5/18 Principles of Microbiology Pre-History/Early History and disease Hunter gatherers: 10,000 years ago (refer to graph A on handout 1) Low incidence of high blood pressure, heart disease, organ cancers, obesity, acute diseases (bacterial or viral) If they did get a disease there was a rapid onset and it was often fatal (plague*, anthrax*, small pox**, influenza**, thyphus*, malaria***) (*=bact.) (**= viral) (***=protozoan) Suffered from herpes**, TB*, Syphilis*, Leprosy*== Chronic diseases, long term (acute is rapid) Agrarian Societies: 6000-8000 years ago Mesopotamia, Egypt, Indus River Increased food supply Later, domestication of animals: Zoonoses: Passed to humans from animals. Examples: anthrax, brucellosis, salmonella, thyphus 6000 yrs ago, the first cities appeared (~100,000 population) Early City States: When a lot of people are brought together (who can carry acute diseases) Epidemics are introduced Epidemic = ?upon the people? in Greek. Spreads rapidly (ie. Small pox, the plague refer to graph A, handout 1) Refer to B, handout 1: spread of The Black Death Problems with cities: Where to dispose waste? Rats and mice mean lice, fleas, ticks? Reservoir of plague == OVERALL POOR HYGENE Sanitariums: started 200 years ago. If ppl were sick with TB they were rounded up and taken to facilities to keep them away from society II. Origin of Disease Disease and the Ancient World Superstitions: Ancient Greeks would think death was caused by ?the gods?. Aristotle and Socrates thought that disease is a part of the natural world. Disease and the 19th Century world Early Microbiological Discoveries Antony Van Leeuwenhoek: First microscope: grinded glass to make glass lenses. Lenses were spherical imbedded in a piece of wood Named bacterial structures: Rod shaped: Bacillus Spiral Shaped: Spirillius Spheres: Cocci Hooke: invented the first microscope, did not see bacteria Hooke?s microscope: Compound Animalcules: Spontaneous Generation Prevailing the 19th Century hypothesis: ?if you leave meat in the window, you will create maggots? Nutrient broth: boil beef, filter out filtrate = filtrate-broth. Viewed clear beef broth saw no living organisms. If left alone, the broth would go cloudy and animalcules would be swimming around Louis Pasteur: from paris, postulated that fermentations were reactions probably caused by animalcules and not from decomposition. Worked with tubes with nutrient broths, thought that dust settling in the tubes would cause growth of animalcules?animalcules travel on dust. Put cotton plug in tubes and broth remained clear for weeks ?Vital force? was air. Pasteur experiment prevented air from touching broth. Created swan-neck flask to show that air was still involved in the reaction but dust was prevented, thus no growth was due to lack of dust (D on handout 1) Cause and effect relationship: if spontaneous generation does not exist, then what causes diseases? Germ Theory of Disease Robert Koch: From Germany, Taught by Henle in the 1840?s. He was able to get involved in these discoveries and do work of his own. Pasteur was working on the same material in France. Sterile potato cut in half, boil it, the next day you will find dark growths on the potato. Looked at these dark growths under a microscope and found that they were animalcules. Take the growth on the potato, let it form on a new sterile potato in a jar?all the same cellular morphology means it came from one colony. He made a pure culture of a strain of bacteria. Potato represented solid medium that could be sterilized and included the needed nutrients for growth Joseph Lister: Listerine antiseptics 1860?s. Found a way to isolate pure cultures using broths of lactic acid bacteria in a medium of lactose. He could see if acid was produced with an early pH indicator. Proposed that if you take a broth sample and diluted it down to where no fermentation would occur, the last vial that contained growth would be a pure culture of the bacterium growing. Was not certified to find a pure culture because other bacterium may be involved in fermentation Koch created nutrient agar from boiled seaweed. Koch?s Postulates: (C on handout 1) Ignaz Semmelweiss: the behavior of the person sets them up for infection of disease?he disproved the current theories of spontaneous generation Germ: what causes the disease 5/19 Time line of Microbiology (E on handout 1) Louis Pasteur Industrial Biology: Fermentation studies for companies trying to pasteurize products Milk: heat to kill pathogens and bacteria Environmental Biology: Beijernick and winogradsky: Testing water because of Mercury poisoning, mining industry found gold in waste piles, Immunology: Revolves around mammalian physiology: 1980s aids developed?helped learn how the immune system works immunizing herds Clinical Microbiology Treatments for diseases were developed Paul Ehrlich: chemotherapy??magic bullet? concept?kill the bad guys but don?t hurt the good guys. Worked on syphilis (secondary to tertiary) Worked with arsenic, made pills to cure syphilis?numbered his treatments number 700 was the right concoction that wouldn?t kill the host Antibiotics: penicillin (Flemming ~1929) based his work on pasteur?s paper about staph. Noted fungal contamination ended up creating a zone on non-growth of the bacteria around the fungal growth. Flemming grew the penicillin, purified it and tested the compound to treat staph and other infections Made enough penicillin to treat 3 people in a hospital that were going to die, military saw this and invested in it so that it could be mass produced for the soldiers in the war Virology: small pox, influenza?wouldn?t fit the mold of other sicknesses. They are unique because they are caused by viruses Beijerinick: tobacco mosaic disease?when the white disease covers the plant, it dies. He tested it to see if it fit Koch?s postulates. Took one of the leaves, crushed it up and took the sap from the leaf. Spread the sap onto a petri dish in a nutrient poor agar medium. He found 10 diff kinds of colonies and isolated them in pure cultures. He sprayed each culture on the plant and found no disease developed from any of the cultures. Chamberlin: studied water quality microbiology?made a bacterial filter to prevent contamination in water. Worked with Beijerinick to produce a filtrate from the plant and when it was sprayed on the plant it caused the disease every time. Overall provided data to suggest that there was an agent smaller than bacteria that could cause disease Filterable agent: able to be caught in the filter Non-filterable agent: too small and able to pass through the filter Plaques: bacterial viruses?ultimately lead to molecular genetics (found on a petri dish in which growth would occur everywhere except for tiny spots. If the agar was purified at these spots and used to attempt growth of bacteria, growth would not occur) Molecular Genetics: the late 1920s (~1928). The Griffiths found that you could transfer dna from one bacterial cell to another via transformation. Chromosomal dna / plasmids Genetic engineering?can transfer human insulin in e.coli so that insulin could be mass produced for diabetics PCR: Polymerase Chain reaction invented. Use of restriction endonuclease Structure and Function of Cells Eukaryotic vs. Prokaryotic A on handout 2 Size Difference: Euk are larger than Prok. A= Ám2, V= Ám3?Surface/Volume ratio = a/v E. Fishelsoni: cancelled out the idea that euk cells were larger because of their organelles. It is a very large prok cell that does not have organelles Organelles: Murein: structural glycol-protein only found in bacterium/prok cells. Not in euk cells Flagella: 9+2 arrangement: similar to muscle tissue?myosin arrangement 9 around the outside and 2 in the middle. Contract or relax to cause movement. PROK DO NOT HAVE A 9+2 ARRANGEMENT. Euk only Mitochondria Endoplasmic reticulum Vacuoles Ribosomes Nucleus Cytoplasmic membrane Eubacteria: Archaebacteria: Endosymbiotic hypothesis: Mitochondria and chloroplasts were ancient prok cells that were phagocysed (engulfed) by an early proto-eukaryotic cell. They had their own chromosomes to code for rRNA Humans use tetracycline used like antibiotics to treat acne: target the small subunit of bacterial ribosomes (review recording) Prokaryotic Morphologies (back of handout 2) Cocci Bacilli Spirilium Binary fission: cells divide Cell arrangements View handout 2 for more morphologies illustrated Prokaryotic Capsules B on handout 2 Capsules are extracellular accumulation of glycol-proteins or polysaccharides found on some cells, not on all. These are polyanionic?surrounded by negative charge Also called glycocalyx (slime layer) similar to mucus Fxns of a slime layer: Attachment to surfaces in aqueous environment Allows the bacteria to filter the low amounts of nutrients available Provides protection from protozoa against phagocytosis; The polyanionic nature makes it difficult for white blood cells to phagocytise the cell (neutrophil morphology) Can avoid desiccation/dehydration (5/23) Prokaryotic Pili / Fimbriae Composition Hollow tube of protein?about half the width of a cell long Attachment to cell: helps attach to surfaces (ie intestine wall) Geobacteria: some pili have metals like copper and iron in the pili?allows for electrical conductivity. Can be used as a bacterial battery (discovered 5 yrs ago) Could possibly use these to make nano-wires Function: Attachment to a cell and VIRULENCE factor (Gonorrhea without pili will not attach and will not infect the host) Virulence: ?measure of intensity of a disease? Capsules are similar to pili in regards to virulence factor Prokaryotic Cell Wall Cell wall is found on all bacteria?capsules and pili are not Gram Positive vs. Gram Negative Negativity or positivity based on the chemical composition of the cell wall. Linked to work of Christian Gram (test developed in the 1880?s-1890?s) Gram stain used in a clinical setting to determine the treatment for infection?determines what kind of antibiotic should be used. Stain depends on lipid content in the cell wall. Petidoglycan: a polymer made up of 2 repeated components: N-Acetyl Glucosamine (NAG) N-Acetyl Muramic acid (NAM) 4 amino acids attached The cell wall of bacteria is similar to the cell wall of a plant cell (cellulose) same except plant does not have muramic acid or amino acids Cross Linkage: (d on handout 2) aka Pentoglycan Bridge: prevents ballooning of cell if it takes on additional liquids 95% of NAM?s are cross linked?5% is because the linked amino acids are bent at the point where they attach Penicillin blocks metabolic pathway of the cross linkage, destroying the cell Lysozyme: cuts the beta ╝ linkage of the NAM unit Cells that are gram negative cannot make these bridges because the bonds do not allow sharing (e on handout 2) ? thought to be a weak cell but it is not Cell Wall Function Osmatic pressure: if lots of solutes in a place = high pressure Hypotonic environment: Less solutes around, water moves in. Cell balloons Hypertonic environment: More solutes around, water moves out. Cell shrivels The cell wall is rigid and protects the cell from swelling or shriveling HELPS RESIST CHANGES IN OSMATIC PRESSURE Gram Positive Structure: (f on handout 2) Peptidoglycan layer: can dbl in thickness w/ age Teichoic Acid: sugar polymer that sticks through peptidoglycan layers Lipoteichoic Acid: links cell wall the the cell membrane Wall associated proteins: recognition fxns permease will break down proteins in the environment so they can go through the cell wall and cell membrane Gram Negative Structure: More complex Lipopolysacharide: (LPS) toxin to human body. Endotoxin causes several problems including septic shock Porin: hollow tube from outer membrane to the cell wall. Allows solutes to move through the cell Periplasm: has periplasmic proteins to help soluble substances in or out of the cell Can tolerate marine environments better, able to adapt to changes in the environment?can live in places with poor nutrients Prokaryotic Cell Membrane Cell membrane is a phospholipid bi-layer. It is selectively permeable b/c solutes can go in but cannot go out needs a protein conduate. Aka permease has the ability to transform?folds inside of itself if something binds to it and deposits the solute into the cell (then will assume normal form) Transmembrane proteins and Peripheral proteins: can make metabolic chains/pathways. Bacteria do not contain mitochondria but components of it are found in the cell membrane Fxns of Cell membrane: Transport: Involves permeases (transmembrane proteins) Passive Transport: no energy needed. Chemical gradient permease act as a gate. Once the cell gets into equilibrium the permease starts spitting out solutes Active Transport: ATP binds to permease on inside (ADP+pi) causes the protein to shift. Expenditure of energy to bring solute inside cell (g on handout 2) Complex Active Transport: paraplasmic binding proteins in the cytoplasm. As soon as they bind, a knob forms. The knob is formed in a way that can attach to a permease. ATP expended and solute is brought into the cell through the cytoplasmic membrane. Effective at working at lower concentrations of nitrogen If introduced to an environment rich with glucose, the transport mechanism changes to passive transport. Active transport stops working. Group Translocation: (find in textbook) found in mainly gram negative bacteria to help take up 5 types of sugars. Feeds into glycolosis, spends ATP Secretion Locomotion Pertains to cell flagellum (hollow protein tube) connects to the cell wall and cell membrane. Cell membrane components make this work (H on handout 2) Goes through rings (L and P ring) which prevent the flagellum from being loose? The MS rings surrounded by transmembrane proteins?flagellum is bound to the MS ring. MS ring surrounded by mot proteins which push up against the ring. Reqs energy, works similar to a muscle in 9+2 Formation. Filament of flag is hollow?moves by spinning the hook of the flagellum Taxis: chemotaxis: the flagellum produces a counter-clockwise spin which produces smooth swimming (directional) Tumble: clockwise spin Energy Transformation Biosynthesis End of quiz 1 (5/24) Prokaryotic Cytoplasm Cytoplasm No compartmentalized organelles Chromosome Called the nucleoid Comprised of a single chromosome that is haploid Inclusion body Sometimes membrane bound Used for food storage, bacterial fat Contains polyhydroxy byterate Ribosomes Plasmids Extrachromosomal circular DNA Endospores Early developmental stage of a bacterial spore that has not been released from the cell Spore formers BACILLUS = mostly aerobic B. anthrasis = anthrax B. cereus = gastroenteritis CLOSTRIDIUM =obligate anaerobe= grows only when no oxygen around C. diffical = nosocomial infection, big problem in hospitals C. botulinum = Botulism = toxic chemical C. tetani = Tetanus Spores provide resistance to Absence of water Freezing Boiling (121░ C for 15 minutes will kill spores) High or low pH High or low [salt] High UV radiation Van Helmonz Thought that spores in a suspended state could be the way that life transferred across the universe His theory was discredited for a long time but more recently research has been done on this idea Amber with honeybees in it from 25 million years ago has been found to have bacillus spores that are very similar to the spores of bacillus bacteria that are found on honeybees today There has been a protein found on bacterial chromosomes that corrects the problems caused to the chromosome due to UV radiation right as the spore is revegetated Has been projected that spores could survive for 2 billion years BACTERIAL ENERGY METABOLISM Why do cells need energy? To maintain order and resist entropy All things naturally go from organized to disorganized (fall apart) In order to resist entropy must have a constant supply of energy Energy Metabolism as a Part of Bacterial Physiology Physiology is the study of the totality (growth and reproduction) of an organism Metabolism is a series of chemical reactions that serve as part of physiology Anabolism Biosynthesis = how a cell takes smaller entities and bring them together to make a polymer (cellular components) Ex. Use glucose to make N-acetyl glucosamine to make part of the cell wall Catabolism Release of energy in some useful form for cell reactions Energy can be used for Movement Growth Waste products Adenosine Triphosphate = ATP Energy transfer from catabolism to anabolism For bacteria the reactions occur in the cytoplasm E. coli can synthesize 2.5 million molecules of ATP in one second Microbial Strategies for Generating ATP Energy Sources vs Carbon Sources (see fig. 9.1 in text) Energy sources Chemoorganotrophs Use organic molecules ?plant eater? Chemolithotrophs all 3 produce ATP Use inorganic molecules ?rock eater? Prototrophs Use light Carbon sources Heterotrophs Preformed organic molecules (C is reduced) Glucose, nucleic acids, etc? Autotrophs Use carbon dioxide (C is oxidized) Electron Sources ( to reduce carbon dioxide so they can use it) Organotrophs = organic molecules Lithotrophs = inorganic molecules Mechanisms to Conserve Energy Substrate level phosphorylation = the synthesis of ATP from ADP by phosphorylation coupled with the exergonic breakdown of a high-energy organic molecule Exothermic reaction A reaction where energy is released in the process of the reaction Endothermic reaction A reaction where energy is needed to complete the reaction ADP + Pi = ATP There are two places in glycolysis and one place in the Krebs cycle where substrate level phosphorylation can occur out of the total 17 steps of the two processes, therefore there is not much energy conserved this way In order for SLP to occur, an endothermic and an exothermic reaction must be linked together simultaneously. The endothermic reaction is the formation of ATP from ADP + Pi. The exothermic reaction is where ATP is used. There must be at least 10 kcal/mol of energy released from the exothermic reaction in order for SLP to occur. If there is less energy than that releases, then it is released as heat in the organism. Oxidation/Reduction and Chemiosmosis Oxidative Phosphorylation CoA NADH O2 H2O Glucosepyruvic acid CoA Pi 6 CO2 ADP ATP NAD Peter Mitchell 1960?s Mechanism for how oxidative phosphorylation worked It was a chemiosmotic mechanism Reduced compound = extra hydrogen or extra electron on it Oxidized compound = lack of electrons or hydrogens NAD+=oxidized NADH + H+=reduced NADP+=oxidized with P Mitchell discovered that NADH + H+ had a great affinity for bacterial cell membrane and mitochondrial membrane, closely tied with the electron transport chain in the membrane The electron transport chain is organized so that the protons move outward from the cytoplasm as electrons are transported down the chain and into the periplasmic space The chain is comprised of multiple proteins embedded within the cell membrane There is a net positive charge outside the cell due to the presence of extra H+ in the periplasmic space and a net negative charge within the cell There is also a concentration gradient of protons that is built up outside the cellular membrane with the higher concentration being outside the cell The ETC is able to move the H+ across the membrane by oxidizing NADH + H+ to NAD+ Sometimes the e- is put in the form of a free radical that can react with anything and other times the e- can be put in a ring Proton Motive Force = PMF Comprised of the combined chemical and electrical gradients outside of the cellular membrane Used to perform work when the protons flow back across the membrane down the concentration and charge gradients back into the cytoplasm The protons (H+) are able to flow back down the gradient coming through the very small channel in an ATPase that is within the cellular membrane The channel is only big enough to allow a proton through As the proton flows through ADP + Pi is made into ATP and the positive charge is brought back into the cell Uses of PMF = credit to do different things Production of ATP Move sodium out of the cell Bring sugar into the cell Bring amino acids into the cell Drive protons out of the cell by spending ATP All of these functions are carried out by the H+ flowing back through a pore in a transmembrane protein that will cause a conformational shift in the protein so that the Na+, sugar, etc. can be brought back into the cell. Once the molecule is brought into or out of the cell then the protein shifts back to its original state. A good example of this is the conformational shift that the Mot protein goes through when protons are brought through it that allow the flagella to spin. May 25, 2011 Terminal Electron Acceptors (TEA) FOOD e- NAD+ TEA e- NADH The NAD+ is an oxidized e- carrier that must be recycled because it is found in finite amounts within the cell TEA can be in three different forms that lead to three different processes O2 = aerobic respiration pyruvic acid = fermentation/anaerobic oxidized inorganic compounds (NO3, SO4, metals) = anaerobic respiration WASTES of these three processes Aerobic respiration CO2 accounts for total food given H2O as well Fermentation Some CO2 but it does not account for all the food given Alcohol And/or organic acids Anaerobic respiration CO2 accounts for the total food given Other waste products If NO3 is the TEA then the wastes will be NO2, N2O, NO and N2 (denitrification) If SO4 is the TEA then the wastes will be H2S = sulfate reducers If CO2 is the TEA then the wastes will be CH4 which are methanogens The Case for Microbial ?Scatology? ?biologically oriented study of excrement (as for taxonomic purposes or for the determination of diet? Metabolic Production of Wastes Identification of Microbes May 26, 2011 Examples of Microbial Energy Metabolism Chemoorganotrophy: Aerobic Glucose Metabolism Embden Meyerhof Parnas Pathway = Glycolysis Type example = E. coli Overall reaction GLU + 2ADP + 2Pi + 2NAD+2PYR, 2NADH, 2ATP Involves two endergonic/endothermic reaction Enter-Doudoroff Pathway (also aerobic) Type example = Pseudomonas group of bacteria Overall reaction GLU + 2NADP+(or 2NADP+) + ADP + Pi2PYR, 2NADPH(or 2NADH), 1ATP With this pathway fermentation cannot occur because only one ATP is produced, whereas with glycolysis 2 ATP?s are produced so fermentation can occur The bacteria that use this pathway lack the enzyme 6-phospho fructo kinase therefore they cannot go through glycolysis Kinase = enzyme that adds a phosphate group Only produce 1 GAP or 1 PYR versus 2 GAP with glycolysis Pentose Phosphate Pathway Lactic acid bacteria use this pathway all the time General reaction GLU + NADP+PYR + CO2 Fructose 6-phosphates are made that are recycled back to 3-glucose E. coli can also use this pathway when inorganic phosphate is limiting in the environment. They shift their metabolism from glycolysis (which requires 2 Pi) to the pentose phosphate pathway (which requires no Pi). Using the pentose phosphate shunt the E. coli create a proton motive force that allows them to spin their flagella in order to swim somewhere where there is more glucose available. During this time, E. coli cannot reproduce. Krebs Cycle Up until this point, the glycolysis, Enter-Doudoroff, and Pentose Phosphate pathways have all worked to prime the glucose in order to enter the Krebs Cycle to further complete the process of respiration. Overall reaction 2PYR + 2ADP + 2FAD + 8NAD+ +2Pi6CO2 + 2ATP + 2FADH2 + 8NADH+ Still have reduced coenzymes (2FADH2 + 8NADH+) so respiration is still not complete, so now the electrons from both of these molecules must be taken to the Terminal Electron Acceptor PYR is converted into CO2 There are some groups of bacteria that can feed citrate directly into the krebs cycle. There is a permease that allows these cells to take up the citrate but they can also leak the citrate back out of their cells. This can be a disadvantage to these types of cells. Also, these citrate positive bacteria lots of CO2 because they are not starting with pyruvic acid, and this leads to a change in the pH of their environment. Electron Transport System The electron transport chain must be across the cell membrane in order to create a proton motive force There is a diagram on the handout that shows an example of an electron transport chain for E. coli Basically, the NADH comes into contact with an iron/sulfur compound that is within the cell membrane and it reduces the NADH to NAD+ and transfers that e- to a quinone in the cellular membrane. From there the electrons are moved in one of two directions. In high oxygen environments the electrons are transferred to a cytochrome that transport the H+ out of the cell into the periplasmic space 4 at a time In low oxygen environments when the cell is in its stationary phase, the quinone transfers the e- to a cytochrome that spits the protons out of the cell into the periplasmic space 2 at a time In both cases, a proton motive force is created. The high oxygen environment creates a higher PMF than the low oxygen environment does *END of AEROBIC use of GLUCOSE* Chemoorganotrophy: Anaerobic Metabolism Fermentation (see Fig. 10.19 in the text for a summary of all types of fermentation) Wastes = reduced organic compounds 2 categories Alcohols Organic acids PYR is the Terminal Electron Acceptor for fermentation Pathways for PYR include lactic acid, ethanol, proprionic acid, etc? The products are alcohols or organic acids that accumulate in the environment where the cells grow. These end products can inhibit the growth of the cells because they can be toxic to the bacteria There are some bacteria that can use these reduced organic compounds as their starting fuel because they can oxidize them to produce more energy Lactic Acid Fermentation Homo Lactic Acid Fermentation Only one product = lactic acid If you add lactic acid to milk you get fermented dairy products such as yogurt Hetero Lactic Acid Fermentation Produce CO2, ethanol, and lactic acid Gives you yogurt with a kick Both forms of lactic acid fermentation are economically important to the dairy industry Ethanolic Fermentation Bacteria ferment glucose to ethanol, CO2 is a waste product Can produce a beverage (wine, etc..) or a fuel ?grass-o-line? is a project at UTK where they are trying to find the most efficient way to convert switchgrass into ethanol. Requires the addition of cellulose, which is the limiting step in the equation. However, the energy output of the glucose fermented from the switchgrass is five times that of what it takes to grow the grass which gives it the potential to be very cost efficient if a cost effective way can be found to ferment it. In South America they are also using sugarcane to produce ethanol When you make ethanol from fermentation, the ethanol has to be distilled before being added to gasoline in order to remove the water from it. Distillation can be an expensive process. Mixed Acid Fermentation Glucose is broken down into many different acids such as acetic acid, succinic acid, lactic acid This lowers the pH in the test tube where these cells are growing, pH will usually be around 4.5 Conversely, if there are alcohols in the tube then the pH will be around 6.6 Anaerobic Respiration TEA?s and their wastes NO3 is the TEA and its wastes are NO2, NO, N2O, and N2 NO3NO2 is a nitrate reducer NO3 reduced all the way to N2 is a denitrification SO4 is the TEA and its waste is H2S = sulfate reducers CO2 is the TEA and its waste is CH4 = methanogens Oxidized metals and electrodes can also be TEA?s but there are no waste products involved with these TEA?s Nitrate Reduction (figure 10.17 on handout) There is a series of membrane proteins as well as periplasmic proteins and cytochromes that reduce NO3 to N2 The structures of the anaerobic electron transport chain are very different from aerobic ETC?s Facultative Anaerobe Ex. E. coli Prefers oxygen as TEA but if oxygen disappears then it will use NO3 as its TEA, and then when all of the NO3 is gone it will switch to fermentation Sulfate Reduction Sulfate reducers are obligate anaerobes because they evolved in an environment where there was no oxygen They do not use glucose They feed on the fermentative wastes of plants that have fallen in the wetlands Ex. Lactic acid, ethanol, acetate Photosynthesis Anoxygenic Much more ancient, were the first cells to convert light energy into cellular energy Chlorophyll takes in light and H2S is oxidized to SO4 which dumps its electrons onto the chlorophyll The chlorophyll will send the electrons in one of two ways It will use the electrons to produce a PMF through one ETS(electron transport system) to be used for various functions Or, it will use another ETS to shunt the electrons down to NAD reductase which will reduce CO2 into carbohydrates for the cell Oxygenic Came along a billion years later These type of bacteria are called cyanobacteria Developed different chlorophyll Shifted their source of electrons form H2S to H2O, otherwise it used the same pathways to create a PMF or carbohydrates Chemolithotrophy Some use CO2 or organic molecules as their food source Nitrobacter Uses nitrate as its electron source See tables 10.3 and 9.24 on the handout Chemolithic autotroph Will produce PMF through on ETS pathway Will reduce NAD to produce the reducing potential they need Electron acceptor is O2 Live at the edge of an anaerobic and aerobic environment because they need anaerobic reduced compounds as well as oxygen for their terminal electron acceptor I. Bacterial Evolution A. Evolution Theory and Microbes B. Origin of Life Hypothesis i. Panspermia: ?Universality of life? ?Von Helmholtz Comet holding bacterial spores crashed on the earth at a time when earth was cool enough to support life. Liquid water must have been on the surface Bacterial spores in the ice core of the comet. Grew in the melted ice (if comet fell in the ocean) and began life for earth Geologists found that the most ancient rock had chemicals that were reduced= the atmosphere of the earth was reducing in ancient times. No oxygen, had hydrogen, ammonia Had an abundance of organic matter?aquatic environments had abundance of organic matter 1920?s biochemistry: discovered catalytic molecules w/I the cells?enzymes. Played a critical role in a living cell?s life Autotrophic Origins: (1920?s) Russian named Trolland came up with autotrophic origins Proposed that there were catalytic molecules that were prob present in the ocean/aquatic environments that were able to react with organic matter and became the basis for cell metabolism What is missing? Membrane Cell= cellularity of life requires selectively permeable membrane Chemical evolution: Oprain and Haldane (~1930) What kind of organic matter was in the ancient oceans. Came up with ?organic soup? idea. Listed potential organic compounds found in organic matter Miller and Ureg (~1950) Came up with an experiment (a on handout 4) Started with sterile water?crystal clear?after 10 days the water had oily film that was black. Had amino acids, sugars, fatty acids, and small levels of nucleic acids. Polymers: proteins, polysaccharides (carbs), lipids, RNA Made organic components of life Some people varied the sources of energies, chemical compounds (in atmosphere) always ended up resulting in amino acids, sugars etc. Phospholipids if mixed in water will make phospholipid spheres?look like a cell membrane ?liposome? 2003 liposomes were formed by mixing clay + fatty acids + carbs = produces phospholipids B handout 4 one hypothetical sequence of events that might have led to the first living cells (1981) Ribozymes discovered (rna sequence and catalysis) Gilbert (1986) ?RNA world? got to where you had ribozymes and needed enzymes Support for this comes in 2 main forms: ATP is a ribonucleotide?ancient parts of cells (that obviously works) would be passed down throughout generations Could produce a world of micr. Cells leaving out DNA ? all info needed stored in RNA?rna coded for proteins. RNA interference (RNAi): pieces of RNA that help modify and regulate the transcriptional process and other genes around them. Helps detect and fix errors in transcription. So critical that if there was an RNA world it must have carried throughout generations Viruses: may have made a better heritable material than RNA?could have helped originate DNA by working with an infected cell Microscopic Fossils? Shark bay: studied stromatolites: pillow shaped masses of biofilms make a microbial mat of phototrophic bacteria. Will hold cells of all metabolic types Caused by climate?half of the year is dry, half the year is wet?local rivers erode sands, calcium carbonite caps off stromatolites. During the dry season the rings of bacteria grow. Layers include oxygen PS, anoxic PS, sulfate reducing, fermenter Found fossilized stromatolites around the world ? 3.8 billion years old. Suggest that there were living orgs growing Chemical assessments: reassess what the environment was like when the bacteria was alive ? older than 2billion years ago the atmosphere was reducing (did not contain oxygen) Evolution of cellular Diversity/ Modern Environment C on handout 4 Anaerobic bacteria been around since existence of first bacteria, aerobic only came around when oxygen started showing up in atmosphere, unicellular algae only came around about 1 billion years ago. Indirect vs. direct fossil: direct = bones, indirect = molds/footprints D on handout 4: Bacterial Taxonomy Why classify? To know what you are sick with, to tell different groups apart, evolutionary history Identification: early taxonomists (monks) had plants and animals to study, didn?t consider smaller organisms. Wanted a better understanding of God?made the church focus on the living world. Evolutionary Relationships: convergent evolution was occurring?noticed that animals paired in the same family were actually very far apart in morphology Early Taxonomic Systems: Plants, Animals, Protists, Fungi, Protists} monera, protozoa Modern Taxonomic Structure Monophyletic: Based on evolutionary history, genotypes/DNA. Comparison of gene sequences Polyphyletic: Based on phenotype?runs into problems of similar animals being paired in the same family when they are far apart. Categories were too broad. Modern use of Molecular Taxonomy Carl Woese: Question: are there any particular genes that are conserved in all life on earth that could be used to make a comparison? Basic physiology?which genes are universal? (for all life) Protein synthesis (ribosomes): made of proteins + rRNA Ribosomes are coded from the DNA Small subunit: Eukaryotes: 18S Ribosomal RNA Prokaryotes: 16S ribosomal RNA Sequences of dsDNA are conserved Three Domain System: (e on handout 4) Bacteria, Archaea, Eukarya ?House keeping genes? genes that are important and conserved for the basic physiology of cells Bacterial Classification Bacterial Species Type strain: kept in labs. If there was a new strain found it was a new type strain.. no way labs could hold all the strains Culture Collections: American Type Culture Collection. ATCC # = exact culture shipped to a lab. Need permission to order cells. Preserve cultures: (spore formers would survive regardless) Freeze cultures rapidly in liquid nitrogen or dry ice and kept them in a bath of dry ice or freezer Freeze dry cultures, bacteria will survive. Mix cultures in the matrix (not TSB, more like sterile skim milk) put into liquid nitrogen and then hook up to a vacuum tube. Cells undergo sublimation. If ice crystals form it will kill the cell ?Lyophilization? Classification Criteria: Growth characteristics Biochemical comparisons Carbon sources used Genetic characterization (%G of C) Hybridization studies PCR reaction results/primers used Gram reaction Bergey?s Manuel: Standardized the testing process and published a vicarious comparator?standardized protocols for tests. Look in the book and compare results to determine the taxa of unknowns Most recent edition has 4 volumes Now is all done online, need to subscribe to view For purpose of recreating experiments, lists media used (cookbook of medias), temp of incubator Lists of results of standard tests on known-type strains Computer based systems: Computers helped by holding data about bacteria. Helps doing the comparison of hundreds of diff strains of unknowns Prokaryote Eukaryote Size Organelles Flagella Cell Wall Nucleus smaller Yes, no 9+2 Contains murein _ Larger Yes, 9+2 Does not contain murein +
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