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:
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)
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 self-transmissible 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 self-transmissible 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 mutantF’ that
carries mutant / #1 / #2 / #3 / #4 / #5
#1 / White
Normal
#2 / Red
Mucoid / White
Normal
#3 / Red
Mucoid / White
Normal / White
Normal
#4 / White
Normal / Red
Mucoid / Red
Mucoid / White
Normal
#5 / Red
Mucoid / Red
Mucoid / Red
Mucoid / 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)