Coli cell injects its dna through their tails as seen

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 phagespecific mRNA 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 phage

Bacteriophage 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 T4

Replicates DNA

Synthesizes viral proteins

Assembles particles

Host lysis
~
Lysogenic pathway:

DNA inserts into host chromosome & replicates with host DNA

Prophage – integrated state of l chromosome


Lysogenics state = phage proteins not expressed

Occasionally l prophage excises from host chromosome & enters lytic pathway

Bacteriophage l lifecycle:
~
Lysogenic pathway:

Integration into host chromosome

Replication as part of host chromosome

Capable of reactivation lytic pathway
~
Lytic pathway:

Replication

Assembly

Cell Lysis

l DNA integration occurs by site specific recombination:
~
Attachment sites:

AttP on l chromosome

attB on bacterial chromosome

mediated by l integrase (l int gene)

recombination occurs through 15 bp sequence common to both attP &
attB
-
GCTTTTTTACTA
-
CGAAAAATGAT

Excision 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 genes

Present in single or multiple copies (10 – 20)

Single or multiple different plasmids/cell

Replicate independently of bacterial chromosomes

Use bacterial enzymes for replication

Can transfer between bacteria

Express 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 metabolite

Example: wildtype Leu
+
(prototroph) & mutant Leu (auxotroph) for leucine
synthesis
~
Mutants resistant to drugs & antibiotics
