Microbiology Ii

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DNA

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Deoxyribose sugar and phostphates linked to nucleotides--nitrogenous bases a,t,c, and g. A and G are purines (bigger), t and c pyramidines (smaller). H bonds form btwn the bases--two btwn a and t and 3 btwn g and c. DNA has a 3' and 5' end.

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Dividing Bacteria by Metabolism

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Metabolism=all biochem processes in cell. Inc. catabolic and anabolic.
Cata=breaking down molecules, releasing e.
Ana=Building molecules, using e.
Prokaryotes can be divided by Carbon Source and Energy Source.
C Source= Can be from organic compounds (heterotrophs), inorganic (like CO2, autotrophs).
Energy S=Light (phototrophs), Inorganic comps (chemolithotrophs), or organic comps (chemoorganotrophs).
Common Combos= Chemolithotroph/Autotroph, Photoroph/Autotroph, Chemoorganotroph/Heterotroph...
Uncommon "mixotrophs"= Inorg.compounds for e w/ org. for C.
Autotrophs and Heterotrophs=both can either be phototrophic, lithotrophic or organotrophic.

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Energy Conservation

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Energy put into ATP with catabolic reactions. PMForce stores pot. e.

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Redox Reaction

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An e- is donated, oxidizing the donor, and accepted, reducing the acceptor. This releases energy. Amount depends on difference btwn the donor and acceptor's potentials. Glucose and O, together, one of most energetic. High reduction pot=Very likely to be acceptor. Low=donor.Measured in Volts.
"Electron source"not same as "energy source." E- source is the redox reaction.

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Aerobic Respiration

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A redox reaction w organic C source glucose as donor and Oxygen as final e- acceptor.
Glucose->glycolysis and +ATP->Pyruvate->Citric Acid Cycle (TSA)->E- carriers NADH->E- transport chain->E- to Oxy, + Generates PMForce by moving protons from inside to outside, making ATP indirectly by ATP Synthase in memb.
Anaerobic Resp. is the same basiclly except diff final e- acceptor.

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Fermentation

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Produces little energy.
Happens when no external e- acceptor. Accptr=broken down donor.
Only substrate-level phosphorilation=no e- tranprt chain or PMForce.
Glucose->pyruvate. Pyruvate then broken down into acids, alcohols, or gases. O2 may be made. Break down pyruvate cause if it builds up it can stop or reverse fermentation.
Doesn't use O2 but can be done in its presence.

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Phototrophy

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Energy source=light, which drives the redox/movement of e- down trnsprt chain.
C Sources= can be photoautotrophic (CO2) or photoheterotrophic (Org cmp.)
Oxygenic=cyanobacteria. Bacteriorhodopsin-mediated phototrophy.
Non-oxygenic=Purple and green sulfur bacteria.

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Bacteriorhodopsin-mediated photolysis

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Some phototrophs do this as a supplement to other energy harvesting sys.
A memb protein that is a light-driven proton pump for PMF.
Absorbs green light.. Binds retinol, which is what gets excited by light.Energy released when retinol comes down, pumping H+ across.

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Photolysis

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Some phototrophs do this.
Light driven removal of an e- from a molecule->trnsprt chain.
CAN be coupled to CO2 fixation (photosynth.).
Chloropyll excited by light, energy->reaction centre, reaction center excited->hydrolysis=an e- is taken from a donor and brought to e- t.chain, generating PMF. Also e- reduces NAD to NADH.
Photosystem II: Bacteriochlorophyll=absorb mostly light not visible. After e-tchain and PMF, e- RETURNS to the bacteriochloropyll. No NADH created. Only Photoheterotrophs.

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Oxygenic photolysis

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Photosys I and II are anaerobic. This is a combo of I and II, phtsis II to I.
H2O is the e- donor, O2 is produced.(Imp to oxygenation of earth's atmos.)
Cyanobacteria do it.

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Chemoorganotrophy

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Organic comps for energy source
Can be auto or heterotrophic for C source.
Respiration or fermentation.
B/c simple sugars not common in environment, many bact can digest pectin, starch, cellulose, etc.

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Fermentation products

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Cheese (ferm amino acids and sugars)
Chocolate
Butter, meat, dairy (lipids)

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Dissimilatory Metal Reduction

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Metals are final e- acceptors for anaerobic resp.
Since many oxidized metals are insoluble, the e- is transferred w special memb components/surface proteins

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Lithotrophy

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Inorganic e- donor.
Since these e- donors usually suck, O2 is almost always the acceptor.
With H2 as donor, produces methane. H2 is a strong e- donor, so can be anaerobic or aerobic. H2 is produced from some fermentation, so these can be found where that occurs.

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Nucleoid

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Prokaryotes have this.
Holds DNA in the cytoplasm. No memb. Gyrases wind the DNA into supercoils or over-relax it into -supercoils.

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Information flow in the cell

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DNA->transcription->mRNA->translation->Proteins.
tRNA facilitates translation.
rRNA is used in forming the structure of ribosomes
Codon=three nucleotides translated at a time. 64 total poss. codons, only 20 amino acids, so there's redundancy.

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Start codon? ORF?

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AUG=start
ORF=Open reading frame. This is the start, translatable codons, and stop code.

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Transcription

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mRNA is made from 5'->3', so it is going to match the 5'->3' DNA strand.
RNA Polymerase looks for promoter sequence. Different sigma factors look for diff promoters.
As soon as one RNA polymerase moves past the promoter, another starts.
Termination: Intrinsic: inverted repeats in the sequence form stem-loop structures in the mRNA product. This causes the RNAPoly to fall off.
Rho-Dependent: Sequence in the mRNA product signals a Rho protein, which binds to the mRNA. When the Rho and the RNAPoly meet, the RNA poly stops transcribing.

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Promoter

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-35 sequence. Some are better at getting RNA polymerases than others. This helps regulate expression.
____
TATA box.

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Prokaryotic mRNA

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Requires no extra processing.
Can be polycistronic, where the RNAPoly can transcribe multiple genes into one mRNA.

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Translation

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Ribosome reads from 5'->3' on mRNA. The mRNA goes through the ribosome. Mult ribosomes can translate 1 mRNA at the same time.
tRNAs with the appropriate amino acids come and ribosome catalyzes peptide bond formation btwn the amino acids.
To begin translation: 30s subunit of the ribosome binds to Shine-Delgano seq on the mRNA (a seq in the 16sRNA of the ribosome is complementary).
Other parts of the rbsme bind, then 50s subunit binds.
Elongation: Adding amino acids to the growing peptide chain.
Termination: Ribsme reaches STOP codon. Instead of a tRNA, a release factor comes and releases the ribsme by cutting the pptdchain from the last tRNA.
Subunits of rbsme disassociate.

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Ribosome structure

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50s and 30s subunits. 30s is a 16sRNA and a bunch of proteins, 50s is also made of RNAs and proteins.

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Overview of a polycistronic sequence

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Promoter, SD, Start, translatable, stop, SD, start, translatable, stop, SD, start, translatable, stop, terminator.
So, to find genes in a bacterial genome, look for ORFs with SDs.

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Bacterial genomes

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Circular, plasmids, or linear chromosomes.
Smaller than all but most archaea and viruses.
Most is coding. Size of genome corresponds to # of genes.
Parasitic or symbiotic bact. have smaller genomes.
Can try to ID function by comparing similar seqs.

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Plasmids

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Small, circular, non-essential but often helpful genes.
Replicate separately form chromosomes. Do need cell's DNA to replicate and express proteins.
Fplasmids can do conjugation, Rplasmids carry antibiotic resistence genes, Pplasmids carry genes that cause pathogenesis.

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Why sequence a genome? Why genomics?

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Seq genomes to find functions of genes by comparison, study metabolisms--how they can be used for humans or how they work in environment, compare genomes to study evolution of bacteria, compare related species--why is one pathogenic, one not? (or resistant or not?)

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Why do some bact. have larger genomes?

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Usually live in less stable environments (like free-living) so they need more abilities to cope with change.
Ex: streptomyces has a big one. Forms towers with spores on top, look filamentous like fungi, can produce many antibiotics.

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Metagenomics

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All the genomes in a communtity.
Sometimes you get whole genomes, sometimes you dont.
You can see what processes can be done in a community, or collect all 16srRNA seqs and see what species (ideally) are present.
Ex: Metagnmcs of gut flora have been studied to see if there is a correlation btwn cert bacteria and obesity.

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Transcriptomics and Proteomics

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T: see what mRNA is actually made, shows you what genes are actually being transcribed.
P: all the proteins present in a cell.

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Bacterial inheritance

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inheritance is asexual, division makes clones, replication errors->mutation and ^ variation.
Horizontal gene transfer: one prokryte to another, often regardless of species. Can happen via transduction, conjugation, or transformation.
Transduction: Gene transfer thru a virus.
Transformation: Genes are picked up from the environment. Only some can do this, they are called "competent."
Conjugation: mediated by an F plasmid, so all required genes are on the Fplas. Replicates whatever is attached to the F plas. (So cell's chromosome will only be transferred if the F plas is integrated into it.) WIll only occur btwn an F+ and F-. Rolling circle replication of Fplas. Imp Fplas genes involved=F-pilus, Tral ("nick" forming), and memb proteins. Pilus extends, grabs F-, pulls it in. One strand of Fplas nicked, replication begins at 5' end. Cytoplasmic channel btwn cells made. New strand is drawn into F-, where a new double strand is made, now two F+ cells.

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Hfr Strains

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High frequency recombination strains have an integrated Fplasmid. Integration can happen if there is a completmentary sequence. Fpla can pop in, and back out again. When in, it's no longer self replicating, but its genes can still be expressed and conjugatin occur. Usually the whole chromosome won't transfer b/c conjugtn is delicate, the genes on either side of the Hfr are most likely to be transferred, depending on orientation of the 5' end.

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Selective media

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Allows growth of some bact but not others. Ex: certain nutrients, antibiotics.

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Auxotroph and prototroph

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Auxo:A cell that requires a cert nutrient.
Proto: A cell that can synthesize the nutrient in question.

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How do we define a "dead" bact

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Even in conditions where the bact should grow, it doesn't. Does it matter if it can stil grow under diff conditions?

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Antibiotic, chemotherapeutic.

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Antibiotic: Chemical produced by a microorg that kills other microorgs. (strict definition).
May be synthetically altered.
Or, totally synthetic ones.
From fungi, like penicillin, bact, like streptomyces.
Usually produced in stationary phase, when there are limited nutrients.
Min. Inhibitory Conc=min needed to prevent growth.
Bacteriostatic=inhibits growth but doesnt kill. When antibiotic is gone, immune sys needs to kick in or bact will return.
Bacteriolytic=lyses bact to kill them.
-cidal=kills bact.
Chemotherapeutic: Chems that are safe to be used internally to treat infections.
Spectrum: Broad or narrow, how many types o bact can it kill?

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So what can antibiotics target that is in prokaryotes and not human cells?

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Cell wall--peptidoglycan synth (penicilin, Beta-lactams)
Ribosome- TET and CHL
DNA replication enzymes
RNA synth machinery
Metabolic processes

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B-lactams

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Antibiotic, bacteriostatic. These have B lactam rings, which resemble transpeptidase and is able to out compete it to inhibit cell wall formation.
Cells can become resistant to it with B lactamase.
Ex of B lactams=penicillin, -cillins.

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Sulfa Drugs

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Prokaryotes are prototrophic for folic acid, aukaryts are auxophilic. Sulfa drugs inhibit production of facid. Resistance by picking up a gene that makes them auxotrophic (so they can scavange for it).

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Antibiotic resistance methods

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enzymes that modify the drug
change of the target site
change of the metabolic needs.
change permeability
efflux pump---tend to be very non-specific, so can pump out many antibiotics

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Alternative functions of natural antibiotics?

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Maybe main function is communication.
Amount in soil=v low.
in low doses, affects gene expression.
What if the resistance strategies are part of the process, just helpful when there are too much antibiotics?

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Viruses and esp bacteriophages

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Obligate intracellular parasites
Line btwn cells and viruses is iffy, but so far no viruses with their own ribosomes.
Usually wya smaller genomes than cells.
Basically genetic material in protien coat, sometimes a lipid bilayer. Cant' replicate on own.
Very symmetrical, regular in structure.
Icosahedral viruses=20 sides.Easiest to make with even just 1 type o protien. (efficiency is imp for viruses).
Filamentous viruses.
T4 bactriophge is complex. Capsid, tail with peices for attachment, injects genetic mat into cell. Capsid remains outside, attached.
Some have DNA, some RNA, single or double stranded. baltimore scheme=7 types but genetic mat type. +sense=genmat read in same dir as cell RNA.no transcrptn ness.
-sense=need to have reverse comp copies (thru regular trnscriptn) made b4 transltn.
Reverse transcriptase=makes DNA from viral RNA
Retrovirus=needs revtranscrptse to make DNA so the DNA can go into host's genome.

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Replication of viruses

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Bctriophge: Attachment. t4 tail fibers bind the polysachharides of the lps layer. Inject enzyme to make hole in cell. Contracts, allowing genmat to be injected. in the cell, 1st proteins degrade host dna. next help w viral rollingcircle replication. next are structyral. Finally more lysozyme made to cut cell wall and release.
General virus: Dont grow, are assembled.
Attchmnt to cell depends on cells surface proteins.
entry across cell memb or walls, at least of genmat.
viral genes expresed to make protiens.
Viral genome replicated.
new virons assembled.(self-assembly. Genome can be pulled into capsid until full (t4) or can be made around the genmat.
Release from cell.
Generalized transduction: Esp. with viruses that degrade their hosts' DNA, frags that are similar to the viral sequence can get into new virons. if that one infects another cell, (AND ONLY HAS HOST DNA) transduction occurs (nd no new virons are made)

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Temperate phages

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Can package both host and viral DNA. Do the lysogenic cycle, where they are incorporated into the host's DNA and lie dormant, being repicated w host's genome. WIll pop out and start lytic cycle if host is not repilcating enough.Ususally this is done accurately but if not, host DNA can be taken along. Then new virons have host DNA. (Specialized transduction.)

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Influenza

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-Can weaken you, leaving you prone to other infections. Usually old and v young die.
Pandemic=has become worldwide. 1918=worst in recorded hist. But mostly young ppl died-like 20s.
Virus is filamentous capsid. Lipid bilayer taken from host cell upon intial release.Studded with viral protiens (2 types). DNA frags, 10 genes total. Minus sense RNA genome. RNA genomes have high mutation rate b/c errors are common w no proofreading (unlike in DNA transciption).
Replication: Surface proteins bind to cell surface. Attachment triggers entry of viron, but it takes lbilayer w it, so its in a vesicle. When the pH in the vesiccle drops naturally, vesicle disintegrates. Virus releases RNA, which enters nucleus, replicates. Genes are expressed. The two surface proteins accumulate on cell wall, new virons leave and take lbilayer w them.
Immune response=antibodies attach to the surface proteins, inhibiting attachment.

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antigenic drift vs shift

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antigenic drift. antigens change, leaving you w only partial immunity at best.
shift=cause of pandemics. big change in virus. For flu, a new combo of HA and NA surface prtns. NOT THRU MUTATION...but ressortment.
Reassortment=when multiple flus infect one cell, a new virus can emerge w a combo of their genes. H1N1=a triple reassortmnt! This time fewer old ppl died bc they had patial immunity from 1918 flu, which was similar.

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what can affect whether the virus will become more or less symptomatic?

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how long it can last outside a body
how its transmitted.
our behavioural changes/reactions to it.

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why do some ppl get sick and not others?

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the nature of the virus.
genetics and immune sys of host
transmission--mode, environment, pop density, amount of virus you were expsed to.
environment--sun, humidity, who knows.

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1918 flu experiemtnt

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was it the flu or the environ that made it so bad?
flus in general cause damage to lung cells, causing immune fluid to leak into lungs. ^immune resp=more fluid.
1918flu infected monkeys died much faster and more often than normal flu monkeys. 1918 flu was found to cause way more damage to cells, and thus more leakage. The younger ppl with greater immune responses were dying BC of their immune responses. Maybe this flu replicates faster or more often.

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viruses in lab

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need to grow a host to grow a virus.
for euk, can get cells form tissue, which will grow as a monolayer on top of dish. viruses replicate and are released into media.
Count them by doing an assay of PFUs (plaque forming units). Serial dilution, count the HOLES in a stained plate--these r the unstained dead cells that have rounded up, detached, shrunk.

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Winogradski column

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good for bact that dont grow on plates.
can do metagenomics to see diversity, metabolic funtions of a community.

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Kirby-bauer test

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antibiotic resistance assay. measure diameter of no growth. have a contorl of a susceptible bact to make sure your antibiotics are working.