PMB/MCB 112 Midterm 1, Fall 2011

Name:

1A) You are studying a bacterium that can grow using either glucose or xylose as its sole carbon source. You want to study xylose catabolism by isolating mutants unable to use xylose. After mutagenizing a wild-type population of cells, describe your process for isolating these mutant strains. (3 pts) Plate mutants on glucose medium, then replica-plate to medium containing xylose only (first) and glucose (second). Select mutants that grow on glucose but not on xylose.

1B) You find four mutants unable to live with xylose as the sole carbon source. If you have created your mutants using an alkylating agent, how will you determine which gene is mutated in each strain? (2 pts) Transform each mutant with a genomic DNA library made using wild-type DNA. Plate transformants on medium containing xylose only. Sequence plasmid insert to identify gene. 1C) The gene mutated in one of your Xyl- mutants encodes a protein, XylT, which has homology to a family of membrane transporters. You think it may be used by the bacteria for xylose uptake. In an assay of radiolabeled xylose uptake, you see these results when you graph the rate of uptake vs. xylose concentration in wild-type cells. Does the graph support your hypothesis? Why or why not? (2 pts) Yes, because the uptake is saturable, which is a characteristic of protein-mediated transport.

Rate of 14 C-xylose uptake

[xylose]

1D) Can you conclude from the graph above that XylT performs active transport? Explain your reasoning. (2 pts) No, because both facilitated diffusion and active transport are protein-mediated and saturable. No information is given about energy use or the absolute concentrations of xylose inside and outside the cell.

2A) The MinD and MinC proteins are located in the shaded area of the cell below. Draw the location of MinE in this cell. (1 pt) MinE is a ring at the medial edge of the MinCD zone.

PMB/MCB 112 Midterm 1, Fall 2011

Name:

2B) Given the shape of the wild-type cell in 2A, draw the shape of mutant in which MinD cannot hydrolyze ATP. (1 pt) filamentous 2C) What experimental observation led investigators to believe that a second mechanism (nucleoid occlusion) also affects placement of the division site in E. coli? (2 pts) Even when the Min genes are absent, cell division does not occur in an area where DNA is present, and chromosomes are not cut by the septum.

3) You need a 500 ml culture of Caulobacter crescentus at 5 x108 cells/ml tomorrow morning at 10 am, and it is now 7 pm. You have a small log-phase culture at 109 cells/ml. Both your small culture and the large flask contain minimal medium. The Caulobacter doubling time in this medium is 2.5 hours. What volume of the small log-phase culture must be inoculated into the large flask so that the desired cell density is achieved tomorrow morning? (3 pts) N = N0 x 2n N = 500 ml x (5 x 108) cells/ml = 2.5 x 1011 cells n = t/g = 15 h/2.5 hours/generation = 6 generations Solve for N0 = 3.9 x 109 cells Volume = 3.9 x 109 cells/109 cells/ml = 3.9 ml

4) List the two most likely reasons(other than trivial errors in pipetting, mixing, etc) why a direct microscopic count of an environmental sample might give a higher number of cells/ml than a viable count (plate count). (2 pts) dead cells in environmental sample some cells in environmental sample dont grow on medium in your viable count assay

5A) What two reactions in peptidoglycan biosynthesis occur outside the cytoplasmic membrane? (2 pts) transglycosylation and transpeptidation

PMB/MCB 112 Midterm 1, Fall 2011

Name:

5B) What reaction is blocked by penicillin and HOW is penicillin able to do this? (3 pts) Penicillin mimics D-ala-D-ala and is attacked by transpeptidase enzymes rather than their normal substrate. This creates an irreversible intermediate between penicillin and the PBP.

6A) Describe the cellular phenotypes of two genetic mutants (and NAME the mutated genes) that support the idea that bacterial cell shape is partly generated by a cytoskeleton. (3 pts) mreB depletion mutants become misshapen and lyse ftsZ temperature-sensitive mutants become filamentous at the non-permissive temperature creS mutants of curved bacteria become straight

6B) In general, how are cytoskeletal proteins thought to affect bacterial cell shape? Explain one experimental finding that supports this argument. (2 pts) They position PBPs and other enzymes that synthesize peptidoglycan. MreB and FtsZ colocalize with PBPs. When MreB is depleted, PBPs are mislocalized. MreB and FtsZ are found in large protein complexes with PBPs.

10A) List the steps of the Gram stain, starting after the sample has been heat-fixed. (2 pts) crystal violet, iodine, alcohol (decolorization), safranin (counterstain)

10B) Explain how and why the staining procedure above differentiates between Gram-positive and Gram-negative cells. (2 pts) Decolorization/alcohol dissolves the outer membrane of Gram-negative bacteria, releasing most of the crystal violet stain. Gram-positive cells have no outer membrane, so the crystal violet bound to the peptidoglycan.

PMB/MCB 112 Midterm 1, Fall 2011

Name:

7) You are studying Myxococcus xanthus, which has one type of pilus that it uses for gliding across surfaces. Using transposon mutagenesis, you isolate a nonmotile mutant and observe the cells by electron microscopy. Surprisingly, the mutant cells have even more pili than the wildtype parent. 7A) What type of protein, with what function and biochemical activity, is likely to be affected in your mutant to cause the observed phenotype? (2 pts) ATPase that functions in pilus retraction

7B) What is the fastest way to identify the affected gene in this mutant? (1 pt) sequence out from the transposon into the surrounding genomic DNA

7C) You later isolate a new nonmotile mutant using UV mutagenesis. In electron microscopy, this cell has no pili. You want to determine the relationship between your two genes/mutants. Describe the steps you would take to determine which mutation is epistatic to the other. (3 pts) Attack the transposon mutant with a transducing phage and harvest the phage. Attack the UV mutant with these phage at low multiplicity of infection and plate the cells on medium to select for the transposon. Examine double mutants by EM to determine if they have many pili or none.

8A) Describe the steps leading from DNA damage to the SOS response. (3 pts) DNA damaging agents expose ssDNA, which is bound by RecA RecA-ssDNA complex stimulates autocleavage of LexA SOS genes formerly repressed by LexA are now transcribed

PMB/MCB 112 Midterm 1, Fall 2011

Name:

8B) Explain how UV radiation causes heritable mutations. (3 pts) UV causes pyrimidine dimers. When these are in the template, they cannot be replicated by normal DNA polymerase. UmuCD, an SOS gene, is a polymerase that can use templates with pyrimidine dimers. It places random bases across from the pyrimidine dimers, creating mutations in the replicated strand.

11) The figure below depicts cell growth and -galactosidase production by wild-type cells and a Lac- mutant. You know that the genes for -galactosidase (lacZ) and lac permease (lacY) are NOT mutated in this organism. 11A) Based on your knowledge of lac 1 = WT cell operon regulation, explain how mutations density in two different proteins could generate the observed mutant phenotype, including 2 = mutant the normal activities of each protein and cell any small molecules that they bind or density produce.(4 pts) 3 = WT -gal production 4 = mutant gal production 1 2 3 1) Point mutation in lac repressor so that it no longer binds the inducer, lactose. The mutant repressor will remain bound to DNA even when lactose is present and block B-gal transcription.

2) Knockout of CAP so that it can never activate lac operon transcription. CAP normally activates lac transcription when glucose is absent, and it bind the coactivator cAMP. (Could also have point mutation so that CAP doesnt bind cAMP) 3) Knockout of adenylate cyclase, so that cAMP is never made, and CAP cant activate transcription of lac operon.

11B) Are both of the proposed mutations equally likely if your screen started with transposon mutagenesis? Why or why not?(2 pts) It is possible to obtain knockout mutations using a transposon, but to obtain point mutations affecting only a small molecule binding site, one must use a mutagen that makes microlesions.