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Microbial Genetics Review for final • Vertical gene transfer DNA - Transmitted to progeny during cell division – Binary fission – After replication • Horizontal gene transfer - Parasexual processes • Conjugation • Transformation, • Transduction Lateral Gene Transfer • Unidirectional transmission of DNA between bacterial cells – Narrow or broad range of recipients • Extra-chromosomal elements – plasmids • Chromosomal DNA – mobilized by F factors, phage, etc. – transposons: hitch-hike with other mobile DNAs Lateral Gene Transfer • Transformation – uptake of free DNA • natural or induced • Conjugation – cell to cell DNA transfer • F+, Hfr, F’ • Transduction – DNA delivered by bacteriophage • generalized, specialized Lateral Gene Transfer • Natural transformation – DNA adsorbs to bacterial cell surface • receptor-mediated – One strand taken up • remaining strand degraded • complementary strand of ssDNA synthesized – Replication • linear DNAs: recombination into chromosome • plasmids: independent origin of replication Lateral Gene Transfer • Conjugation – Self-transmissible or mobilizable plasmids • oriT, tra genes (Dtr, DNA transfer and replication; Mpf, mating pair formation) • ssDNA strand transferred (relaxase) – Cf Rolling Circle replication • complementary strands synthesized in donor & transconjugant – Cell-cell attachment and contact (pilus) • Cytoplasmic continuity via channel or pore Lateral Gene Transfer • Conjugation – F+ Self-transmissible plasmid • oriT, tra genes Lateral Gene Transfer • Conjugation – Hfr Lateral Gene Transfer • Conjugation – Hfr • After transfer, Hfr fragment recombines into recipient chromosome. Lateral Gene Transfer • Conjugation – F’ F DNA incorrectly excised from Hfr chromosome, taking host DNA with it. Transfer results in merodiploid Lateral Gene Transfer • Generalized transduction – Bacterial chromosomal DNA packaged into phage heads – After lysis, phage particles inject this DNA into new host – Homologous recombination: donor DNA incorporated into recipient genome • DNA replacement Lateral Gene Transfer • Specialized transduction – When lysogen is induced to excise from bacterial chromosome, taking some host DNA (that flanks integration site) with it – Specialized phage can integrate donor host genes into recipient cell chromosomes • int-mediated recombination – merodiploid • homologous recombination – DNA exchange Evolutionary consequences • Extensive lateral gene transfer has occurred among bacteria – Transmission of antibiotic resistance, virulence & pathogenicity factors – Transfer of new genes or gene homologues • Genomic stability: housekeeping functions – Transfer of traits in one step or multiple steps – Novel phenotypes adaptation • Second-order selection • Population advantage vs. deleterious effects to most recipients Plasmids • Autonomous replicons with limited, nonessential genetic functions – Substrate utilization – Antibiotic resistance – Pathogenicity • Wide range of size and copy number Plasmids • Circular plasmids – theta replication – rolling circle replication • Rep protein nicks strand to provide primer to DNA polymerase at DSO • extension of DNA from 3’ end displaces 5’ end • displaced linear ssDNA is recircularized by Rep protein • ssDNA replicated from SSO • Linear • ssDNA • ori region Plasmids – Regulate plasmid copy number • Relaxed vs. Stringent • RNA:RNA interactions of transcripts from ori region – ColE1 plasmids, counter-transcribed RNAs that modulate availability of primer RNA – R1 plasmids, anti-sense RNA modulates Rep protein expression – Iteron plasmids: repA promoter and iterated R sequences in ori region • ori region Plasmids – Determine plasmid host range • Narrow vs. Broad – Influence plasmid incompatibility • Two plasmids of same Inc group can’t coexist in same cell – due to common replication and partition systems, and randomization of plasmids to daughter cells at cell division Plasmids • Plasmid maintenance (stringent plasmids) – plasmid addiction • Multiple systems – multimer resolution • Site specific recombination – partitioning • par site on plasmid bound by Par proteins • plasmids moved towards cell poles • cytokinesis divides cell between poles, with one plasmid in each daughter cell Bacteriophage • Lytic phage – Infect bacteria • • • • • Multiplicity of Infection take over cellular functions ensure replication of phage DNA production of phage proteins release to environment by lysis of cell – plaques, burst size – Early, Middle, & Late genes Bacteriophage • Lytic phage – Diverse genomes • small – large • circular, linear, ssDNA • Diverse modes of replication – Host defense: restriction-modification • Phage T4 & genetics of rII locus – The gene: minimum genetic unit • Divisible by recombination – Complementation and deletion mapping Bacteriophage • Temperate phage: phage lambda – Lysis/lysogeny • bacteria: lysogen; phage DNA: prophage • induction: prophage excises and carries out lytic cycle – Genetic control • Lytic: anti-termination and early-middle-late genes • Lysogeny: repression of phage genes by cI protein • Lysis/lysogeny – Competition between regulators cII and Cro for operator sites » Host growth conditions Recombination • Homologous – Breakage and reunion between DNAs with tracts of sequence (near) identity – Precise exchange of genetic material: alleles – Strand breakage • Invasion and branch migration • Heteroduplex formation • Holliday junction – Resolution to parental or recombinant products Recombination • Homologous – RecA protein • pairing of homologous DNAs – RecBCD pathway • initiates recombination – RecF pathway • similar to RecBCD – Ruv proteins • Recombination machines: branch migration – Resolution of Holliday structure Recombination • Site-specific – Recombinase enzymes • recognize short, specific DNA sequences – Bacteriophage lambda integration/excision • integrase, excisionase – Bacterial development • Bacillus sporulation • Cyanobacteria nitrogen fixation • Salmonella flagellar variation Transposition • Insertion sequences (IS) – Transposase – Flanking inverted repeats – Target site direct repeats • Transposons – IS elements flanking central DNA • encoding antibiotic resistance, etc. • Mechanisms – Conservative – Replicative – DNA cleavage, ligation, repair • single or double stranded Transposition • Regulation – Transposition events occur rarely • Movement of transposons from cell to cell – Conjugation: Hfr or F’ – Transduction – Transformation • Mutagenesis – Gene inactivation – Recovery of transposon and targeted locus Transcriptional regulation • Initiation and termination • Repression and Activation – DNA binding proteins • LacI repressor and lacO operator – RNA Polymerase Sigma factors • Promoter specificity Transcriptional regulation • Repression and Activation – DNA binding proteins – Negative regulation • LacI repressor and lacO operator • Catabolite repression • Trp repressor and operator – Anti-termination – Positive regulation • AraC activator and araI • LuxR and luxI Operons • Functionally integrated genetic units – Structural genes • Polycistronic mRNAs – Regulatory elements – Coordinated control of activities required for a cellular activity Global Regulation • Regulons – Adaptive response to environmental conditions • • • • Catabolite repression: CAP-cAMP SOS DNA repair: LexA Adaptive response (DNA repair): Ada TrpR regulon: trp and aroH operons, trpR Global Regulation • Mechanisms – – – – Transcriptional regulators Sigma factors Regulatory RNAs Two-component systems Global Regulation • Nitrogen assimilation and Ntr regulon – Glutamine synthetase expression • Sigma-54 • NtrB sensor histidine kinase • NtrC response regulator: transcriptional activator • Heat shock – Expression of chaperones and proteases • Sigma-32 – Auto-regulation • Ribosomal RNAs and proteins – Auto-regulation – Stringent control • Amino acid starvation leads to decrease in tRNA & rRNA expression Comparative Genomics • Microbial phylogeny & evolution • Lateral gene transfer – virulence (pathogenicity islands), antibiotic resistance – Gene functions • unknown; species-specific • functional and regulatory networks – transcriptome, proteome – Diversity of niches and cellular functions • Genome size, GC content • Gene duplication, rearrangement, & loss