5.) Name and describe one gene product in E.coli that is associated with performing each step in the recombination process. (6pts)

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1 Student ID# Bacterial Genetics, BIO 4443/6443 Spring Semester 2001 Exam II 1.) 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? (6pts) 2.) What general role do the products of the fino and finp genes have in the conjugation of the F plasmid? What fortuitous role did these genes play in the discovery of conjugation? (5pts) 3.) Naturally transformable cells take up DNA from outside the cell. Exactly why it would be advantageous for a cell to take up non-self DNA is something that is still debated extensively. Describe three possible roles (or functions) that transformation has been proposed to play in the bacterial life cycle AND for each possibility, describe one piece of evidence that supports or fails to support that idea. (6pts) 4.) Most recombination models propose that enzymes are required to catalyze at least four fundamental functions (or steps) during the recombination process. In general, what steps are required during the recombination process? (6pts) 5.) Name and describe one gene product in E.coli that is associated with performing each step in the recombination process. (6pts) 1

2 6.) Some Insertion Sequences transpose by using a Replicative mechanism of transposition. Other Insertion Sequences utilize a Cut and Paste mechanism. Describe two observations that differentiate between these two mechanisms of transposition? (5pts) 7.) 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. For each substrate, describe why you would, or would not, expect to obtain ampicillin resistant, histidine deficeint transformants? (8pts) 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. 2

3 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 rif R 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 rif R 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 rif R mutation. You infect the mutant rif R pro + lac + E.coli with P1 bacteriophage and make a P1 lysate. You then use the lysate to infect a rif S pro - lac - recipient and select for rif R transductants. Of the rif R transductants, you find that 10% are pro +. and 60% are lac +. 8.)What are the two possible orders that these three genes could be in? (4pts) 9.) You repeat the infection, this time selecting for lac + transductants. Of the lac + transductants, 60% are also rif R but none are pro +. Where does the rif R gene map in relation to pro and lac? (4pts) 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 - nal R (nalidixic acid resistant) strain of E. coli that you have in your lab and test whether the pathogenic strain contains a selftransmissible plasmid. 10.) 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) (3pts) 11.) 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) (3pts) 3

4 12.) 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 (9pts). 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. 13.) 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 early). You obtain the following numbers of recombinants individually. 10 cells out of every100 donor cells are trp + nal R 53 cells out of every 100 donor cells are his +. nal R 5 cells out of every100 donor cells are leu +. nal R 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. 14.) What type of experiment would this be called? Is it a time of entry experiment or a gradient of transfer experiment? (2pts) 15.) Draw the map order of trp, his, leu, pen R (penicillin resistance), and red (red/mucoid). (10pts) 16.) If you wanted to know the distance between genes rather than their relative order, what type of experiment could you do? (3pts) 17.) What can you conclude about the pen R gene and the red gene(s)? What does this imply about the acquisition of the new traits in the pathogenic strain? (5pts) 4

5 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 for 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 #2 #3 #4 #5 F that carries mutant #1 #2 #3 #4 #5 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) 5