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麻省理工大学课件：系统微生物学（笔记）20.106J – Systems MicrobiologyLecture 24Prof. SchauerFinal Exam next weekThe main focus of the final exam is going to be our last eight lectures: the topics listed belowo The format will be similar to the format of the testso Open-ended questionso You need to know the concepts –you do not need to memorize specific detailso There will also probably be a couple of questions about the lectures from earlier in the course, given by Prof. DeLong Topics:o1: Growth ControlPhysical growth controlHeat and autoclavingThe kind of cell death that occurs with heatChemical growth controlOutside of the bodyStrategies of controlling microbes on surfaces, etc.AntibioticsInhibition of cell wall synthesis, ribosomesBeta-lactam antibioticsKnow why these don’t interfere with protein synthesis inour own cellsAntibiotic design requires targeting a feature that is unique to bacterial cells, and not human ones. Asking you toformulate a hypothetical new antibiotic would a be areasonable question on the test.Antibiotic resistanceKnow that the existence of antimicrobial resistancepredated our own medical use of antibiotics. Why is that?o2: Microbe-host InteractionsHealthcare-associated infections (HAI)Know about the acquisition of drug resistance by some ofthese microbes in peopleVRE, MSRAThe fear of developing a superbug that we have to drug to treatCommensal microbiota (ecology, models)You should be familiar with the concept that there aremicrobes that live in every living thing on earthKnow about the development of a climax community ofmicrobiota in the human gut as people grow up frominfants to adultsGnotobiotic animals: in the lab, people can create rabbits,mice, or pigs grown in a sterile environment so that theyhave no microbiota inside them.You might be able to make some comparisons between our endosymbionts and those of aphidso 3.4: Immunology I and IIImmune cellsBe familiar with the main components of the immunesystemKnow the main components of lymphocytesKnow what B and T cells do once they become activated,what they secreteInflammation, phagocytosisNatural immunityGetting microbes out of the injured siteAdaptive immunity (Ag, MHC, T, B)Know the way that antibodies recognize antigens, ascompared to the way that T cells can recognize themB cells bind to conformational antigensAffinity maturationo Creates a little extra diversity in a B cell responseo However, this does not happen in T cells, because itwould be catastrophic to have T cells that recognizedifferent things –you don’t want them to startattacking your own cells. They need to recognizeyour own MHCVaccines (types)Know about the general types of vaccinesYou won’t have to name specific vaccinesUnderstand the range of successful vaccines that have been usedFor example, there’s the TB vaccine, which only protectsagainst childhood TB. You can’t give a vaccine like that to immuno-suppressed people – it could kill themAnergy (tolerance)HypersensitivityType I: IGEType IV: TB test is an exampleo5: Diagnostic MicrobiologyExotoxins: A-B toxins, SAg (Super antigens)Super antigens bind to the conserved parts of the TCR and the APCo They stimulate large numbers of T cells that sharecommon variable regions of the Beta chaino Whole sale stimulation, release of large quantitiesof cytokinesSelective, differential mediaSelective media to inhibit growth of commensalsYou can also make differential media, such as adding sugars or pH sensitive dyesMAb, serologyo6: Person-to-person TransmissionTB (risk factors, pathogenesis)Many people get exposed, many people develop latent infectionsIf they become immuno-suppressed, they can develop active TBInfluenzaAntigenic shifto Large, sudden changesAntigenic drifto Small changes in the proteins compromise the ability of your system to protect you from the virus Hp Only a small proportion actually develop peptic ulcer diseaseo7: EpidemiologyTermsIncidence, prevalence, control, transmission Emerging infectious diseaseso8: Arthropod-borne and Zoonotic DiseasesPlague (epidemiology, pathogenesis)Wild rodents, transmission through fleas on a sporadic basisBe familiar with how plague affects the flea life cycle, causing it to bite more peopleBubonic, Systemic, and Pneumonic forms of plague。
20.106J – Systems MicrobiologyLecture 4Prof. Schauer¾Reading for today: Chapter 6 – On Growth¾Problem set due today¾Today: Growth – in microorganisms it’s different from in metazoans – increase in number of organisms instead of sizeo Binary fissiono Other methods:Organisms that replicate their DNA many times over, than splitinto many parts at once¾Next week: metabolic regulationBinary Fissiono Time from bacterium to bacteria is a generationo Generation time is how long it takeso20 minutes is a rather fast generation time. 8 minutes is the world record.o We look for bacteria that can replicate fast, or that can replicate in extreme conditions.o Cell content replicates before division.Fts proteins and the “divisome”o FtsZ aligns before divisiono The most intense signal occurs at the center edges due to the 3-dimensional shapePeptidoglycan synthesiso Peptidoglycan needs to be extgended for the cell to growo The balance needs to be right, so cell integrity isn’t compromisedo Antibiotics bind to DNA binding proteins like FtsI, so that those enzymes aren’t available for the peptidoglycan synthesis, and the bacterium lyses.(Autolysins without autolysis)o The FtsZ ring leaves a scar in the cell wall, which you can see later Peptidoglycan structureo Two planes with cross-links in between. These cross-links give it its integrityo MreB allows a variety of shapes -- not just spheresExponential Growtho Because bacteria undergo binary fission, they can replicate into mind-boggling numbers very fast (exponential rate)o After two days of unregulated growth one bacterium’s offspring would weigh more than the earth (assuming a 20 minute generation time) o Make a logarithmic plot of change in numbers as a function of timeGrowth Parameterso Write out equationso There will be homework problems relating to this growtho Related growth parametersThe growth cycleo Why aren’t bacteria always doubling? What limits their growth?They exhaust their nutrients, causing the growth curve to level offBuild up of toxic waste productso The cell has to replicate everything before it dividesTherefore if you move a cell from a bad medium to a good one,there’s a lag before it begins to grow.o Stationary phase – in a batch culture, for the most part things stay the same.o Death – in bacteria, this is exponential, like growth (very important)It’s not clear what’s going on here – people have speculated.Total cell counto Demonstration: Prof. Schauer shows the class a counting chamberGrid etched on with a laserTwo raised ridges – glass coverslip fits directly over, allowing you to measure the space between the platform and the coverslip –count through a microscopeThe same concept and method is used for bacterial, blood cells,environmental samples, etc.o Problems with this method:Not very preciseHard to seeDoesn’t distinguish live cells from dead onesRequires phase contrast microscope to count unstained cellsDilute samples must be concentratedViable counto This is the more common method – dilute sample many times overo Demonstration: Prof. Schauer displays samples of test tubes with successive dilutions – each test tube is progressively less cloudy.o Then you plate the resulting tubes and wait for colonies to appearo You want to count a plate with between 30 and 300 cellsOtherwise the error becomes too higho Demonstration: Prof. Schauer displays agar plates resulting from each successive dilutiono This kind of evaluation is difficult for slow-growing bacteria – you have to leave the plate to grow for up to a month.o This method doesn’t work for bacteria that can’t make coloniesThese bacteria might be viable, but clump (you can use detergents to try to fix this problem)Some organisms don’t separate, but come in chainso Plating methodsSometimes putting the agar on top is useful, because it stops thebacteria from moving aroundTurbidity as an indirect measureo Light scattering off of organismso Depends on morphology of organisms – larger organisms scatter more lighto You can quantify organisms by measuring the light scatteringPhotometersThis is advantageous because you can still keep using the sample Chemostat cultureo Instrument called a chemostat – bioreactor of sorts – you grow bacteria in ito Open systemo Number of bacteria and rate of growth are kept constanto It enables you to control both the bacterial concentration and the doubling time.Cardinal temperatures: extremophileso Temperature as an environmental condition – controls rate and yieldo For every organism, you can determine maximum, optimum, and minimum temperatures for growtho The optimum is always closer to the maximum than it is to the minimumo Classes of organismsSome organisms can grow in up to 113o COrganisms can grow anywhere that there’s watero PsychrophilesIt’s very clear why organisms can’t grow at very hightemperatures: proteins denature, etc.However, it’s less clear why they can’t grow in low temperatures: you lose hydrogen bonding, but that’s about all that changesTrue psychrophiles, that prefer very cold temperatures, are rareThose organisms can’t handle warmer temperatures – thereforethey live only in areas where it’s cold all year round: the North andSouth Poles, glaciers.o HyperthermophilesMost of these are archaeaArchaea probably originated at very high temperatures: thermalvents, magmaThey grow in superheated, high pressure water, over 100o CThey have positive supercoiling of DNA – everything else on earth has negative-coiled DNAProblems with membrane stability – remember, archaea havedifferent membranes from us (eukaryotes can never grow above50o CThermophileso Important source of enzymes for biotechnologyDifferently colored band at Yellowstone: each colored band is adifferent thermophileExtremophiles of pH and osmolarityo They maintain their internal cell environmentThey don’t, for example, have such low pH or such high saltconcentration inside the cell as they do outsideo Accumulate inorganic ions or make organic soluteso Compatible soluteso Note: freezing is similar to dehydration: what kills cells as they freeze is the loss of H2O as it forms into crystalso Demonstration: Prof. Schauer shows the class a device for creating an anaerobic atmosphere for growtho Toxic forms of oxygen。
20.106J – Systems MicrobiologyLecture 2Prof. DeLong¾ Reading for next week: Chapter 5 (Biol. Energy, Couscku)¾ Problem Set #1 due next week¾ Reading for today: Purcell, Berg, and Pace• Related to the last lecture:o Life’s history on Earth – EvidencePhylogenetic treeThe same machinery for making proteins – with ribosomes – isused all over Earth.You can map how different the ribosomal RNA is in each species on Earth.• In this way we can compare microbes to eukaryotes.A lot of the Eukarya tree (our own tree) is dominated by microbes – Archaea.Chloroplast RNA falls right next to cyanobacteria on that tree• This supports the endosymbiont hypothesis – chloroplastsderive from cyanobacteria.Similarly, mitochondrial RNA falls by agrobacteria – αproteobacteria.o Life on Earth today: the foundationCO 2/O 2 cycle• To be covered today: Structure, Function, Motilityo The nature of being smallo Cell membranes and cell wallso Flagella• Shape and Appearance – not where the interesting stuff is regarding microbeso They don’t bring in solid food – they bring in dissolved substrates.Surface area to volume:r r r V SA 334432==ππ o “Prokaryote” vs. EukaryoteIn eukaryotes, there are organelles and a nucleus – quite a lot ofcommunication and transport is going on.In prokaryotes, transcription and translation all occur together inthe cytoplasmHowever, “Prokaryote” is in quotes because it is only a negativedefinition – they are defined only by the lack of a membrane-bound nucleus.One group of microbes – Archaea – are a lot more like eukaryotes than they are like bacteria.•Their informational machinery – RNA polymerase,promoters – are more similar to those of eukaryotes.Hence there are Three large branches of life: Bacteria, Archaea,and Eukarya (the two-branch representation of life as prokaryotevs. eukaryote is less accurate).•Cell membranes: phospholipid bilayero Main permeability barriero Embedded integral membrane proteins – communication, transporto Membrane structureBacteria, eukaryotesArchaeao Archaea can still make lipid bilayers – though sometimes they hook them directly together, making a lipid monolayer.This is much more structurally rigid.This is never found in bacteria or eukaryotes.o Membranes act as a protein anchor.o Also energy conservation – protein motive force.o Membrane permeability to various molecules:Simple transport: let a proton down the gradient in order to move things.Group translocation: chemical modification of transportedsubstance driven by phosphoenolpyruvate.ABC system: periplasmic binding proteins are involved and energy comes from ATP.o Transport method:Uniporter: one thing comes in.Antiporter: one thing in, one out.Symporter: two in at once.o Gram-positive bacteria have one phospholipid bilayer.With a thick peptidoglycan layer outside.o Gram-negative bacteria have two bilayersThere is periplasm in between.•Most of the binding proteins are located here.The outer membrane (lipopolysaccharide and protein)•Antibiotic resistance occurs here – resistance thus occursmore easily in gram-negative bacteria.There is a peptidoglycan layer in the middle of the periplasm, but it’s very thin.•It forms a net-like structure, with a single molecule ofpeptidoglycan that acts as a nylon stocking.• This maintains structure, shape, and integrity.Lipopolysaccharide chains outside – can often make people sick o In penicillin, lysozyme chews up peptidoglycanThen water all rushes in, causing lysisPenicillin inhibits the crosslinksTherefore penicillin only works on cells that are growingo Archea – S-layers, pseudo peptidoglycan• Motilityo Flagella – moves like a propeller in bacteria, not like a whip – they’re rigidVideo clip: E. coli moving with rotating flagellaηνρa ForcesViscous Forces Inertial ≈← Fluid Density ← Fluid Viscosity The movement is dominated by viscosityo Clamshell hypothesis: reciprocal motion doesn’t work at low Reynolds number – instead it’s a rotary motorProton motive force turns a ring that drives the motoro Flagella are hollow on the insideMade of one protein: flagellinIt grows from the inside-outVery complexo Going counter-clockwise they drive the cell forwardGoing clockwise, they fly out in a tumbleo By changing the frequency, you get longer or shorter runs。
