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Modern Genetic Analysis Chapter 9. The Genetics of Bacteria and Phages
Working with Microorganisms

Individual bacteria are too small to be seen with the naked eye, although they can be seen under powerful light microscopes. How can such small organisms be manipulated for genetic analysis? The bacteria have to be cultured in such a way that phenotypes can be discerned and genotypes deduced. Although individually invisible to the unaided eye, bacteria are visible as colonies (see Figure 9-1)opaque masses of cells, each derived by asexual cell division from a single progenitor cell. Hence most bacterial phenotypes are classified and analyzed at the colony level. Bacteria can be cultured in a liquid medium or on a solid surface, such as an agar gel, as long as basic nutritive ingredients are supplied. In liquid medium, the bacteria divide by binary fission: they multiply geometrically until the nutrients are exhausted or until toxic waste products accumulate to levels that halt population growth. For solid-medium culture, a small amount of an actively growing liquid culture is pipetted into a petri plate containing a solid agar medium, and the cells are spread evenly on the agar surface with a sterile spreader. This process is called plating (Figure 9-1). Each cell then divides by binary fission to make two daughters, and the daughters in turn divide, and so forth, until a large number of descendent cells are produced. Because the cells are immobilized in the gel, all the daughter cells remain together in a clump. When this mass reaches approximately 107 cells, it becomes visible to the naked eye as a colony. The members of such a colony are known collectively as a clone, a group of individual cells derived asexually from one original cell and therefore all genetically identical with one another and with the original cell.

Phenotypes

What are some specific bacterial characters that can be studied? One category concerns ability to synthesize essential biochemical compounds. Wild-type bacteria are prototrophic: in other words, they can produce colonies on minimal mediuma solution containing only inorganic salts, a source of energy such as a sugar, and water. From these simple substances, the bacteria synthesize all the molecules of life. Some mutant clones can be identified because they are auxotrophic: they will not grow unless the medium contains one or more specific nutrientssay, adenine, threonine, or biotin. Such a phenotype is indicative of the loss of the cell's ability to make that specific compound. The two phenotypes of prototrophy and auxotrophy for a specific nutrient have proved to be very useful markers for genetic analysis. As with discrete phenotypic variants in eukaryotes, these alternative bacterial phenotypes are often determined by a pair of allelesfor example, ad+ (prototrophic) and ad (auxotrophic). A second character used by bacterial geneticists also concerns nutrition but in a fundamentally different way. Bacteria need to be opportunists in using energy sources in the natural environment. For example, wild-type bacteria can utilize the sugar galactose as an energy source. However, mutations arise that inactivate this function and so galactose cannot be used (other energy sources must be found). Here the determining alleles are gal+ (galactose utilizing) and gal (galactose nonutilizing). Another useful phenotype concerns resistance. Whereas wild types are susceptible to various inhibitors, such as streptomycin, resistant mutants differ in that they can form colonies in the presence of an inhibitor. Hence, in this case, the two alternative phenotypes that can be studied genetically are resistance and sensitivity to the inhibitor, determined by the alleles str r and strs.

For many characters, the phenotype of a colony can be determined readily through visual inspection or simple chemical tests. The colony phenotype can then be assigned to the original cell that started that colony, and the frequencies of various phenotypes in the pipetted sample can be determined. Table 9-1 lists some more examples of bacterial phenotypes and their genetic symbols.

Selective Systems

We have seen that all genetic analysis is based on genetic variants, generally mutations, and the same is true in bacterial genetics. As usual, the rarity of mutations is a problem if an investigator is trying to amass a collection of a specific type of mutation for genetic study. One solution is to use a selective system, an experimental protocol designed to allow the desired mutant types, but not the wild types, to grow.

For bacterial mutations causing the reversion of auxotrophy to prototrophy, there is a direct selective system. Take an adenine-requiring auxotroph, for example. Grow a culture of such a strain in an adenine-containing medium. Then plate a large number of cells on a solid nutritive medium containing no adenine (minimal medium). The only cells that can grow and divide on this medium are adenine prototrophs, which must have arisen by reverse mutation in the original culture, symbolically ad ad+ (Figure 9-2).

For forward mutations from prototrophy to auxotrophy in bacteria, a direct selection technique is available. Many species of bacteria are highly sensitive to the antibiotic penicillin (isolated from the fungus Penicillium), but this sensitivity is expressed only during their proliferating stage. If penicillin is added to a suspension of cells in liquid minimal medium, all the prototrophic cells are killed because they start to divide, but the auxotrophic mutations survive because they cannot divide without supplementation. After treatment, the penicillin can be removed by washing the cells on a filter. There will be many different types of auxotrophic mutations in the culture but, if the washed cells are plated on a range of different media, each supplemented with one specific chemical, the specific requirement of any given auxotroph can be deduced.

Armed with these techniques for manipulating bacteria, we now return to the analysis of recombination, starting with conjugation.

© 1999 by W. H. Freeman and Company. All rights reserved.