BIO 208 - Microbiology - Unit 2 - Lectures 7-8 - Microbial ...



Unit Two – Microbial Growth and ControlIn Lecture 7 we will be considering how microbes grow, what they need to be able to grow, and how they get what they need.We will be reviewing several figures from your text. It may be helpful to have the text with you and open to Ch. 6 so you can mark those figures.I. Microbial Growth (Chapter 6)A. Basics“Growth” – increase in number of cells, increase in size of population.1. Bacterial Division (Fig. 6.12, 6.14)binary fission2. Generation Time = time required for a cell to divide (or a population to double).varies greatly with speciescell number = 2n, n = # of divisions (or generations)Ex. start with 5 cells and they each divide 9 timesB. Equation for Cell GrowthNUTRIENTS + INFORMATION + ENERGY POLYMERS MACROMOLECULES NEW CELLsource of nutrients - source of information – source of energy - 1. Energy (E):E from chemicals – 1) organic (-C-C-) chemicals - chemo organo trophEx. C6H12O6 + 6O2 6CO2 + 6H2O2) inorganic chemicals – chemo litho trophEx. 4FeS2 + 15O2 + 14H2O 4Fe(OH)3 + 8H2SO4Thiobacillus ferrooxidans E from light - Ex. Chlamydomonas nivalis2. NutrientsMacronutrients: need in large quantities: Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous, Sulfur (CHNOPS)1). Carbonsources:CO2 – auto trophsorganic (-C-C-) chemicals – hetero trophs*note that organic chemicals can serve as both energy and carbon sources2) Hydrogen source –3) Nitrogensources:amino groups (-NH2)ammonia (NH3)nitrate (NO3-)atmospheric nitrogen (N2)4) Oxygen source – 5) Phosphoroussources:phosphate (PO43-)organic molecules6) Sulfursources:sulfate (SO42-)sulfide (S2-)Micronutrients: K, Mg, Ca, Ferequired in small but sig. amts.act as cofactors for many enzymes (enz)are important in cell structuresTrace elements: Co, Zn, Curequired by a small number of enz.C. Growth of Bacteria in CultureAssume cultivation in:liquid mediuma closed systemaffected by:nutrient shortageswaste accumulationPhases of Growth -4- (Fig. 6.15)1. lag phase - 2. log phase - 3. stationary phase - 4. death phase - D Growth of Bacteria “in the Wild” Affected by:Biofilm - A multilayered bacterial population embedded in a polysaccharide matrix and attached to some surface.Also see Fig. 6.5Cell to cell communication - a. quorum sensing – AHL (aka HSL) – acylhomoserine lactoneb. regulating cellular processesBiofilm lifestyle allows these structurally simple yet physiologically diverse microbes to coexist in an environment --and the activity and growth of the community exceeds what is possible for an individual.E. Measurement of Microbial Growth – most of this will be covered in lab, so we won’t cover much in lecture1. Cell Numbersa. Direct countsadvantages - quick, cheap, cell size & morphdisadvantages - pop. needs to be large, can’t tell living from dead*b. Plate countsDiluted sample dispersed over solid agar surface - each microbe grows into 1 colonyOriginal # viable cells in a sample can be back calculatedEx. dilute 1 ml sample into 100 ml water (a 1/100 dilution) plate incubate count 150 colonies# coloniesxinverse of dilution = # original per ml150x100= 15,000 CFU/mladvantages - simple, sensitivedisadvantages - have to know how to culture the microbeModification - counts from membrane filters2. Cell Massa. turbidity – cells interfere with transmission of light, looks cloudy. The lower the light transmission, the greater the mass of cells.b. metabolic activity – e.g., ATPase activity.c. dry weightIn Lecture 8 we will look at the effects of the physical environment on the growth of microbes.F. What Affects Microbial Growth?1. Nutrientsa. presence in the environmentb. ability to transport across plasma membrane2. Physical environment: water, pH, temp., oxygen – for each physical parameter, microbes will display a range of tolerances (too low, optimum, too high), which will vary from species to species.optimumtoo lowtoo higha. Water1) effects of water imbalancehypotonic - hypertonic - 2) adapted to hypertonic environmentsHalotolerant – habitat - Ex. Staphylococcus aureusFacultative halophile - habitat - Extreme halophile - habitat - Ex. Halobacterium (Archaea)b. pH internal pH of cell is protozoa & most bact. preferfungi & algae prefer1) effects of being in the wrong pHdisrupts plasma membraneinhibits transportinactivates enz2). pH classifications Neutrophile: 5.5 - 8 most microbesAcidophile: 0 - 5.5some molds, some bact.habitats - ore mines, bogs, stomachEx. Helicobacter pyloriuses for humans – enzymes to function in low pH industrial environmentsAlkalophile: 8.5 - 11.5habitats - soda lakesuses – enzymes to perform in alkaline laundry detergentc. Temperature1) effects of being at the wrong temperatureinfluences membrane fluidityaffects enzyme functionminimum – optimum – maximum - 2) temperature classificationsPsychrophile – optimum - Ex. Chlamydomonas nivalisPsychrotroph – optimum - Ex. Listeria monocytogenesMesophile - optimum -Ex.Thermophile – optimum - Ex. Thermoplasma (Archaea)Hyperthermophile – optimum - Ex. Pyrolobus fumariid. Oxygen1) evolution of Earth’s atmosphere4.8 billionno O2 (reducing = anoxic)2.25 billionoxygenic photosynthesis O22 billion1% O2Today21% O22) why is oxygen bad?O2 - accepts e- and becomes reduced to H2OO2 + e- O2-O2- + e- + 2H+ H2O2H2O2 + e- + H+ H2O + OHpull e- off of other molecules (DNA, plasma membranes)3) what protects O2 respiring cells from bad effects of O2? - superoxide dismutase (SOD)2O2 + 2H+O2 + H2O2 catalase (cat)2H2O22H2O + O24) Oxygen tolerance classification –we will not go over in lecture but you will need to know these termsObligate anaerobe – does not require O2 for growth, will not survive exposure to oxygen, has neither catalase (cat) nor superoxide dismutase (SOD).Aerotolerant anaerobe – does not require O2 for growth; will survive exposure, has SOD only.Microaerophile – needs a little O2 for growth, but less than amount present in air.Facultative anaerobe – can grow with or without O2, has both cat and SOD.Aerobe – requires O2 for growth, has both cat and SOD.AssignmentsCh. 6 – Read entire chapterReview – 1- 3, 5-8MC – allCT – 1, 4CA – allIn Lecture 9 we will explore control of microbial growth, including the use of antibiotics.II. Control of Microbial Growth (Chapter 7 and pp. 554-558)A. Terminology1. sterilization/sterilize - destroy all viable cells, spores, viruses2. disinfection/disinfectant - kill pathogens on inanimate surfaces3. antisepsis/antiseptic - kill pathogens on living tissue4. de-germ – mechanical removal5. sanitization/sanitize - lower # of pathogens to acceptable levelsB. How do we kill microbes?1. Nonspecific – work against almost all microbes in the same waya. Physical methodsb. Chemical methods1) phenols - denature proteins, disrupt membranesEx.2) halogens - oxidation of cellular materialExs.3) alcohols - denature proteins, dissolve lipid membranes - Ex. 2. Specific – specifically kill some types of microbes, others are left unharmed.Antibiotics (pp. 554-558)Antibiotic –natural substance produced by one microorganism that inhibits the growth of anothera. How were antibiotics discovered?Alexander Fleming (1928)Penicillium notatum (Eukarya – fungi)b. How do antibiotics work?Bactericidal - killBactriostatic – inhibit*Selective toxicity – no harm to host**c. Cellular Target Sites of Antibiotics – 4 - ImportantFig. 20.2cell wall synthesisprevent synthesis of new peptidoglycanwork only on growing cellsselective - how? - least toxicExs. penicillin, methacilllin, cephalosporinplasma membrane integrity and/or function alter permeabilityselective - how? - Eukarya – Bacteria – Exs. polymyxin B, nystatinnucleic acid synthesisinterfere with enzymes gyrase and polymeraseselective - how? - Ex. rifampin, quinolones like ciprofloxacinprotein synthesistarget 70S ribosomegreater toxicity – why?Exs. tetracycline, chloramphenicol, erythromycin3. Antibiotic Resistancea. history1940s - 1969 - 1980s - 1980s - 1990s and on - b. how did we get in to this predicament?amount manufactured - number of prescriptions - Antibiotic resistance can develop extremely rapidly - even in a patient receiving treatment in a hospitalNotes from clinical case:AssignmentRead Chapter 7 and pages 554-558Review 1, 2, 5, 7- 9MC 1,9,10CT 1-3CA 3FYIQ. How do you know if an antibiotic is going to work?A. You should feel better within 24-48 hours of starting antibiotic treatment.If you do not feel better:You have a viral infection and not a bacterial infection ORYou have a bacterial infection but the antibiotic prescribed is not effective against the bacteria you have ORYou have a bacterial infection but bacteria are resistant to the antibiotic that was prescribedThen you should contact your doctor and let her/him know that the antibiotic is not working.What can you do to reduce the likelihood that bacteria will become antibiotic resistant?Take the correct dosage of your antibiotic and always take the entire prescription. If you don’t, infectious bacteria that have not yet been killed off may survive, reproduce, and cause a more severe relapse, one that may not be treatable.Ask the doctor to tailor the prescription to fit your schedule so that you don’t miss a dose.Ask if you can take the antibiotic for the shortest amount of time possible.Ask the doctor to prescribe a narrow-spectrum antibiotic, one that works specifically against a few strains of bacteria, rather than a broad-spectrum antibiotic that targets more strains. The more bacteria exposed to antibiotics, the greater the chance that a strain will develop antibiotic resistance.Use the antibiotic only for the prescribed illness. Never take antibiotics that you have left over from a previous illness. Never take antibiotics that were prescribed for someone else (not even your mom).Review - Important Concepts for Lectures over MetabolismI know that you have had an introduction to the basics of metabolism in BIO 110. The metabolism you learned was focused on the types of metabolism that animal and plant cells carry out --aerobic respiration. The microorganisms are tremendously more diverse and complex in metabolic patterns than are Eukarya and I want to spend our time emphasizing what microbes can do, not just covering what you have already had in other courses.My focus in metabolism is that as microbes create and store and use energy for transporting nutrients, making their cellular components, growing, and moving in their environment. Importantly, as a consequence of their metabolism, they can profoundly change that environment!So, if you do not remember the basics of metabolism you will need to review. The following pages should serve as a reminder. If it doesn’t all come back to you then read Chapter 5 in the text. If you have not had chemistry you will also need to read Chapter 2.Review of nutritional patterns:Source of energySource of carbonChemicals = chemotrophmake it (CO2) = autotrophsOrganic = chemoorganoeat it (organic molecules (-C-C-C-)) = heterotrophsinorganic = chemolitho – you were not exposed to this concept in BIO 110Light = phototrophMost common combinations of Energy gaining strategy plus Carbon gaining strategy – this terminology was not used in BIO 110, but you were exposed to the concepts behind the terms “chemoorgano heterotroph” and “photo autotroph”)Chemoorgano heterotrophsChemolitho autotrophsPhoto autotrophsPhoto heterotrophsYou should also know definitions of metabolism, anabolism, and catabolismYou should know what ATP is and does. ATP (Adenosine Tri Phosphate) connects reactions that produce energy with reactions that use energy. It is made to store energy for later use – it is the energy “currency” for the cell.During catabolism – ATP ADP + Pi + energyDuring anabolism – ADP + Pi + energy ATP(Pi = inorganic phosphate; ADP stands for adenosine diphosphate)adenineriboseAdenosine diphosphate (ADP)Adenosine triphosphate (ATP)unstable bond = high energyAdenosine monophosphate (AMP)ATP can be formed in 3 ways:1. by substrate level phosphorylation – the simplest, oldest, and least-evolved way to make ATP - a high energy phosphate is removed from a substrate and is added to ADP to make ATP. Ex. C-C-C~P + ADP C-C-C + ATP2. by oxidative phosphorylation, aka electron transport phosphorylation – electrons are transferred from organic compounds to electron carrier molecules and then to final electron acceptor molecules. The transfer of electrons releases energy that is used to convert ADP ATP.3. by photophosporylation – occurs in photosynthetic cells only. Light energy is converted to ATP.You should understand basics of energy productionAll molecules have energy that is associated with the electrons that form bonds between atoms. The electrons can move around in a cell from molecule to molecule, transferring energy as they move. The molecules are changed as they either gain or lose electrons.Oxidation – Reduction Reactions (redox)In biological systems:HH++e-Hydrogenprotonelectron atomOxidation = a loss of an e- (and in biological systems, usually a loss of the H+ as well)Ex.H2O - 2e- - 2H+H2 + ?O2Ex.NO2-- 2e- - 2H+NO3-Reduction = a gain of an e- (and in biological systems, usually a gain of the H+ as well)Ex.?O2 + 2e- + 2H+H2OEx.NO3-+ 2e- + 2H+NO2-+ H2ORemember as LEO the lion says GER (Lose of Electron is Oxidation, Gain of Electron is Reduction)Redox reactions are always balanced.e- donor = reducing agent – causes its partner molecule to become reduced, to gain e-e- cannot exist free in a cell, it must go somewhere. So if are e- removed from one molecule they are added to another.e- acceptor = oxidizing agent – causes its partner molecule to become oxidized, to lose e-In biological molecules it is usually the entire H atom (electron and proton) that is lost or gained, but not always. Sometimes the electrons are separated from the proton and only the electrons are lost or gained; and sometimes it may be one H atom + 1 electron (from a second H atom) that are lost or gained.Ex.C3H4O3C3H6O3pair one pyruvatelactic acid oxidizedreduced 2e- + H+ NADH + H NAD+pair two reducedoxidizedNADH passes 2e- and 1 H+ to C3H4O3, as soon as C3H4O3 accepts the e-s and H+, it becomes C3H6O3 and NADH + H becomes NAD+In any pair of molecules you can distinguish which molecule is in the oxidized state (has lost an e-) and which molecule is in the reduced state (has gained an e-):Oxidized stateReduced state:Contains more oxygen atoms ORContains fewer oxygen atoms ORfewer hydrogen atoms ANDmore hydrogen atoms ANDtherefore has fewer electrons and istherefore has more electrons and isless negative or more positivemore negative or less positiveExample pairs:GlucosePyruvateC6H12O6C3H4O3NAD+NADHSulfateHydrogen sulfideSO4H2SEnd ReviewIn Lectures 10-13 we will explore how microbes create, store, and use energy. Through these processes microbes can profoundly change their environment.While we will be covering the topics outlined in Chapter 5 of your text, we will be doing it in a very different manner from how it is presented in the text. Please be prepared to take careful notes in class.III. Patterns of Metabolism in the Microbial World (a.k.a. how do microbes make a living – and why should we care?)A. The Basics: quick review of basics from BIO 110Metabolism = sum of all chem. rxns occurring within a living organismAll cells need a source of energy for:Catabolism- breaking bonds in molecules – Ex. glucose to carbon dioxide and waterAnabolism – creating bonds - B. Patterns of Energy Production Among Living OrganismsBasic informationPatterns among Eukarya:alcohol fermentation (yeast)lactic acid fermentation (muscle cells, neutrophils)aerobic respiration (mold, protozoa, animals)oxygenic photosynthesis (algae, plants)Bacteria and Archeae do all the above plus:anaerobic respiration: uses inorganic molecules other than 02 as a final electron acceptor lithotrophy: use of inorganic substances as sources of energy photoheterotrophy: use of organic compounds as a carbon source during bacterial photosynthesis anoxygenic photosynthesis: photophosphorylation in the absence of O2 methanogenesis: uses H2 as an energy source and produces methane light-driven nonphotosynthetic photophosphorylation: converts light energy into chemical energy We will explore only a few of theseThere are 2 initial sources of usable energy:1. sunlight – 2. chemical bonds of molecules - Heterotrophs - energy is created by breaking bonds in a molecule and harvesting the electrons released from the H atoms in:Organic molecules - Inorganic molecules – The more electrons a molecule has, the more energy it is capable of releasing. This initial molecule is called the ____________________________________________.Ex. glucose (C6H12O6) has a lot of H atoms (12) and therefore a lot of electrons, the oxidation of glucose will release a lot of electrons. **Glucose is a high energy electron donor.The electrons released from a molecule such as glucose have to go somewhere - they get passed from the initial donor of released electrons (electron donor) to intermediate electron carrier molecules.Example intermediate electron carrier - NAD (Nicotinamide Adenine Dinucleotide) - accepts 2e- (and 1 proton) and becomes reduced toNAD+NADHOxidized statemissing an H, (which means missing an e-)Reduced state(complete with e-)NAD = Nicotinamide Adenine Dinucleotide a mobile, cytoplasm soluble electron carrier PROBLEM: NADH can’t accept anymore electrons. If energy production is going to continue, the NADH must be converted back to NAD+, which means NADH must transfer the electrons somewhere.We will examine some of the solutions to this problem by seeing what chemoorgano heterotrophs do during carbohydrate catabolism (beginning next page)Many types of molecules can undergo catabolism to release energy:Proteins amino acidsLipids glycerol + fatty acidsCarbohydrates sugarsOne Example – generation of energy via carbohydrate catabolism – specifically the carbohydrate glucose – by a chemoorgano heterotrophwatch movement of e- and regeneration of NAD+watch for formation of ATP (energy storage molecule)Glycolysis (via Embden-Meyerhof Parnas (EMP) pathway) occurs in the cytoplasm – the initial electron donor is glucoseGlucose + 2 ATP2 Pyruvate + 4 ATPnet ATP production = C6H12O62 C3H4O3initial e- donorAt end of glycolysis - Need NAD+; NADH needs to get rid of e-First strategy:Fermentation –pass e- from NADH to an organic molecule, NADH becomes NAD+ - fermentation reactions occur in the cytoplasm of the cell. organic molecule(pyruvate)2 C3H4O32 NADH + 2H+2 C3H6O32 NAD+(lactic acid)End of fermentation - Inefficient process– Alternative (to fermentation) strategy:Respiration – pass e- from NADH (becomes NAD+) along a series of intermediate electron carrier molecules, ultimately to a final (or terminal) electron acceptor molecule.Occurs in 2 steps:Step 1 – Tricarboxylic acid (TCA) cycle (also known as citric acid cycle or Krebs cycle) – occurs in the cytoplasm - harvests the energy still within the bonds of pyruvate, but transfers even more e- to NAD+ (so more NAD+ is converted to NADH). Doesn’t solve the shortage of NAD+ problem.At end of TCA:For each pyruvate (C3H4O3) since we get 2 pyruvate per glucose…For each glucose (FAD is another intermediate electron carrier that functions so much like NAD that for the purposes of this course we will consider them equal)Step 2 – Electron Transport Chain – the soluble NADH and FADH2 carry e- from the cytoplasm (where glycolysis took place) to the cytoplasmic membrane and pass them off to a series of membrane associated proteins (when NADH passes off the e- it becomes NAD+). These proteins function as intermediate electron acceptors, accepting e- and becoming reduced, then passing the e- off to the next protein in the chain, becoming oxidized again, ending with the final or terminal electron acceptor (which accepts the e- and becomes reduced).This final electron acceptor may be oxygen – Final e- acceptor (oxidized state)accepts e- and becomes reduced toaerobic respiration1 molecule of C6H12O6 oxidized completely to CO2 coupled to reduction of oxygen to water (aerobic respiration) can yield up to a max of 38 ATP.ORThe final electron acceptor is an inorganic molecule other than oxygen – anaerobic respirationExamples of final e- acceptors for anaerobic respiration:Final e- acceptor (oxidized state)becomes reduced to:Fe3+Fe2+Iron respirationferric ironNO3-NO2-, N2O, N2Nitrate respirationnitrateSO42-HS-Sulfate respirationsulfate CO2CH4Methanogenesiscarbon dioxidemethaneS0HS-Sulfur respirationsulfurYield of ATP by cells undergoing anaerobic respiration is greater than the 2 ATP produced by glycolysis (and maintained in fermentation), but fewer than the 38 ATP produced by aerobic respiration.C. Some Exciting Implications of Microbial Activity:Metabolism of the Human Intestinal Microbial CommunityWhere does your gut microbial community come from?At birthProgression of your gut community if you were a breast-fed babyDay 1 - First colonizer was Escherichia colifacultative anaerobechemoorgano heterotroph (gets both C and E from organic molecules)Where did E. coli come from?What organic compound does E. coli use as a C and E source?How does E. coli get C and E from galactoseEntner-Douderoff2-keto-3-deoxygluconic acid 6-phosphatepyruvate glyceraldehyde 3-phosphatelactoselactoseglucoseglucose 6-phosphate2 pyruvate2 acetyl CoA lactic acidformic acid CO2 + H2succinic acidacetic acidethanolCO2CO2 Embden-Meyerhoff-Parnasfructose 6-phosphate2 glyceraldehyde 3-phosphateTCA cycleElectron transportoutsideinsideLactose utilization by E. coliDay 3 – 2 more bacteria joined you gut communityEnterococcusobligate but aerotolerant anaerobeschemoorgano heterotrophsobligate fermentative metabolismBifidobacteriumLactic acid bacteria (named from their final fermentation end product)e-1 Glucose NAD+glycolysis2 ATPe-2 Pyruvate NADHfermentation2 Lactic acidNAD+ Soon after added:Enterobacter- facultative anaerobeClostridium - obligate (aerotolerant) anaerobeButanediol fermenterse-1 GlucoseNAD+glycolysis2 ATPe-2 PyruvateNADHfermentationethanolAcetoinCO2 + H2NAD+ acetic acidlactic acidsuccinic acid 2,3-Butanediol + CO2Microbial gut community from 1 week to ~ 3.5 months if you were a breast-fed infant:E. coliEnterococcusBifidobacteriumEnterobacterClostridium3.5 months to weaning99% Bifidobacterium infantis –When meat was introduced to your diet:Gram-negative anaerobes:BifidobacteriumClostridiumFusobacteriumEubacteriumRuminococcusPeptococcusPeptostreptococcusBacteroides – 30% of total adult communityBacteroidesobligate anaerobeextremely oxygen sensitivefermentative metabolismb. What are the benefits of a stable, mature gut microbial community?2) and 3) in supplemental info at the end of this unit, p. 38. We do not have time to cover them in this class Nutritional Prevents colonization by pathogensTrains the immune system1) Nutritiona) Gut microbe metabolism converts complex polysaccharides to volatile fatty acids (vfa) – goodBacteroides is the key player in this processHost and dietary carbohydrates – complex carbs, starch, cellulosesaccharolaseshydrolasesfermentationby gut communityshort-chain volatile fatty acids*acetic acidbutyric acidpropionic acidreabsorbed through the large intestineused by you as an energy sourceprovide a significant proportion of your daily energy requirement (540 kcal)* These products in brown are good for your health metabolic by-productsb) Gut microbes can metabolize dietary fats too – not so goodBacteroides is the key player in this process alsoDietary fatsLiverBile acidsAbsorbed by small intestine----------------------------------------------------------------------------------------------------If fats and bile acids are not reabsorbed by small intestine but make it to colondeconjugated deoxycholic acid intermediate productslithocholic acid Bacteroides thetaiotomicronethyl ester*These products in purple are mutagenic, carcinogenic products; they can induce cancer – bad for your health productsc) Gut microbes can also metabolize dietary protein – can be badBy the combined activity of the colonic microbial communityDietary proteinPeptidesAmino acidsAbsorbed by small intestine----------------------------------------------------------------------------------------------------If peptides are not reabsorbed by small intestine but make it to colonAmino acids R+H3N – C – C – O- H – O deamination decarboxylationaromaticsulfuramino acidsamino acids reduced tophenolicSO4H2S gascompounds anaerobic respiration by fermentation sulfate-reducing bacteria by many microbesammoniaH2branched chainvolatile CO2fatty acidsfatty acids reduced toCH4 gasanaerobic respiration by Methanogens (which are Archaea)brown and purple as explained before. red are final electron acceptors in anaerobic respirationAutotrophy - BackgroundAll microbes need:a source of energy (electrons = ATP)a source of C to build macromolecules (-C-C-)Heterotrophs get C to make –C-C- by recycling the C contained in organic molecules. (all heterotrophs get energy [e-, ATP] to make –C-C- from breaking chemical bonds)**Autotrophs – get C to make –C-C- from CO2But there are 2 sources of energy that can be used to turn CO2 -C-C- and the source defines 2 groups of autotrophs:1. Photo autotrophsenergy from sunlight (C from CO2) (I’ll leave this for Botany, but lots of microbes do this too)2. Chemolitho autotrophs energy is generated from inorganic chemicals (C from CO2)Many different inorganic chemicals can serve as electron donors to provide the energy for microorganisms via aerobic respiration (notice the presence of O2 as electron acceptor in all the equations following – therefore all chemolitho autotrophs are obligate aerobes):a. Hydrogen gas as an electron donore- donore- acceptordonor reduced toH2 +1/2 O2H2OHydrogen bacteriaEx. Alcaligenes faecalis (from Lab 8)b. Sulfur compounds as electron donorse- donore- acceptordonor reduced to2S + 2H2O+3O2 2H2SO4Ex. Sulfur bacteria like Thiomargareta namibiensis or the bacteria that form snot-tites in caves.c. Nitrogen compounds as electron donorsNitrifying Bacteria2 groups of Nitrifying Bacteria:e- donore- acceptordonor reduced to2NH3 +3O2NO2 + 2H2O + 2H+Ex. Nitrosomonase- donore- acceptordonor reduced to2NO2 +2O22NO3 + 2H+Ex. Nitrobacterd. iron as an electron donore- donore- acceptordonor reduced toFe2+ +1/2 O2 + 2H+Fe3+ + H2OEx. Iron bacteria like FerroplasmaTwo scenarios where chemolitho autotrophs are very important:Metabolism of Wastewater TreatmentHow do we go from toilet water to treated water? (stay tuned, we will discuss this in Unit 4 )Metabolism of the Deep - What? No photosynthesis???Deep sea hydrothermal vents – provide all the necessary inorganic chemicalsBlack smokers – vent hydrogen, sulfur, iron (electron donors for energy), and CO2 (for carbon) from the Earth’s core Sea water contains dissolved oxygen (electron acceptor for aerobic respiration)Everything that is needed for chemolitho autotrophs to grow.Chemolitho autotrophic metabolism turns CO2 and inorganic chemicals into bacterial biomass, with excess energy to spare!Animals (chemoorgano heterotrophs)Giant tube wormswith endosymbiotic chemolitho autotrophsGiant musselsBrittle starsLimpetsWormsCrabsVent fishSharksAssignmentRead Chapter 5Review 4, 5b,c, 6,7,9MC 1,4,6CT 1,3,5This ends the lecture material for Test 2.Supplemental Information – If we have any extra time I may cover some of these topicsAside - How can 2 people eat the same foods, 1 person gains weight and the other stays lean?colonization of gut by microbes increases glucose uptake in the intestine↓microbial fermentation↓resulting in substantial elevations in serum glucose and insulinresults in production of short-chain fatty acidsbothstimulate lipogenesis in the liver↓triglycerides into the circulation↓taken up by adipocytes (fat cells)The composition and operation of your gut microbiota influences your energy balance.Relatively high-efficiency gut microbial communities would promote energy storage (weight gain), whereas lower efficiency communities would promote weight loss.Small but long-term differences between energy intake and expenditure can, in principle, produce major changes in body composition.Ex. if energy intake exceeds energy expenditure by +12 kcal/day, >1 lb of fat could be gained in a year; this is the average annual weight gain experienced by Americans between ages 25 and 55.1. b. Metabolism of Human Intestinal Microbial Community continued from p. 322). Mature gut microbial community prevents colonization by pathogens – pathogens like Salmonella, Shigella, Campylobacter, the pathogenic strains of E. coli, etc. that cause intestinal disease. a. competition for attachment sites – the gut epithelium is so densely colonized by normal microbiota, nowhere for pathogens to attach.b. competition for nutrients – if pathogens do attach, they have to fight normal microbiota for a share of nutrientsc. antimicrobial chemicals – and then the normal microbiota secrete antimicrobial chemicals that kill pathogens.Ex. E. coli – produces a chemical called colicin3) Mature gut microbial community trains the immune systemThe primary barrier between the outside world and you is a single layer (1 cell thick) of gut epithelium. This barrier is tight, but not impenetrable.Microvilli – where adsorption takes placeEpithelium-914400131445SubmucosaMuscleThe surface of the intestinal epithelium is protected by your immune system – the antibody IgA, and white blood cells called T and B lymphocytes, and phagocytic macrophages.-114300175895The gut epithelium tests the contents of the gut lumen (open cavity) and can directly sense the antigens of microbes using “pattern recognition receptors” (PRRs) – the epithelium recognizes conserved structures of bacteria and viruses and then alerts the host to the potential of infection.Normal microbiota of the gut and dietary antigens in food are tolerated (should not stimulate an immune response).1. continued.c. How does what you eat influence your gut community and in turn your health? –So very cool!!1). Over stimulation of microbial growth and metabolismEx. Lactose intoleranceIn babies, the enzyme human lactase is secreted by the small intestine and will break milk lactose into glucose and galactose. By the age of weaning, humans stop secreting human lactase.After the age of weaning if lactose is consumed in dairy, it will pass undigested to the large intestine. In the large intestine E. coli will secrete the enzyme ?-galactosidase, which will now break lactose in to glucose and galactose. The E. coli will use the glucose as a carbon and energy source to support rapid population growth. As a result of their fermentative metabolism on this bounty of glucose, E. coli will produce a lot of 3 carbon fermentation end products, and a lot of CO2 gas. The 3 C end products increase the osmotic pressure in the large intestine, which combined with the CO2 will results in the symptoms of bloating and diarrhea Adult lactose intolerance is the normal state for humans. People who as adults can tolerate lactose had ancestors that acquired a mutation that allows them to continue to secrete human lactase in to adulthood.2). Diet can upset immune system trainingThe gut immune system has the challenge of responding to disease-causing microbes but not responding to food antigens and the normal gut microbial community.In developed countries like the U.S., this discriminatory ability appears to be breaking down.High-fat, high-sugar, low-fiber diet changes gut community composition, which upsets immune training resulting in allergies and/or chronic inflammationEx.1. Allergies Children w/ allergies have a higher chance of having bad Clostridium difficile and Staphylococcus aureus and lower prevalence of good Bacteroides and Bifidobacteria in their gut.Ex. 2. Chronic inflammationCrohn's disease and ulcerative colitis (UC)? breakdown in tolerance to Bacteroides initiates an autoimmune reaction?Experimental txtt - whipworms3). Diet can promote abnormal cell growth – i.e., cancerExamples of suggested links between microbial metabolism and cancer:High fat diet – go back and look at diagram of what happens to fat in the gutconjugated secondary bile acids – are carcinogensHigh protein diet – go back and look at protein diagram againprotein fermentations may be sources of systemic toxinsHeterocyclic amines (HCA) are converted into carcinogens.phenolics from aromatic amino acids may enhance production of mutagens.reduced sulfur compounds (like H2S) may be toxic to the colonic epithelium.3. Alcohol consumptionacetaldehyde toxicityLook again at diagram of lactose utilization by E. coli. See where ethanol is produced by mixed acid fermentation? An intermediate molecule in the pathway Acetyl CoA ethanol is a toxin called acetaldehydeAcetyl CoA acetaldehyde ethanol Part of this pathway also runs in the reverse direction: oxidation mitochondria in the liver cellsethanol acetaldehyde (bad)acetic acid (good)alcohol dehydrogenasealdehyde dehydrogenaseIf there is a lot of ethanol being converted to acetaldehyde, the hepatic mitochondrial enzyme aldehyde dehydrogenase cannot keep up, and acetaldehyde levels build in the liver and blood. This causes symptoms of hangover in the short term, in the long term the acetaldehyde causes mutations in DNA that can lead to cancer.Prebiotics are complex carbohydrates that you cannot digest, such as fructo oligosaccharides (FOS). They pass to the intestines where they stimulate the growth and activity of intestinal bacteria that secrete beneficial metabolic end products. Fruits and vegetables contain oligosaccharides; bananas and artichokes are especially high.Probiotics are living bacteria from genera that produce favorable end products, such as Bifidobacterium and Lactobacillus.Review – Metabolism BasicsAll cells need:1. A source of carbon for making cellular molecules.There are two strategies for obtaining carbon:a. recycle the C already present in some organic (-C-C-) moleculeb. use CO2 from the atmosphere2. A source of energy for performing all cellular work (building molecules, transport across the plasma membrane, locomotion, etc.)Energy is created by harvesting the electrons (e-) present in:a. Organic molecules. (specifically the e- in the H atoms in the molecules)Hydrogen – showing the proton and electronlike a sugar or an amino acid ORb. Inorganic molecules. e- in molecules like ammoniahydrogen sulfideThe more electrons a molecule has, the more energy the molecule is capable of yielding – so look at glucose compared to hydrogen sulfide – which molecule should yield the most energy? (glucose has 12 H vs. 2 in H2S)The electrons that are released when bonds are broken have to go somewhere, so they get passed from the donor (the molecule that you started with that had all the electrons) to intermediate electron carriers.NAD+ is a soluble carrier present in the cytoplasm. It is lacking 1 electron (1 H) and so it can accept 1 electron (1 H). As it accepts the electron, it is reduced to NADH. Oxidized statefewer H, fewer e-more positive (NAD+)Reduced statemore H, more e-more negative (NADH)NAD+ is in limiting quantities in the cell and it must be converted back to NADH if energy production is to continue. There are 2 ways convert NADH back to NAD+:NADH passes the electron to an organic molecule like pyruvate – this process is called fermentation - as NADH loses the electron it becomes oxidized to NAD+ again. As pyruvate accepts the electron it becomes reduced to acetic acid or to ethanol, etc., which are excreted from the cell, carrying waste electrons with them. Acetic acid, ethanol, etc. still have electrons, so potential energy is lost in the fermentation strategy.2. NADH travels to the cytoplasmic membrane and passes the electron off to the electron transport chain. This process is called respiration. (NADH then becomes NAD+ )electrochemical gradient - energyFig. 5.16The electrons are passed along the chain, generating two types of usable energy along the way – electrochemical gradient and ATP - until they reach a final electron acceptor, an inorganic molecule which can be:a. oxygen (aerobic respiration)ORAs oxygen accepts e- it will become reduced to H2Ob. some other inorganic molecule (anaerobic respiration) like nitrateor sulfatebecomes reduced to nitrite (NO2)becomes reduced to hydrogen sulfide (H2S)Note – fermentation is NOT anaerobic respiration. By definition respiration requires both an electron transport chain and an inorganic terminal electron acceptor. Fermentation does not employ an electron transport chain and the terminal electron acceptor is an organic molecule. Fermentation takes place in the absence of oxygen, it can occur in anoxic (no oxygen but has nitrate) and anaerobic (no oxygen, no nitrate) environments, but it is not respiration!Comparison of Respiration vs Fermentation in ChemoorganotrophsRespirationFermentationInitial electron donor:organic moleculeorganic moleculeexamples:carbohydrates, amino acids, lipidscarbohydrates, amino acids, lipidsIntermediary electron carrier(s):NADH, FADH2, carriers in the electron transport chainNADHFinal electron acceptorinorganic moleculeorganic moleculeexamples:O2CO2, NO3, SO4pyruvatefinal electron acceptor reduced to:H2OCH4, NO2, H2Slactic acid, acetic acid, ethanol, etc.example organismsMitochondria, E. coli, Pseudomonas,S. aureusMethanogens, E. coli, Pseudomonas, Sulfate-reducing bacteriaBifidobacterium, Lactobacillus, E. coli, Clostridium, BacteroidesPotential net ATP yield:as many as 38 if starting with 1 glucose by aerobic respiration with an electron transport chain containing all the cytochromes – but often far fewer than 38 - but still more than 2.2Comparison of Respiration in Chemoorganotrophs vs ChemolithotrophsChemoorganotrophChemolithotrophInitial electron donor:organic (-C-C-) moleculeinorganic moleculeexamples:carbohydrates, amino acids, lipidshydrogen gas, ammonia, nitrate, hydrogen sulfideElectron donor oxidized to:CO2water, nitrate, nitrite, sulfuric acidFinal electron acceptorinorganic moleculeinorganic moleculeexamples:O2 (aerobic respiration)CO2, NO3, SO4 (anaerobic respiration)O2 (aerobic respiration)electron acceptor reduced to:H2OCH4, NO2, H2SH2Oexample organismsMitochondria, E. coli, Pseudomonas,S. aureusMethanogens,E. coli, Pseudomonas, Sulfate-reducing bacteriaAlcaligenes, Nitrosomonas, Nitrobacter, Thiomargarita ................
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