plasmids and bacteriophages

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Plasmids and Bacteriophages. Plasmids : autonomously replicating extrachromosomal DNA molecules present mostly in the bacterial cells. Bacteriophages (phages) : bacterial viruses. Plasmids. General properties of plasmids: dsDNA, mostly circular Size: 3 kb-150 kb - PowerPoint PPT Presentation

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  • Plasmids and BacteriophagesPlasmids: autonomously replicating extrachromosomal DNA molecules present mostly in the bacterial cells.Bacteriophages (phages): bacterial viruses.

  • Plasmids General properties of plasmids:dsDNA, mostly circularSize: 3 kb-150 kbCopy number: low, intermediate, or highHost range: narrow v.s. broadStabilityIncompatibilityTransfer: self-transmissible;mobilizable, nonconjugative;nonconjugative, nonmobilizable

  • Features of selected plasmids of E. coliPlasmidSize (kb)

    Copy number

    ConjugativeOther phenotype

    ColE1

    6.6

    1020

    No

    Colicin production and immunity

    F

    95

    12

    Yes

    E. coli sex factorR100

    89

    12

    Yes

    Antibiotic-resistance genes

    P1

    90

    12

    No

    Plasmid form is prophage; produces viral particles

    R6K

    40

    1020

    Yes

    Antibiotic-resistance genes

  • Plasmid replication:Mode of replicationCairns intermediate ( or butterfly form)Rolling circleRequirements for host enzymes

  • Control of copy numberNegative controlInhibitor-target mechanismIteron-binding mechanism

  • Replication of ColE1

  • Evolution of pUC18/19

    ColE1

    pBR322

    pUC18/19

  • AmplificationFor ColE1-related plasmids, like pBR322By chloramphenicol treatmentCopy number increases up to 1,000XMechanismsReplication of host chromosomal DNA decreases, while the machinery for plasmid replication is more stable.Rop concentration decreases, and that way blocks the negative control of plasmid copy number.

  • Plasmid R1 control circuit for replication

  • Iteron-binding mechanism

  • Stability of plasmidsPartitioningLethal segregationResolution of plasmid multimers(e.g., Xer and cer of ColE1)

  • Centromere-like functionRegions involved in partitioning of plasmidsThe binding of ParA and ParB to parS serves as a unit for interaction with the host components involved in segregation.

  • parB-mediated post-segregational killing of plasmid free cells of R1 plasmidh: hok (host killing) mRNA; s: sok (suppression of killing) mRNA.Targets of poisons: cell membrane, gyrase, DnaB, unknownAntitoxins:mRNA (type I)Protein (type II)

  • IncompatibilityIncompatibility groups: IncA-IncZ

  • Factors determining host rangeStable mating pair formation and mobilizationRestriction enzymes in the recipientReplication defect

  • F plasmid

  • Cell contact via the sex pilusDNA mobilization and transferDNA replication while transferringSurface exclusion:The F+ strains do not usually serve as the recipient

  • F plasmidF plasmidHfr strainIntegration of F plasmid into host chromosomeAberrant excision of F plasmid from host chromosomeIntegration of F plasmidExcision of F plasmid from host chromosome

  • E. coli Hfr strains

  • Transfer of chromosomal DNA from Hfr into a F- recipient

  • Time of entryHfr x F-Hfr: str-s; a+, b+, c+, d+, e+F- : str-r; a-, b-, c-, d-, e-Transconjugants

  • Construction of the genetic map of E. coli by conjugation

  • PhagesBacterial viruses; replicate only within a metabolizing bacterial cell.Structures Coat Nucleic acid (ds- or ss-DNA or RNA; linear or circular)

  • Lytic (virulent) phages

    Lysogenic (temperate) phagesLife cycle of phages

  • Multiplicity of infection (moi)Poissons law P(n)=mne-m/n!m: moi; n: no. absorbed phagePlaqueInfective centerBurst sizeInfective centers (fraction of yield)Min. after infectionOne step growth curve of phageFactors contributing to host specificity:ReceptorsAbility of bacterial RNA pol to recognize phage promotersHost restriction and modificationProperties of a phage-infected bacterial culture

  • Properties of several phage types

    Phage

    Host

    DNA; RNA

    Form

    Lysoginize

    Mode of release

    fX174

    E

    DNA

    ss, circ

    -

    Lysis

    M13, fd, f1

    E

    DNA

    ss, circ

    -

    Extrusion

    Mu

    E

    DNA

    ds, lin

    +

    Lysis

    T7

    E

    DNA

    ds, lin

    +

    Lysis

    l

    E

    DNA

    ds, lin

    +

    Lysis

    P1

    E

    DNA

    ds, lin

    +

    Lysis

    SPO1

    B

    DNA

    ds, lin

    +

    Lysis

    T2, T4, T6

    E

    DNA

    ds, lin

    +

    Lysis

    MS2, Qb, f2

    E

    RNA

    ss, lin

    -

    Lysis

    CTXf

    VC

    DNA

    ss, cir

    +

    Extrusion

  • Lytic cycle

  • Temporal control of SPO1 transcriptionRegulatory cascade in lytic cycle

  • Control of phage l life cycleLytic cycleEarly gene expressionExpression of late genesExpression of repressorLysogenic cycle

  • Genetic map of phage l

  • Temporal control of transcription during lytic infection by phage l

  • Gene expression regulation by antitermination(nutL)

  • N protein is an RNA-binding protein (via an Arg-rich domain), recognizing a stem loop formed at the nut sites. Host proteins are involved in antitermination.N causes antitermination at both r-dependent and r-independent terminators by restricting the pause time at the terminator.Antitermination by N protein

  • Q binds to the qut site, which overlaps PR, alters the RNA pol in a way that it resumes transcription and ignores the terminator, continuing on into the late genes.Late gene expressionAntitermination by Q protein

  • DNA replication and maturationModes of replication: q and rolling circleCutting and packaging of DNA (38-51 kb) terminase (Ter system)two cos sites

  • Lysogenic cycleGeneral properties Turbid plagues

    Immunity InductionMechanism of immunity CI repressor; OR and OL Homoimmune and heteroimmune lcI -; lvir

  • Site-specific recombinationgalbioProphage integrationatt sites (attP and attB)Integrase (Int)IHF (host factor)

    Prophage exision att sites (attL and attR)IntegraseExisionase (Xis)IHF

  • Synthesis of cI: promoters PRE and PRMCII acts on PRE, PI and Panti-QCIII protects CII from degradation by HflAcyL and cyR mutations prevent establishment of lysogeny in cisPRE

  • OL and OR contains three repressor-binding sitesAT-rich spacers allow DNA twist more readily which enhances the affinity of the operator for repressorBlocks access of RNA pol to the promoter

  • The lytic cascade requires Cro (the repressor for lytic infection)

  • Fate of a l infection: lysis or lysogenyCI:OR1 > OR2 > OR3 OL1 > OL2 > OL3Cro: OR3 > OR2 = OR1, OL3 > OL2 > OL1The key to the fate is CIICII is degraded by host protease, and stabilized by CIII

  • Induction of l prophage

  • TransductionSpecialized transduction (e.g. l transduction)Generalized transduction (e.g. P1 transduction)

  • Methods to determine plasmid copy numberQuantification of gene productsActivity of enzymesFluorescence (GFP)Rate of segregationQuantification of nucleic acidsWithout separation/isolation of plasmid DNADot blotSequence-specific assay/ILAPCRWith separation/isolation of plasmid DNAHPLCDensity gradient centrifugationAGE and densitometryAGE and image analyzerAGE and Southern blotPCR and TGGECGEFIA/FIPFriehs K. 2004. Adv Biochem Engin/Biotechnol. 86: 47-82

  • Methods to avoid segregational plasmid instabilityFriehs K. 2004. Adv Biochem Engin/Biotechnol. 86: 47-82Adding an antibiotic to the mediumComplementation of chromosomal mutationsPost segregational killing of plasmid free cellsInfluences of plasmid size and formActive plasmid partitioningHigh copy number and plasmid distributionLowering the difference in specific growth rates by the internal factorsInfluences of cultivation conditions (pH, O2, phosphate, etc.)Integration into the chromosome

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