Detection And Isolation Of Mutants

Mutations often arise spontaneously and provide genetic diversity, which include survival during adverse or changing environmental conditions; thus mutations are of value to microorganisms.

Mutation are also of practical importance to microbial geneticists.

Mutant strains have been used to reveal mechanisms of complex processes such as DNA replication, endospore formation, and regulation of transcription. They are also used as selective markers in recombinant DNA procedures.

To study microbial mutants, they must be readily detected, even when they are rare, and then efficiently isolated from wild type organisms and other mutants that are not of interest. Microbial geneticists typically increase the likelihood of obtaining mutants by using mutagens to increase the rate of mutation. The rate can be increased from the usual one mutant per 10⁷ to 10¹¹ cells to about one per 10³ to 10⁶ cells. Even at this rate, carefully devised means for detecting or selecting a desired mutation must be used.

Mutant Detection

When collecting mutant of a particular organism, the wild type characteristics must be known so that an altered phenotype can be recognized. A suitable detection system for the mutant phenotype also is needed.

The use of detection system is called screening.

Screening for mutant phenotypes in haploid organisms is straightforward because the effects of most mutations can be seen immediately. Some screening procedures require only examination of colony morphology. For instance, if albino mutants of a normally pigmented bacterium are being studied, detection simply requires visual observation of colony color. Other screening methods are more complex. For example, the replica plating technique is used to screen for auxotrophic mutants. It distinguish between mutants and the wild-type strain based on their ability to grow in the absence of a particular bio synthetic end product.

Mutant Selection

An effective selection technique uses incubation conditions under which the mutant grows because of properties conferred by the mutation, whereas the wild type does not. Selection methods often involve reversion or suppressor mutations or the development of resistance to an environmental stress.

Methods for selecting mutants resistant to a particular environmental stress follow a similar approach. Often wild type cells are susceptible to virus attack, antibiotic treatment, or specific temperatures, so it is possible to grow the microbe in the presence of the stress and look for surviving organisms. Consider the example of a phage-sensitive wild-type bacterium. When it is cultured in medium lacking the virus and then plated on selective medium containing viruses, any colonies that form are resistant to virus attack and very likely are mutants in this regard.

Substrate utilization mutations also can be selected. Many bacteria use only a few primary carbon sources. With such bacteria, it is possible to select mutants by plating a culture on medium containing an alternate carbon source. Any colonies that appear can use the substrate and are probably mutants.

Mutant screening and selection methods are used for purposes other than understanding more about the nature of genes or the biochemistry of a particular microorganisms. One very important role of mutant selection and screening techniques is in the study of carcinogens.

Reference: Microbiology, Prescot