Download E. coli(λ) - UCSF Biochemistry

1. λ lysogens are immune to superinfection by λ, but not with other
lambdoid phages such as 434. λimm434 (a lambdoid hybrid phage that has
mostly λ genes except for cI and the surrounding “immunity” region of 434
phage) also can grow on E. coli(λ).
a. If you take a 1ml culture of E. coli(λ) (this is wild-type E. coli) and
superinfect with λimm434 at an MOI of 1, the tube will clear after about an
hour. Describe how you would determine whether the released phages from
this infection are λimm434 or λ (induced from the prophage). Do you
expect there will be more λimm434, more λ, or equal numbers of both
phages in the lysate? Way more λimm434.
b. You perform infection experiments using wild-type E. coli and E. coli(λ),
infecting them at an MOI = 1 with either λ, 434, λimm434, or λimm434 Ram
(an amber mutant in the R gene) and get the following result:
λ
E. coli
+
E. coli(λ)
+ = lysis, - = no lysis
Infecting Phage
434
λimm434
+
+
+
+
λimm434 Ram
+
Explain, in detail, why λimm434 Ram is able to lyse the λ lysogen but not
wild-type E. coli. It doesn’t lyse wild-type because it is deficient in R,
one of the two lysin genes. The reason why infection of the lysogen leads
to lysis is because the R gene on the prophage is transcriptionally
activated and translated, complementing the mutant. The reason why R
is transcribed, as well as all of the late genes, is because Q is supplied in
trans by λimm434. The important thing here is that while PR and PL are
directly repressed by cI, PR’ is not and RNAP able to bind and initiate
transcription there, even in the prophage state.
c) Do you expect there are viable phages in the lysate from the infection of
E. coli(λ) with λimm434 Ram and, if so, what phage genotype will be
present? λimm434 Ram