Ames Test

The fact that microorganisms are susceptible to chemi­cal mutagens can be used to determine the ability of various chemicals to increase the rate of mutation, or mutagenicity. In the Ames test procedure, a strain of the bacterium Salmonella typhimurium is the test organism for determining chemical mutagenicity (see Figure). This bacterial strain is an auxotroph (nutri­tional mutant) that requires the amino acid histidine. These organisms are exposed to increasing amounts of the chemical being tested on a solid growth medium that lacks histidine. Normally, the bacteria cannot grow and in the absence of a chemical mutagen no colonies can develop. If the chemical is a mutagen, lethal muta­tions will occur in the areas of high chemical concentra­tion and no growth will occur in these areas. At lower chemical concentrations along the concentration gradi­ent, however, fewer mutations will occur and some of the cells may revert through mutation to nutritional types that do not require histidine for growth. Such mu­tants are able to grow and produce visible bacterial colonies on the medium. The appearance of bacterial colonies, therefore, demonstrates that the chemical is a mutagen and the absence of colonies indicates that it is not.

The Ames test procedure also is used to screen chem­icals to determine if they are potential cancer-causing agents, or carcinogens. The theoretical basis for this use of the Ames test is that nearly all carcinogens that act di­rectly by attacking DNA are also mutagens. Rather than screening chemicals directly for carcinogenicity, Bruce

The Ames test procedure is used to screen for mutagens and po­tential carcinogens. The auxotrophic strain used in this proce­dure, generally a histidine-requiring mutant of Salmonella ty­phimurium, will not grow on a minimal medium. Mutants that revert to the prototrophic wild type will grow on this medium. The number of colonies that develop after exposure to a chem­ical indicates the effect of that chemical on mutation rate and therefore its degree of mutagenicity. The development of many colonies indicates that the chemical is highly mutagenic.

Ames and his co-workers thought it would be better to test them first for mutagenicity. They recognized, though, that some chemicals are chemically modified in the body and, in particular, some chemicals are inad­vertently transformed into carcinogens in the liver in an apparent effort by the body to detoxify these chemicals. Therefore, in testing for potential carcinogenicity, once a chemical has been found to be mutagenic, it is incu­bated with a preparation of rat liver enzymes to simu­late what normally occurs in the liver. Various concen­trations of this preparation are then incubated with the Salmonella auxotroph to determine whether any prod­ucts that would cause mutations are formed. The chem­icals that do not produce mutations are assumed to be noncarcinogenic or are carcinogens that are not detected by this procedure. Those shown to be mutagenic are subjected to further testing.

Although the Ames test does not positively establish whether a chemical causes cancer, determining whether a chemical has mutagenic activity is useful in screening large numbers of chemicals for potential mutagens, be­cause it is highly probable that a chemical that is a mu­tagen is also a carcinogen. Since this test can be com­pleted in 24 hours, rapid identification of a mutagen is possible. Today, the use of this bacterial assay greatly simplifies the task of screening many potentially dan­gerous chemicals, permitting us to recognize potentially carcinogenic compounds. Using bacteria in the Ames assay also allows scientists to avoid animal testing in many cases.

204 CHAPTER 7 MICROBIAL GENETICS: REPLICATION AND EXPRESSION OF GENETIC INFORMATION

FIG. 7-13 Various chemical and physical agents increase rates of mutation. UV light and dioxin are mutagens that cause formation of mutants (pink cells).

Factors Affecting Rates of Mutation

Naturally occurring rates of mutation are relatively low, about one in a million times the rate of DNA replication. Various physical and chemical agents, however, can modify the nucleotides within DNA, increasing the rate of mutation (FIG. 7-13). Agents that increase the rates of mutation are called muta­gens.

Exposure to high-energy radiation such as X-rays can cause mutations. Such high-energy ionizing radi­ation produces breaks in the DNA molecule. The time and intensity of exposure determines the num­ber of lethal mutations that occur. Exposure to gamma radiation, such as that emitted by an isotope of cobalt, “Co, can be used for sterilizing objects, in­cluding plastic Petri plates, because sufficient expo­sure results in lethal mutations and the death of all exposed microorganisms. Gamma radiation from “Co has also been used to kill microorganisms on the

surfaces of some foods, thereby delaying the spoilage of the food.

Exposure to ultraviolet light also can result in thymine dimer formation, that is, the covalent link­age of one thymine to another thymine. Covalent linkages are formed between two thymines on the same strand of DNA. A thymine dimer cannot act as a template for DNA polymerase, and the occurrence of such dimers, therefore, prevents the proper func­tioning of DNA polymerase. Exposure to ultraviolet light can cause lethal mutations and is sometimes used to kill microorganisms in sterilization proce­dures. Sometimes ultraviolet light is placed above a work surface when it is not in use to maintain steril­ity of the surface. Also, air is sometimes passed over ultraviolet lights in hospitals treating patients with respiratory diseases to kill airborne pathogens.

Radiation causes lethal mutations and can be used

for sterilization of some materials.

EXPRESSION OF GENETIC INFORMATION

The expression of genetic information involves using information encoded within DNA to direct the syn­thesis of proteins. In a cell, DNA stores and transmits the complete hereditary information called the geno­type ['ʤenəutaɪp]. Proteins mediate functional activity—called the phenotype ['fēnəˌtīp] — that is, the actual appearance and activ­ities of the organism. For example, proteins (en­zymes) catalyze all the metabolic activities of cells and produce the observable characteristics that dis­tinguish one microorganism from another.

Proteins mediate ['miːdɪət] functional activity (phenotype); DNA mediates the informational capacity (genotype) of the cell.

The sequence of nucleotides within the DNA mol­ecule ultimately codes for the sequence of amino acids in proteins. Because proteins are the "action" molecules within cells and nucleic acids dictate the formation of proteins, it is the ordering of nucleotides within DNA that provides the information for estab­lishing, controlling, and reproducing cell structure