Lecture 6: Bacterial virulence

  • Created by: chandanee
  • Created on: 04-01-19 11:29
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  • Bacterial Virulence Determinants
    • Post transcriptional reg of GE
      • RsmA (csrA)
        • RNA binding proteins, homologs in bacteria, bind to RBS/Shine-Dalgarno of mrna and inhibit translation or facilitates mRNA decay, reg effect on glycogen biosynthesis, catabolism, biofilm, QS
        • How its discovered? P. syringae - add drop to plant = necrosis. Found GacS mutant which didnt kill plant. GacS/A = sensor and DNA binding protein. S senses signal, conf change, phosphotransfer, goes to GacA, conf change = triggers VF. So they made fusions - placed into WT and Gac mutants = not by Gac. Hydrogen cyanide gene - transla reporter fusion - controlled by gac. Transcription fusion of +1 = not controlled by gac - so they replaced promoter and studies RBS = controlled by Gac
        • Then discovered RsmA = acting as RBS. RsmA binds to RBS and prevents protein made > GacA activates , transcibes ncRNA >folds into loops > takes away protein so reg rna has more protein
        • How discovered? Bioinformatics= regions, characteristic Experimentally - sRNAs, copurify RsmA look for GGA = signature of RBS
      • Hfq
        • Discovered as E.coli host factor needed for replication of bacteriophage QB
        • Abundant RNA binding protein, regulates RpoS and RpoE. Regulates sRNA in Vibrio cholerae e.g. micx sRNA, 6 subunits that binds dsRNA.
        • +REF = 1. mRNA forms stem loop with itself, masks RBS, dsRNA binds and stabilises. When it needs protein, expresses sRNA, RBS freed = protein made. 2) mRNA subject to RNase degradation, bacteria makes ortY >transcribes sRNA, Hfq stabilises so RNase cannot degrade
        • -REG = Orf2, asRNA binds to RBS, stabilises by Hfq so orfZ not made. Rnase chops off sRNA = irreversible Example = Hfq protection binds and prevents RNase E cleavage. RpoS = when mRNA made, Hfq binds to RBS = no translation. When you need Rpos, dsrA made, comp to rpos, stabilised by hfq so RBS free = translation
    • PART 1: Bacterial Operon
      • Functional unit of clustered genes which transcription is controlled by a single promoter
      • E.g. lac operon 1941: Monod came up with the diauxic growth theory for glu and lac as part of gene reg. Revealed B-Gal production. 1961: Monod and Jacob - lac operon - lac present, no glu.
      • No lactose, lacl binds to operator = stops transcription. Allolactose (lots of lactose) binds to lacl, makes it let go of the operator so RNA pol can transcribe operon. Operon turns on = when no glu - cAMP binds to CAP, binds to DNA, then RNA Pol can attach to promoter. Yes lac - lacl released so RNA pol can transcribe,
      • 1. Lysogeny = only strains lysogenic for prophage produce DT, structural gene for tox on phage genomes and expressed under certain conds.           2. Regulation of toxin formation = Tox gene, Fe3+, toxin made after Fe exhausted, Fe regulates transcription by interacting with reg. prot. 3. Mode of action = pure DT = 0.1 ug/kg lethal, DT kills cells by blocking protein synthesis, DT converted to enzyme that inactivates EF-2 (translocation)
      • Toxin Structure: AB toxin A = enzymatic and B = binding and entry. They inhibit PS, heavility regulated by DtxR (represses exp of B-gal - iron dependent) and restore reg of dip toxin and siderophore GE
      • Recog and Commun. needed for - sexual exchange, niche colonisation, combat host defences and population migration
      • Example: Aliivibrio fischeri and Hawiian bobtail squid - understand how QS works. Pathway regulates genes that cause luminescence, high density of cells in light organs of squid.
      • Lux operon = invCDABEG and luxR. LuxI = synthesises autoinducer, HSL. LuxR = codes for transcription factors which respond to HSL. High dens = so HSL builds up and binds to luxR = bioluminescence. Luciferase = uses O2 to oxidise products - one emits blue photon. Squid cannot control reaction but can control if they show light.
    • PART 2: Gene Reg in Bacteria
      • 3 ELEMENTS: 1) Promoters = elements of DNA that bind RNA pol and other prot to initiate transcription. 2) Operator = recognise repressor proteins that bind and inhibit transcription 3) + Control elements - bind to DNA and incite higher levels of transcription
        • Untitled
      • Sigma factors = specialised bacterial proteins that bind to RNA pol and start transcription initiation. Act as mediators of sequence-specific transcription, can be used for HK genes/genes in response to stimuli e.g. stress/starvation
      • 1) INITIATION: regulated by ribosomes, comp to 3' end, stretch of purine residues are located upstream of initiation codon (AUG, GUG, UUG), polymorphism has + and - effects of efficiency of base pairing and prot. exp. Reg by initiation factors - IF1 binds to 30s subunit - conf change = binding of IF2 and 3 (proofreads to prevent random codons starting)
      • Elongation = irreversible > translation efficiency diminised by tRNA pools, required for elongation of polypeptides.
      • Termination = requires coordination between release factor proteins, mRNA sequence and ribosomes. Once termination codon is read, releases RF 1, 2 and 3 - terminates chain. Bases downstream the stop codon affect RF activity.
      • Transcriptional reporter fusions = report activity of the promoter. E.g. Gene X - amplified promoter and fused to lacZ gene = B Gal Pros: best one, reports 100%, no mrna degradation complications Cons = forms del mutant, diff to construct, multiple promoters present
      • Reporter fusions (Quick n Dirty) = cut gene, add premature stop codon then restart translation Pro = easy to make, dont need to find +1/ mul pro, common Cons= forms del mutant, stability
      • Examples: cryIIIA-lacZ = B. thurin endotoxin - made a fusion so they cut gene, studied promoter to see how much B-Gal produced. Found 135bp from RBS 30 nuc important for B-Gal production so they concluded promoter was there. BUT - promoter was far upstream and has extra RBS (stabiliser RBS)
      • 2) Example mc'lacZ = cut gene, fusion with lacZ = 3 x increased B-Gal, could this be an operator? NO. RNAaseIII sites, chops mRNA = stops translation - stabilises mRNA
      • Non-disruptive fusion - dont disrupt gene X, remove terminator and add reporter. Pro = easy to make, no worries with +1/promoters, no mutant. Cons = high risk - stability affected
      • Translational fusions = Interrupt gene X, put frame so codon > lacZ can fuse to anything. Pro = no worries with +1/multiple promoters, reporter production affects transcription/translation Cons =difficult to make bc fusion is neat, forms a del mutant, subject to artifacts
      • E.g. arcDABC (Gamper p. aeru) - Make Abs and studied levels, found ArcA increased more than ArcD (Westerns) and ArcA mRNA increased more than ArcD (Northern) Why? As there was one promoter it didnt make sense. Made translational fusions for ArcA and D - found ArcD increased = oppsosite why? There was a RNase III site at ArcD that cut mRNA = WT ArcA than ArcD
      • When we want to only study RBS in translational fusions? Make fusion, make lac prom constitutiv, measure T efficientcy by WT, mutant, temps PRO= best reporter, 100%, no del mutant CON= most difficult reporter to make, subject to artefacts

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