Download 06MicrobialGenetExamII

Name_______________________________
The plasmid shown to the right has an oriV and oriT at the positions
indicated, and is known to replicate bidirectionally.
1.) Indicate where replication would be expected to terminate
during vegetative replication? (4pts)
2.) Indicate where replication would be expected to terminate
during conjugation? (4pts)
3.) ColE1 plasmids maintain a copy number of
about 16 copies/cell.
How would the copy number be
effected by a mutation in the promoter region
of the following genes or transcripts? Assume
that the mutation prevents any transcription
from occurring at each gene. Explain your
answer. (8pts)
RNAI:
RNAII:
rop:
Microbial Genetics
BI 410/510
Spring Semester 2006
Exam II
oriV
oriT
4.) The single copy F plasmid replicates only once per cell generation. Thus at the time
of cell division, there are only two copies of the plasmid per cell. Assuming that the
plasmid is sorted randomly, calculate (or draw) how often the plasmid should be lost by
chance each generation. (5pts)
5.) By measuring the rate that the F plasmid is cured, you find that only one in every ten
thousand cells actually loses the plasmid. Name two general mechanisms or processes
that could account for the discrepancy between the observed cure rate and your calculated
cure rate. (5pts)
Most mutations that make E. coli resistant to the antibiotic rifampicin are found in the
rpoB gene of the RNA polymerase. You have just isolated a new rifR mutant and to your
suprize, your conjugational mapping experiment suggests that the mutation is on the
opposite side of the chromosome from the rpoB gene. Your mutation does map nearby
both pro and lac, however. Since this represents a possibly novel mechanism of bacterial
resistance, you decide to map your rifR mutation in a little more detail and set up a
transduction experiment.
To the best of your knowledge, your mutant is wild type for all other genes
besides the rifR mutation. You infect the mutant rifR pro+ lac+ E.coli with P1
bacteriophage and make a P1 lysate.
You then use the lysate to infect a rifS pro- lac- recipient and select for rifR
transductants.
Of the rifR transductants, you find that 10% are pro+. and 60% are lac+.
6.) What are the two possible orders that these three genes could be in? (4pts)
7.) You repeat the infection, this time selecting for lac+ transductants.
Of the lac+ transductants, 60% are also rifR but none are pro+.
Where does the rifR gene map in relation to pro and lac? (6pts)
8.) What is the primary difference between conjugative plasmids and mobilizable
plasmids? What genes are typically found on conjugative plasmids that are not found on
mobilizable plasmids? (4pts)
9.) In a transduction experiment, if you used an MOI of 5, what fraction of the bacteria
would not get infected? Use the poisson expression, Pi = ( mi e-m )/ i! and show
your work. (4pts)
We are trying to knockout the chromosomal copy of the hisA gene in a naturally
competent strain of Bacillus subtilis. We have constructed various substrates (shown
below) that contain the ampicillin resistance gene (beta lactamase) inserted into the
middle of the hisA gene. You mix each of the DNA substrates together with your
competent cells.
10.) For each substrate, describe why you would, or would not, expect to obtain
ampicillin resistant, histidine deficeint transformants? (10pts)
ampR
hisA
You add linear single stranded
DNA containing the ampR
gene inserted into the middle
of the hisA gene
You add linear double
stranded DNA containing the
ampR gene inserted into the
middle of the hisA gene
You add a circular double
stranded plasmid containing
the ampR gene inserted into
the middle of the hisA gene
You add linear double
stranded DNA. This time,
however, the hisA gene
remains intact and complete
and the complete ampR gene
is inserted after the hisA gene.
One year later...
Following Stanford’s dominating performance at the NCAA championships,
1,345 fans were hospitalized and seven eventually died from a hemolytic fever which set
in within hours after the game ended. Health officials quickly zeroed in on the arena’s
hot dogs as the culprit. Scientists were easily able to isolate a bacteria from the hot dogs
that appears almost identical to the common nonpathogenic strain of E.coli that is
normally found in our intestines. When culturing the bacteria, scientists found that the
pathogenic strain is resistant to penicillin, a problem that delayed effective treatment for
some patients. Additionally, rather than forming normal round, white-ish, colonies on
plates, this pathogenic strain grows into red, pussy, mucoid colonies.
Hot dog sales around the country have taken a serious plunge and the high profile
case has both scientists and the public worried about where or how this E.coli strain
became pathogenic. Although the colonies look very different from E.coli, the initial
genotyping hasn’t shown any genetic difference between this strain and normal E.coli.
Based on the penicillin resistance and pathogenicity, you speculate that this
variant of E. coli may have acquired a plasmid that confers these phenotypes.
The pathogenic strain of E. coli seems to grow well on minimal plates that contain
only glucose. You decide to make use of a common trpB- hisA- leuC- nalR (nalidixic
acid resistant) strain of E. coli that you have in your lab and test whether the pathogenic
strain contains a self-transmissible plasmid.
11.) Which cell will serve as the donor in your test? What is the genotype of the donor?
(Include trpB, hisA, leuC, nal, pen, and red (for red,mucoid) (4pts)
12.) Which cell will serve as the recipient in your test? What is the genotype of the
recipient? (Include trpB, hisA, leuC, nal, pen, and red (for red,mucoid) (4pts)
13.) Describe how you would test to see if the pathogenic strain contains a selftransmissible plasmid. Be sure to include which supplements and/or antibiotics would be
used in your selection plates (10pts).
Based upon your results, you conclude that there probably is some form of a selftransmissible plasmid in the pathogenic strain.
Interestingly, you never are able to get the entire population to transfer the
plasmid successfully so you decide to examine your transconjugates more closely and
you are suprized to find that several of the transconjugates are now able to grow without
the addition of histidine leucine or tryptophan.
14.) What does this most likely imply about the plasmid in the pathogenic strain of
E.coli? (5pts)
You decide to map the position of this virulence gene(s) in the pathogenic strain.
You repeat your transfer experiment, allowing the mating to go on for several hours. (for
simplicity, we will assume that nal transfers extremely late). You obtain the following
numbers of recombinants individually.
30 cells out of every100 donor cells are trp+ nalR
53 cells out of every 100 donor cells are his+. nalR
5 cells out of every100 donor cells are leu+. nalR
44 cells out of every 100 donor cells are resistant to penicillin
<0.1 cells out of every 100 donor cells are pussy, red, or mucoid.
15.) What type of experiment would this be called? Is it a time of entry experiment or a
gradient of transfer experiment? (4pts)
16.) Draw the map order of trp, his, leu, penR (penicillin resistance), and red
(red/mucoid). (10pts)
17.) What can you conclude about the penR gene and the red gene(s)? What does this
imply about the acquisition of the new traits in the pathogenic strain? (3pts)
Clearly you need more information about the pathogenicity. Several scientists
have shown that the red, mucoid phenotype correlates perfectly with causing
pathogenicity in mice. Using the red/mucoid phenotype as your marker for
pathogenicity, you mutagenize a red, mucoid culture and isolate 5 mutants that are no
longer red or mucoid. After a lot of work, you also obtain F’ factors that contain the red
mucoid region from each mutant.
You transfer each F’ into each mutant and score for complementation of the red, mucoid
phenotype. The color and shape of each transconjugate carrying the F’ is shown.
Recipient mutant #1
F’ that
carries mutant
#1
#2
#3
#4
#5
White
Normal
Red
Mucoid
Red
Mucoid
White
Normal
Red
Mucoid
#2
#3
#4
#5
White
Normal
White
Normal
Red
Mucoid
Red
Mucoid
White
Normal
Red
Mucoid
Red
Mucoid
White
Normal
Red
Mucoid
White
Normal
18.) Which mutations belong to the same complement group(s). At least how many
genes or complementation groups do you suspect are involved in the red (red, mucoid)
pathway? (10pts)