Mutagenesis

Types of Mutations

Four independent mutations were obtained that affect the synthesis or assembly of fimbriae. The properties of the mutations are described in the table below (where + indicates that fimbriae were produced and - indicates that no fimbriae were produced).

a.

Based upon the above results, indicate whether each of the single mutations is a temperature sensitive (Ts) or coldsensitive (Cs) mutation. ANSWER: fim-1 and fim-3 are Cs, fim-2 and fim-4 are Ts.

b.

Based upon the above results, what is the predicted order of the mutant gene products in the pathway of fimbriae synthesis and assembly? ANSWER: First consider three pairs of double mutants separately.

o o o

The results suggest that fim-2 acts before fim-1 because the product if only made if the cellsare first grown at 30¡C where fim-2 is active, then shifted to 42¡C where fim-1 is active Ð hence fim-1 must convert an intermediate made by fim-2 into the product. The results suggest that fim-1 acts before fim-4 because the product if only made if the cells are first grown at 42¡C where fim-1 is active, then shifted to 30¡C where fim-4 is active Ð hence fim-4 must convert an intermediate made byfim-1 into the product. It is not possible to infer the order of fim-1 vs fim-3 because the double mutant is defective under all growth conditions.

Combining these three results leads to the conclusion that the gene products probably act in the pathway in the following order: fim-2 - fim-1 - fim-4

Wild-type Salmonella typhimurium grows on minimal medium plates without addition of fattyacids. Fatty acid auxotrophs require supplementation with a fatty acid (for example, oleate) for growth. Fatty acid auxotrophs are normally rare (less than 10-6 fatty acid auxotrophs are found in a population of S. typhimurium cells). a. How would you distinguish fatty acid auxotrophs from wild-type prototrophic cells? [Specify the phenotype of both types of cells.]

ANSWER: Replica plating onminimal medium + or - fatty acids. Fatty acid auxotrophs will not be able to grow without fatty acids in this screen. b. How could you could enrich for fatty acid auxotrophs? [Indicate the relative proportion of auxotrophs in the population at each step.] ANSWER: One approach would be to do a penicillin enrichment with growth in minimal medium + fatty acids followed by growth in minimal medium +penicillin without fatty acids, repeated multiple times. Each cycle of penicillin enrichment should result in approximately 100-fold enrichment for the mutant cells relative to the wild-type cells. [Note that any other auxotrophs that accumulated would not be able to grow in minimal medium without fatty acids either -- this is one good reason why you wouldn't want to use rich medium for growth betweenpenicillin steps.] c. What is a potential problem with this method and how you could avoid this problem? ANSWER: A major problem with this method is that each enrichment culture is likely to contain siblings. This problem can be avoided by doing multiple, independent enrichments and only choosing one colony from each enrichment. [As described in the text, a second problem may be that lysis ofcells in the cultures with penicillin could feed any penicillin auxotrophs, allowing their growth (and subsequent death) if the enrichment is not done properly.]

In order to identify new targets for antibiotics, a large collection of temperature-sensitive lethal mutations were isolated in Staphylococcus aureus. a. How could you isolate a collection of temperature-sensitive lethal mutations inStaphylococcus aureus (that is, mutants that survive at 30 C but die at 43 C)? ANSWER: Chemical mutagenesis (to increase the frequency of point mutations) followed by diluting and plating the cells on rich medium at 30 C (to obtain isolated colonies), and subsequent replica plating on rich medium at 43 C. The desired mutants would grow at 30 C but not 43 C. By using rich medium you can avoid most...