero 0 pH is calculated as the log H39 in moles 0 pH 0 negative log of 1 mole logl O 0 12M HCl is calculated to be log 12 108 Pyrolobus Temperature maximum is 113 C Lives in the walls of black smokers called chimneys Is an obligate hydrogen chemolithotroph Electron acceptor can be nitrate thiosulfate or oxygen is microaerophilic Can survive autoclaving 121 C for an hour Comparison of upper temperature limits for different energyyielding metabolic pathways For phototrophy is a bacterium hot spring cyanobacterium the rest are Archaea highest eukaryotic temp 48 highest chemolithotrophy 112 chemoorganotrophy 11O phototrophy 73 9 know highest temperatures and the organism cyanobac or archae AND table 1925 before it 9 we used to think it was archae now we know its bacteria
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