Transcript Microbial Genetics - University of Montana

Microbial Genetics
Review for final
• Vertical gene transfer
DNA
- Transmitted to progeny during cell division
– Binary fission
– After replication
• Horizontal gene transfer
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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
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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
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Catabolite repression: CAP-cAMP
SOS DNA repair: LexA
Adaptive response (DNA repair): Ada
TrpR regulon: trp and aroH operons, trpR
Global Regulation
• Mechanisms
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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