86%(7)6 out of 7 people found this document helpful
This preview shows page 59 - 62 out of 75 pages.
T = 0 , cell attachment – T4 bacteriophage attaches to E. coli cell & injects its DNA (through their tails, as seen in electron micrograph)~ T = 2, mRNA synthesis – synthesis of phagespecificmRNA begins~ T = 6, DNA replication – replication of phage DNA begins, host DNA has been degraded by phageencoded nucleases~ T = 14, assembly begins – DNAfilled heads, tails lacking tails fibers, & assembled tail fibers first appear~ T = 17, assembled phage – first intact phage particles assembled~ T = 25, host cell Lysis progeny released – host bacterium lysed, releasing about 300 progeny phageBacteriophage T4 genetics:~ Phage particle can be quantitated – phage count proportional to plaque count~ Gene mapping can be performed by coinfection with 2 different mutants & screening for recombinants~ Temperature sensitive mutants (grow at 25C but not 42C) Bacteriophage Lambda (l ): ~ Coliphage~ dsDNA genome~ Consists of 48 502 bp (~ 50 genes)~ Linear DNA in head circularizes after host entry in tail (immediately)~ Replicates by lytic or lysogenic pathway ~ Lytic pathway:Similar to T4Replicates DNASynthesizes viral proteinsAssembles particlesHost lysis ~ Lysogenic pathway:DNA inserts into host chromosome & replicates with host DNAProphage – integrated state of l chromosome
Lysogenics state = phage proteins not expressedOccasionally l prophage excises from host chromosome & enters lytic pathwayBacteriophage l lifecycle:~ Lysogenic pathway:Integration into host chromosomeReplication as part of host chromosomeCapable of reactivation lytic pathway ~ Lytic pathway:ReplicationAssemblyCell Lysisl DNA integration occurs by site specific recombination:~ Attachment sites:AttP on l chromosomeattB on bacterial chromosomemediated by l integrase (l int gene)recombination occurs through 15 bp sequence common to both attP & attB-GCTTTTTTACTA-CGAAAAATGATExcision also occurs through sitespecific recombination between sequences & requires action of l integrase & l excicase (l xis gene) - Note: excision occurs 1 in every 1000 replications Genetics of Bacteria:~ Escherichia coli (strain K12)~ Model organism for studying bacterial genetics~ Single circular chromosome (4.64 million nucleotides, 4 377 genes) ~ Also harbor separate, small circular DNA molecules called plasmids ~ Plasmids:2 to 10 000 nucleotides, only few genesPresent in single or multiple copies(10 – 20)Single or multiple different plasmids/cellReplicate independently of bacterial chromosomesUse bacterial enzymes for replicationCan transfer between bacteriaExpress genes required for plasmid transfer (F plasmids)Often contain genes providing resistance to cellular stresses (R plasmids) Isolation of bacterial mutants:~ 3 methods~ Mutants unable to utilize specific energy sources
Example: wildtype Lac+& mutant Lac (unable to utilize lactose as sole energy source)~ Mutants unable to synthesize essential metaboliteExample: wildtype Leu+(prototroph) & mutant Leu (auxotroph) for leucine synthesis~ Mutants resistant to drugs & antibiotics