Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
because a mutation in it has caused an observable change in the organism. With the molecular genetic approach, a gene can be isolated and mutated in the test tube without any knowledge of its function. Only after the mutated gene has been returned to the organism does its function become apparent.
Rather than one approach supplanting the other, molecular genetics and classical genetics can be used to answer different types of questions, and the two approaches often complement each other. In fact, the most remarkable insights into biological functions have often come from a combination of classical and molecular genetic approaches.
Bacterial Genetics
In bacterial genetics, genetic techniques are used to study bacteria. Applying genetic analysis to bacteria is not different in principle from applying it to other organisms. However, the methods that are available differ greatly. Some types of bacteria are relatively easy to manipulate genetically. As a consequence, more is known about some bacteria than is known about any other type of organism. Some of the properties of bacteria that facilitate genetic experiments are described below.
Bacteria Are Haploid
One of the major advantages of bacteria for genetic studies is that they are haploid. This means that they have only one copy, or allele, of each gene. This property makes it much easier to identify cells with a particular type of mutation.
In contrast, most eukaryotic organisms are diploid, with two alleles of each gene, one on each homologous chromosome. Most mutations are recessive, which means that they do not cause a phenotype in the presence of a normal copy of the gene. Therefore, in diploid organisms, most mutations have no effect unless both copies of the gene in the two homologous chromosomes have the mutation. Backcrosses between different organisms with the mutation are usually required to produce offspring with the mutant phenotype, and even then, only some of the progeny of the backcross have the mutated gene in both homologous chromosomes. With a haploid organism such as a bacterium, however, most mutations have an immediate effect and there is no need for backcrosses.
Short Generation Times
Another advantage of many bacteria for genetic studies is that they have very short generation times. The generation time is the length of time the organism takes to reach maturity and produce offspring. If the generation time of an organism is too long, it can limit the number of possible experiments. Some strains of the bacterium E. coli can reproduce every 20 minutes under ideal conditions. With such rapid multiplication, cultures of the bacteria can be started in the morning, and the progeny can be examined later in the day.
Asexual Reproduction
Another advantage of bacteria is that they multiply asexually, by cell division. Sexual reproduction, in which individuals of the same species must mate with each other to give rise to progeny, can complicate genetic experiments because the progeny are never identical to their parents. To achieve purebred lines of a sexually reproducing organism, a researcher must repeatedly cross the individuals with their relatives. However, if the organism multiplies asexually by cell division, all the progeny are genetically identical to their parent and to each other. Genetically identical organisms are called clones. Some simpler eukaryotes, such as yeasts, and some types of plants, such as water hyacinths, can also multiply asexually to form clones. Identical twins, formed from the products of the division of an egg after it has been fertilized, are clones of each other. While there are a few examples where mammals have been cloned by transplanting a somatic cell into the ovary, bacteria form clones of themselves every time they divide.
Colony Growth on Agar Plates
Genetic experiments often require that numerous individuals be screened for a particular property. Therefore, it helps if large numbers of individuals of the species being studied can be propagated in a small space.
With some types of bacteria, thousands, mill ions, or even bill ions of individuals can be screened on a single agar-containing petri plate. Once on an agar plate, these bacteria divide over and over again, with all the progeny remaining together on the plate until a visible lump, or colony, has formed. Each colony is composed of millions of bacteria, all derived from one original bacterium and hence all clones of the original bacterium.
Colony Purification
The ability of some types of bacteria to form colonies through the multiplication of individual bacteria on plates allows colony purification of bacterial strains and mutants. If a mixture of bacteria containing different mutants or strains is placed on an agar plate, individual mutant bacteria or strains in the population each multiply to form colonies. However, these colonies may be too close together to be separable or may still contain a mixture of different strains of the bacterium. If the colonies are picked and the bacteria are diluted before replating, discrete colonies that result from the multiplication of individual bacteria may appear. No matter how crowded the bacteria were on the original plate, a pure strain of the bacterium can be isolated in one or a few steps of colony purification.
Serial Dilutions
To count the bacteria in a culture or to isolate a pure culture, it is often necessary to obtain discrete colonies of the bacteria. However, because bacteria are so small, a concentrated culture contains billions of bacteria per milliliter. If such a culture is plated directly on a petri plate, the bacteria all grow together, and discrete colonies do not form. Serial dilutions offer a practical method for diluting solutions of bacteria before plating to obtain a measurable number of discrete colonies. The principle is that if dilutions are repeated in succession, they can be multiplied to produce the total dilution. For example, if a solution is diluted in three steps by adding 1 ml of the solution to 99 ml of water (one in a hundred), followed by adding 1 ml of this dilution to another 99 ml of water, and finally, by adding 1 ml of the second dilution to another 99 ml of water, the final dilution is 10–2 x 10–2 x 10–2 = 10–6, or one in a million. To achieve the same dilution in a single step, 1 ml of the original solution would have to be added to 1,000 liters (about 250 gallons) of water. Obviously, it is more convenient to handle three solutions of 100 ml each than to handle a solution of 250 gallons, which weighs about 2,000 lb!
Selections
Probably the greatest advantage of bacterial genetics is the opportunity to do selections, by which very rare mutants and other types of strains can be isolated. To select a rare strain, billions of the bacteria are plated under conditions in which only the desired strain, not the bulk of the bacteria, can grow. In general, these conditions are called the selective conditions. For example, a nutrient may be required by most of the bacteria but not by the strain being selected. Agar plates lacking the nutrient then present selective conditions for the strain, since only the strain being selected multiplies to form a colony in the absence of the nutrient. In another example, the desired strain may be able to multiply at a temperature that would kill most of the bacteria. Incubating agar plates at that temperature would provide the selective condition. After the strain has been selected, a colony of the strain can be picked and the colony purified away from other contaminating bacteria under the same selective conditions.
The power of selection with bacterial populations is awesome. Using a properly designed selection, a single bacterium can be selected from among bill ions placed on an agar plate. If we could apply such selections to humans, we could find one of the few individuals in the entire human population of Earth with a particular trait.
Storing Stocks of Bacterial Strains
Most types of organisms must be continuously propagated; otherwise, they age and die. Propagating organisms