Download The 43 strains contain deletions that extend from the immunity

7). (18 points). Shimada, Weisberg, and Gottesman (1973; J. Mol. Biol. 80:297-314) isolated a secondary
site lambda lysogen where the prophage had inserted into the E. coli trpC gene as shown below. The
orientation of the prophage relative to the trp operon as well as some lambda markers in the prophage are
shown. All lambda genes are wild type except for the cI gene which is the temperature sensitive cI857
allele. The lysogen was isolated as a tryptophan auxotroph at 30 o C.
A). (6 Points). Why is this strain a tryptophan auxotroph?
The prophage has inactivated the trpC gene which is required for tryptophan biosynthesis.
If a culture of this lysogen is grown at 30o in LB broth and an aliquot is plated on LB plates
which are incubated at 42o C, rare colonies occur at a frequency of about 1 in 107 cells plated.
Colonies are picked and checked for the ability to grow in the absence of tryptophan by streaking
on minimal plates lacking tryptophan. Some colonies are able to grow and some are not.
B). (4 points). What is the most likely explanation for each class of colonies? What phenotype
does this experiment select for?
The prototrophs are the result of excision at attL and attR restoring the integrity of the trpC
gene. The colonies that are still auxotrophs are the result of aberrant spontaneous excision
events. (These events could originate in the prophage and extend either leftward or
rightward or both. Some could leave behind some prophage genes if the deletion endpoint
is in the prophage. Some could extend into adjacent bacterial DNA). All of the survivors
must have the cI gene deleted so they can survive at 42o C.
They also performed the following experiment. The lysogen was grown in LB at 30o C until the
cells were at a cell density of about 5 X 108 cells / ml. The culture was split in half. One half was
incubated at 30o C for 2 hours. The other half was grown at 42o C for 6 minutes and then
returned to 30o C and grown for 2 hours. After the 2 hour growth, the number of prototrophs
(Trp+) colonies was determined for each culture. The one that was transiently heated to 42o C and
returned to 30o C had 1000 times more prototrophs than the culture that was grown at 30o C the
entire time.
C). (3 Points). Why were more prototrophs observed from the second culture? What is the
molecular mechanism for the result?
The culture that was grown at 30o C the entire time was repressed so there was little
expression of int and xis to catalyze excision of the prophage which would make the cell
Trp+. When the second culture was raised to 42o C, the cI857 repressor becomes inactive
and allows expression from PL to allow the synthesis of Int and Xis. The Int and Xis
promote excision of the prophage from the chromosome restoring the Trp+ phenotype.
Since the temperature is lowered to 30o C soon, the repressor becomes active again and reestablishes repression preventing expression of the lytic functions. (Apparently expression
from PR during the 6 minutes is not sufficient to allow the accumulation of Cro to an extent
that would permanently establish the lytic pathway). Int and Xis become repressed when
the cI857 binds OL (and OR) and are diluted as the cell grows. The excised prophage cannot
replicate its DNA or grow lytically and is also diluted out of the population.
They had a collection of mutant lambda phages that had amber mutations in known genes such as
the N (lambda Nam7), S (lambda Sam7) and J (lambda Jam53) genes. These genes are required
for lytic growth or development. The N gene codes for the N protein antiterminator required for
lytic growth. The S gene encodes a protein required for lysis of the host and the J gene codes for
the phage tail fiber. These phage cannot form plaques on suppressor-free strains. When these
phage infect a defective prophage strain lacking the immunity region, they can undergo
recombination with the prophage. If the prophage contains a wild type copy of the gene that is
mutated on the infecting phage a wild-type recombinant can be produced. The recombinant
phage is able to form a plaque on a suppressor-free strain. (This technique is called “marker
rescue”).
They also had a collection of Phi80-trp transducing phages that carry the trp operon. One phage,
Phi80-trpE42 carries the trp operon with a nonsense mutation in trpE. A second phage, Phi80trpA44 carries the trp operon with a nonsense mutation in the trpA gene. When either Phi80trpE42 or Phi80-trpA44 infects a strain that carries a wild-type trpE or trpA gene, Trp+
transducing phages can form by marker rescue.
D). (5 Points). The table below shows the results of marker rescue experiments with the lambda
am phages and Phi80-trp phages when they infect four different strains selected above at 42o C
that are trp auxotrophs. Explain the results for each of the 4 E. coli strains. (You can use the
figure above to illustrate your answer).
The 4 strains contain deletions that extend from the immunity region to various lengths on the right.
The deletions are shown below.