Isolating Auxotrophs, Use of Mutagens and Replica Plating

Isolating Auxotrophs, Use of Mutagens and Replica Plating

Consider the isolation of a bacterial strain requiring leucine as a growth supplement added to the medium. A spontaneously arising Leu^- mutant might exist in a population at a frequency of about 10^-6. One method of finding or isolating such a leucine-requiring mutant would be to dilute cells to a concentration of about 1,000 cells/ml and spread 0.1 ml quantities on the surface of 10,000 glucose plus leucine plates. After these had grown, each of the 1,000,000 colonies could be spot tested for a leucine requirement. By this method one spontaneously occurring Leu^- mutant out of 10⁶ cells could be found. This is not a workable method, however, and when faced with problems like this, geneticists devised many shortcuts.

One way to decrease the work of finding a mutant is to increase its frequency of occurrence. Standard techniques for increasing the fre-quency of mutants in a population are treatment with chemical mu-tagens, exposure to UV light, or the use of mutator strains. The spontaneous mutation frequency in such strains is greatly elevated, for example due to mutations in DNA polymerase. Another shortcut is to reduce the time required to spot many colonies in the scoring steps. Replica plating allows spotting all the colonies from a plate in one operation onto a testing plate. This can be done with a circular pad of sterile velvet or paper which is first pushed against the master plate of colonies. The paper or velvet picks up many cells which can then be deposited onto a number of replica plates.

In some situations the use of a mutagen might be unwise due to the possibility of introducing more than one mutation into the strain. Therefore, a spontaneously occurring Leu^- mutant might have to be found. Even with replica plating, this could entail much work and a method of selectively killing all the Leu⁺ cells in the culture would be most valuable.

Penicillin provides a useful reverse selection for bacterial mutants. Ordinarily, mutants capable of growing in a particular medium may easily be selected. The reverse, the selection for the mutants unable to grow in a particular medium, requires a trick. Penicillin provides an answer. This antibiotic blocks the correct formation of the peptide crosslinks in the peptidoglycan layer. Walls synthesized in the absence of these crosslinks are weak. Only growing cells synthesize or try to synthesize peptido-glycan. Therefore, in the presence of penicillin, growing cells synthesize defective walls and are lysed by osmotic pres-sure. Non-growing cells survive. A penicillin treatment can increase the fraction of Leu^- cells in the population 1000-fold. That is, a penicillin treatment can select for Leu^- cells.

In practice, a culture of the desired cells is grown on minimal medium containing leucine, and then the leucine is removed either by filtration or centrifugation. The cells are resuspended in medium lacking leucine, and penicillin is added. The Leu⁺ cells continue growing and are killed by the penicillin whereas the Leu^- cells stop growing and remain resistant to the penicillin. The penicillin is removed and the surviving cells are plated out for spot testing to identify the Leu^- colonies.