MIC 401 Week 2 Notes
MIC 401 Week 2 Notes MIC 401
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This 13 page Class Notes was uploaded by Christinee on Monday February 8, 2016. The Class Notes belongs to MIC 401 at University at Buffalo taught by Dr. Amy Jacobs in Spring 2016. Since its upload, it has received 227 views. For similar materials see Biomedical Microbiology in Microbiology at University at Buffalo.
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Date Created: 02/08/16
Lecture 3 Cellular Functions Microbial Genetics *No additional notes for this lecture since the professor included his notes on the lecture slides & it was selfexplanatory. Lecture 4 – Bacteriophages *These are just additional notes that aren’t included on the slides that were discussed during lecture. Some examples of lysogenic conversion (slide 4) o Diphtheria – high mortality, can be found in throats however is avirulent unless there’s a bacteriophage that infects it o Vibrio cholerae – diarrheal disease – can lose 20L of fluid in 24 hours, death by dehydration o Bacteriophage called vibriophage that has to infect vibrio cholerae to bring cholera toxin and make it a virulent disease Why are bacteriophages important organisms? (slide 5) o All of molecular biological processes were studied in bacteriophage o Bacteriophages are very very small o 65 nm in size How do we culture & study bacteriophages? (slide 7) o Bacteriophages are parasites – they grow on bacteria, replicate in bacteria, kills bacteria o How do we grow them in a lab? Plaque assay Serial dilutions of phage lysate (slide 9) o Bacteria death plaque Icosohedral (slide 14) o In a lot of eukaryotic type viruses Example: T phages (T4, T35, T55, etc.) (slide 19) o Bacteriophage attaches to bacterium o Injects its DNA inside bacterium o Undergoes a process of replication & make new progeny & released o Bacteriophage lyses the cell T4 phage (slide 20) o Adsorption: bacteriophage binding to receptors on the cell T4 has a receptor type of lipopolysaccharide (major component of gram negative bacteria on a membrane) o Penetration: penetrating the membranes & injecting its DNA from its head through its tail & into the cytoplasm of the cell Phage morphogenesis occurs in a stepwise progression (slide 22) o Lifestyle of T4 – very regulated lifestlye Adsorption Penetrates & injects DNA inside cell First 5 minutes: transcription occurs, there’s going to be some early mRNA’s which translate for early proteins which do some early jobs First thing it does is destroy host cell chromosome – reason being is to make a lot of progeny, need a lot of nucleotides – great place to find that is in host cell chromosome also makes other proteins – start shutting down host protein synthesis (stop making proteins for host, start making proteins for phage) 10 minutes: start replicating DNA End result: long linear pieces of DNA formed 15 minutes: At this point, start transcribing late mRNAs (structural proteins) Start producing proteins for the head & tail Ultimately, the DNA gets fed into the heads Heads & tails will then bind to each other forming the phage particle Late proteins lyses the cell & releases the progeny o Latent Period: time from beginning of adsorption to the phase where you have new progeny culture supinate o Eclipse Period: adsorption – where you have intact phage particles but still contained within the bacterial cell o Difference between the two: lyses Morphogenesis of bacteriophage particles (slide 25) o Larger pins evolved in order to increase radius of surface o High affinity small pins Temperate bacteriophage (slide 27) o Temperate Lifestyle Adsorption Penetration It CAN go through the lytic lifestyle o Actually Adsorption Penetration It can be come quiescent inside the cell – dormant – by integrating into the bacterial chromosome The phage replicates by letting the cell replicate for it o If bacterial cell dies, the phage dies o Phage in temperate lifestyle can switch & come out of the lysogenic lifestyle back into the lytic lifestyle – phage particles end up being very stable during adverse situations since they’re not metabolizing Temperate lifestyle (slide 31/32/34) o Lambda doesn’t bind to LPS, it actually binds to maltose binding protein o Lambda affects E. Coli – E. Coli can use maltose as a source of sugar, as a source of carbon – if maltose is around it’s going to express the receptor o Lambda binds to maltose receptor, penetrates & does something different then what the T4 phage does o Injects DNA as linear DNA, but the first thing it does is circularizes the DNA o How does it circularize the DNA? (slide 32) Linear DNA is double stranded until you get to both ends of the DNA On both ends are a single stranded DNA overhang – about 12 base pairs of overhang, very GC rich Why GC rich? Exact opposites of the other nucleotides Nucleotides bond by hydrogen bonds (AT base pairs have 2 hydrogen bonds; GC base pairs have 3) GC pairs are more thermodynamically stable – maximizes the number of hydrogen bonds o Temperate lifestyle (slide 34) Lambda Adsorption Penetration Circularization Lytic or lysogenic lifestyle? Molecular decision to Lytic or Lysogenic phage depends upon: (slide 35) o Lytic or Lysogenic? Depends on four promoters & two proteins o All of the genes \are expressed from a message from the promoter Pl & Pre o The decision of which lifestyle to choose from depends totally upon which one of those two promoters become active first Regulation of lambda bacteriophage – lysogenic cycle (slide 36) o Lysogenic lifestyle – the promoter Pre becomes activated first, when it does it initiates transcription of this long mRNA On that mRNA is an encoded gene for the protein C1, an unusual protein – a DNA binding protein, it doesn’t bind just to any DNA – it binds to very specific sequences that are very close to the PL promoter & very close to the PR promoter When it binds to those promoters, it represses those promoters – excludes RNA polymerase from reaching them If RNA polymerase doesn’t reach them, you don’t get any of the gene expressions for lysis Regulation of lambda bacteriophage – lysogenic cycle (slide 37) o C1 has a DNA binding activity to PL & PR and shuts them down o Also has a binding activity for the PRM promoter – when it binds to PRM, it activates, makes it easier for RNA polymerase to get to that site o Turns PRM into a very powerful promoter that transcribes a shorter message but a higher amount More C1 expressed – flood the cell Absolutely going to shut down lytic lifestyle Regulation of lambda bacteriophage – lytic cycle (slide 38) o PR promoter recruited RNA polymerase first & when it does this long mRNA containing the lysis genes is being expressed but another gene that’s being expressed is the CRO protein o CRO protein, like C1 protein is a DNA binding protein – doesn’t have any activity at PRE/PL site has a site to bind at PRM Different from C1 which activates this promoter – CRO turns off PRM promoter – don’t get high level of expressions of C1 which means it doesn’t shut down PL/PRE : now get the lysis gene aka the lytic lifestyle Lytic cycle: lambda infection 2 step dna replication (slide 40) o Lambda Infection COS Sites: circularizes at COS sites – cohesive sites Then goes to first step of normal replication Forms two replication fork Bidirectional or theta replication End up with two circular DNA – each one of those can undergo bidirectional replication After a certain amount of bidirectional replications, undergo rolling circle replication Stops theta replication Forms a knick in one strand, sets up replication fork with one strand knicked Replication fork – instead of making a circle, end up making a long linear piece of DNA “roll of tape” Long linear concatemers Theta replication: rolling circle replication (slide 41) o On linear concatemeres – there are COS sites o Between each COS site is one bacterial genome o Empty phage heads have binding affinity for COS sites Once it binds, a protein called TER cleaves that COS site not in the middle but asymmetrically Where single stranded 12 base pair overhang comes from – TER cleavage Once it makes its cleavage, head starts reeling in DNA until it reaches the next COS site When it reaches the next COS site, TER cleaves again What are molecular steps if the choice is for the lysogenic cycle (slide 42) o Lambda goes through chromosomal integration o Depends on 3 different factors Process of lysogeny requires three factors: (slide 43) o attP: nucleotide site, on the phage particle o attB: exists on bacterial chromosome o Int: site specific recombination protein Continuous expression of cI maintains lysogenic state (slide 44) o Linear form genome by the half COS sites will circularize o Once circularized, you have attP site and attB site in bacterial chromosome which are very closely related in sequence – not perfect homology, there are some nucleotide changes o Int protein comes in and causes a recombination between attP and attB o Since they’re not absolutely similar, once you have recombination, you’ve now integrated that circle into the chromosome o But because attP and attB aren’t identical, conjunction sites are a hybrid – attBP Superinfection immunity (slide 46) o attB looking for attP site – there’s a hybrid attBP – INT has no substrate – can’t go lysogenic or lytic = DEATH Excision of lambda (slide 47) o Adverse conditions: reverse integration step, not using INT, by using Xis o Xis: bacterophage protein that recognizes attP & attB, opposite reaction Disintegration (slide 48) o Integrated phage has Xis – Xis recognizes attBP/attPB & disintegrates them back into the circle o Circle does lytic lifestyle and phage survives Lambda (slide 49) o Lambda – lytic or lysogenic o Lysogenic in the chromosome: signals to go lytic – when do you, as lambda, want to go from lysogenic to lytic? under adverse conditions o E.Coli – rec A: can bind to DNA & is a protease & degrades certain proteins – inducible, bacterial cell doesn’t produce rec A all the time, if DNA damaged, signals sent to that cell to make more rec A c1 protein of lambda has a proteolytic sensitive site that’s recognized by rec A – cells have DNA damage bc of adverse growth conditions cell makes rec A: tries to make the repair but starts cleaving and destroying c1 protein – as a result Xis is produced : no c1 protein – start making lytic genes o Lambda makes cell commit suicide Lecture 5 – Genetic Exchange and Variation *These are just additional notes that aren’t included on the slides that were discussed during lecture. Adverse environmental conditions (slide 2) o If one bacterium picks up a mutation, makes it refractory from being picked up by macrophage – it’s going to persist Selective advantage (slide 3) o Once it persists it divides & the new population of cloned bacteria is unable to be picked up by phages Staphylococcus aureus (slide 5) o Transposon: mobile genetic element Mobilizable genetic elements (slide 6) o Staph Aureus: scalded baby syndrome – baby looks like dipped in boiling water – boils Point mutations – 3 types (slide 12) o Missense mutation: substitution of another base pair – substitute T o Nonsense mutation: substitution or addition – single point changes that produce STOP codons o Frame shift mutation: deletion of one base pair – shifts codon Chromosomal mutations: antibiotic resistance (slide 13/14) o Streptomycin: stops protein synthesis by interacting with a ribosome o Streptomycin resistance If you have a ribosome, it binds to mRNA & does all it needs to do Streptomycin binds to ribosome and sterically hinders ribosome itself – kills the cell – stops protein synthesis Lysine encoded in ribosomal protein in position 42 and has an AAA codon Resistant forms: codon changed by single nucleotide change to keep streptomycin from binding to ribosome – now have resistant organism brought by single nucleotide change Neisseria gonorrhoeae & N. meningitides (slide 16) o N. meningitidis – bacterial meningitis – can kill you in 24 hours o Neisseria gonorrhea – sexual transmitted disease o Both are subject to phase and antigenic variation o They make opa proteins that can switch the protein off or switch to a different type of OPA protein – 11 opa genes Neisseria gonorrhoeae (slide 17) o If you take those 811 opa genes in the chromosome of NG or NM, you see regions of high homology – regions of fairly similar nucleotide sequences – encoded with very similar AA, which are normally buried in the membrane of the bacteria (inaccessible to antibodies) o There are regions that stick outside the cell known as hypervariable regions – which are targeted by the immune system when infected o Signal peptide coding regions: signals in mRNA that send these HV regions to the membrane Nucleic acid sequence of an opa gene (slide 18) o Starts with ATG codon o Has signal peptide encoding region o There’s repeats of pentanucleotide CTCTT in Sig. pep. En. Region o Slip strand mispairing occurs here Frameshift mutations regulate Opa expression (slide 19) o DNA polymerase goes to that region – two strands separate o Hydrogen bonds – can snap back together o If DNA polymerase loops out a DNA strand or reads it twice, it creates or loses CTCTT repeats o If you lose 5 nucleotide segment – frameshift mutation occurs o If you lose 3, no problem, just add or subtract an AA o If you add 5, you completely shift the sequence o Good way for Neisseria to turn opa genes on and off Phase variation: all the opa genes are out of frame Antigenic variation of flagellar protein – salmonella typhimurium (slide 21) o Salmonella typhimurium Similar to Salmonella typhi – typhoid fever – lives in the gut where there’s mucous lining Cells are buried under mucous in gut, salmonella has to find a way to get to those cells The only way is to swim to them using flagella – which are made of proteins – however body loves to make antibodies to proteins Can change flagellum First segment: h2 flagellin gene makes h2 flaggelin, h1 repressor gene makes h1 repressor which has binding affinity to OP which houses another promoter & when it binds it turns it off On the first segment that contains the Ph2ON promoter, it’s an invertible region – able to flip, polar Promoter pointing away, OP promoter turns on and gets a different flagellin The invertible Hin region (slide 22) o How does this flip occur? Enzyme called HIN, the invertase, exists on particular DNA that has inverted repeats of nucleotide sequences on either side HIN can recognize those repeats, can catalyze the cleaving of that sequence – holds onto it and flips it around HIN included on fragment – inversion can always occur Streptococcus pneumonia (slide 27) o Griffith’s experiment: SP can infect a lot of different animals including humans Wanted to understand how SP caused disease – used a mouse model On plate, there were two types of colonies – smooth and shiny, flat rough colonies If touched smooth colony and streaked it, he would get thousands of smooth colonies and once in awhile rough colonies & vice versa Colony morphology Seemed to be alleles of the same gene Conducted an experiment Mouse Model Experiment (slide 28) o Used a mouse model o Grew shiny smooth bacteria, injected it into the mouse & the mouse died o Took rough dull bacteria, injected it into the mouse, the mouse lived Small shiny virulent vs. flat dull avirulent o Took shiny smooth virulent bacteria, heat killed it, injected it into the mouse – the mouse lived o *** heat killed virulent form, mixed it with avirulent living form, injected it into the mouse – the mouse died – expected the mouse to live Took this sample and streaked it and found that almost all of it was the small shiny smooth virulent type o Griffith concluded: somehow, dead virulent form was able to transform the avirulent form into a disease causing form o Another experiment: Avery, Macleod, and McCarty 20 years later: Still wondered what genetic materials were – genes were still an idea – most thought it was protein Took bacteria and fractionated them into parts Avery could take the avirulent form & extract all the proteins and injected it into the mouse He did this and to his surprise the mouse lived Took lipids & RNA – injected – mouse lived Took DNA – injected into mouse – mouse died ****** Did it with protease – the DNA still transformed Mechanism of Transformation (slide 29) o Transformation: uptake of DNA in the external area o Bacteria can also take up single stranded DNA as well Streptococcus sp. (slide 31) o Streptococcus produces an enzyme which allows cells to autolyse Neissria gonorrhoeae, Neisseria meningitis (slide 32) o Make adhesins & pili – long rigid rods made with proteins – the ends make it able to attach to different epithelial cells o Pili are even more varied then OPA proteins – hundreds of different variants Pilus switching in Neisseria gonorrhoeae (slide 34) o Look at gene for pilus o Regions that are hyper variable o Regions that produce AA o In between, are OPA genes – region of high homology Chromosome of Neisseria spp. (slide 35) o How do we control antigenic phase variation? o there’s a single expression locus on the chromosome, but there are many silent loci o Silent loci: they have all the information to encode a pilus but there’s no promoter o “vanks” Neisseria gonorrhoeae – antigenic variation (slide 36) o Wherever there are regions that have homology, cells have recombination functions o If you have a expression gene & silent locus – you can have homologous recombination & now have a mixture of different regions – part from original gene and part from silent locus o Able to outrun immune system – antigenic variation Neisseria gonorrhoeae – phase variation (slide 37) o What about phase variation since there’s some NG that don’t produce pilli at all o There will be homologous recombination – but will be aberrant recombination and will cause a shift o Frame shift causes jumbled information downstream – now not expressing pilli at all o NG uses cellular factor (homologus recombination) and cellular mistakes (frameshift) to be able to change productively Slide 40 o Every once in awhile rarely, instead of packing its own genome, the bacteriophage empty phage head can package a piece of chromosomal DNA o when it does this, you have an infectious particle because all the necessary functions for binding to cell to adsorption & penetration are all proteins that are located in the head and the tail o If it injects that piece of chromosomal DNA into the cell (not phage DNA) & this piece of DNA has homology to the bacterial chromosome, it can be recombined in the chromosome o If that chromosomal DNA expresses a gene that’s useful to bacterium, that becomes a selective advantage Plasmids (slide 42/43) o Borrelia – linear plasmids – lime disease o Cryptic – haven’t discovered function yet Conjugation (slide 45) o Cell to cell contact by the sex pilus – has adhesin on the end that can recognize cells that don’t have the conjugal plasmid in it o Donor & recipient o Why the long rigid rod? o Search a wider area for recipient cells Slide 46 o Once attached, pilus starts being degraded at its base o As the pilus gets shorter – the membranes touch & meld into each other o Creates single bacterial cell with two nuclei o The conjugal plasmid forms a knick in one strand , starts replicating the strand, replicates a single strand of DNA on one side, the host cell itself will fill in the second strand o Ends up with a replicate copy of the conjugation plasmid in the opposite cell Transposons (slide 48) o Not JUMPING GENES o Non homologous type of transfer o Tnp catalyzes non homologous regions o Tnp can cause mutations Insertional Mutagenesis by a transposon (slide 49) o If transposon mobilizes into the interior of that gene, you’ve now disrupted that gene sequence (frame shifts) o Ends up prematurely terminating mRNA o Cause drastic mutations 3 types of transposons (slide 50) o Insertion sequence o inverted repeats o only enough DNA to encode the tnp o Composite transposon o larger encoding regions o one carries kanR gene o Complex Transposon o most evolved o lost duplication sequence o only has repeated sequences that it needs for transposition Epidemiology of penicillinaseproducing N.G. (slide 54) o Vietnam: 400,000 young soldiers 1825 years old o Brothel & sex workers o Penicillin was used to treat gonococcus – very effective o Penicillin gonococcus resistance arouse o 34 years, penicillin resistance arouse around the world
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