Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
transfers and replenishing of the food supply, which can be very time-consuming. However, many types of bacteria can be stored in a dormant state and therefore do not need to be continuously propagated. The conditions used for storage depend on the type of bacteria. Some bacteria sporulate and so can be stored as dormant spores. Others can be stored by being frozen in glycerol or by being dried. Storing organisms in a dormant state is particularly convenient for genetic experiments, which often require the accumulation of large numbers of mutants and other strains. The strains remain dormant until the cells are needed, at which time they can be revived. Organisms that are continually propagated will also accumulate mutations, a problem that is eliminated with the proper storage of bacteria.
Genetic Exchange
Genetic experiments with an organism usually require some form of exchange of DNA or genes between members of the species. Most types of organisms on Earth are known to have some means of genetic exchange, which presumably accelerates evolution and increases the adaptability of a species.
Exchange of DNA from one bacterium to another can occur in one of three ways. In transformation, DNA released from one cell enters another cell of the same species (see chapter 6). In conjugation, discrete genetic elements transfer DNA from one cell to another (see chapter 5). Finally, in transduction, a bacterial virus accidentally picks up DNA from a cell it has infected and delivers this DNA into another cell (see chapter 7). The ability to exchange DNA between strains of a bacterium makes possible genetic crosses and complementation tests, as well as the tests essential to genetic analysis.
Phage Genetics
Some of the most important discoveries in genetics have come from studies with viruses that infect bacteria; these viruses are called bacteriophages, or phages for short (see chapter 7). Phages are not alive; instead, they are just genes wrapped in a protective coat of protein and/or membrane, as are all viruses. Because phages are not alive, they cannot multiply outside a bacterial cell. However, if a phage encounters a type of bacterial cell that is sensitive to that phage, the phage, or at least its DNA or RNA, enters the cell and directs it to make more phage.
Phages are usually identified by the holes, or plaques, they form in layers of sensitive bacteria. In fact, the name “phage” (Greek for “eat”) derives from these plaques, which look like eaten-out areas. A plaque can form when a phage is mixed with large numbers of susceptible bacteria and the mixture is placed on an agar plate. As the bacteria multiply, one bacterial cell may be infected by the phage, which multiplies and eventually breaks open, or lyses, the bacterium, releasing more phage. As the surrounding bacteria are infected, the phage spread, even as the bacteria multiply to form an opaque layer called a bacterial lawn. Wherever the original phage infected the first bacterium, the plaque disrupts the lawn, forming a clear spot on the agar. Despite its empty appearance, this spot contains millions of the phage.
Phages offer many of the same advantages for genetics as bacteria. Thousands or even millions of phages can be put on a single plate. Also, like bacterial colonies, each plaque contains millions of genetically identical phage. By analogy to the colony purification of bacterial strains, individual phage mutants or strains can be isolated from other phages through plaque purification.
Phages Are Haploid
Phages are, in a sense, haploid, since they usually have only one copy of each gene. As with bacteria, this property makes isolation of phage mutants relatively easy, since all mutants immediately exhibit their phenotypes without the need for backcrosses.
Selections with Phages
Selection of rare strains of a phage is possible; as with bacteria, it requires conditions under which only the desired phage strain can multiply to form a plaque. For phages, these selective conditions may be a bacterial host in which only the desired strain can multiply or a temperature at which only the phage strain being selected can multiply. Note that the bacterial host must be able to multiply under the same selective conditions; otherwise, a plaque cannot form.
As with bacteria, selections allow the isolation of very rare strains or mutants. If selective conditions can be found for the strain, mill ions of phages can be mixed with the bacterial host, and only the desired strain multiplies to form a plaque. A pure strain can then be obtained by picking the phage from the plaque and purifying the phage strain under the same selective conditions.
Crosses with Phages
Phage strains can be crossed very easily. The same cells are infected with different mutants or strains of the phage. The DNA of the two phages is then in the same cell, where the molecules can interact genetically with each other, allowing genetic manipulations, such as genemapping and allelism tests.
A Brief History of Bacterial Molecular Genetics
Because of the ease with which they can be handled, bacteria and their phages have long been the organisms of choice for understanding basic cellular phenomena, and their contributions to this area of study are enormous. The following chronological list should give a feeling for the breadth of these contributions and the central position that bacteria have occupied in the development of modern molecular genetics. Some original references are given at the end of the chapter under Suggested Reading.
Inheritance in Bacteria
In the early part of the 1900s, biologists agreed that inheritance in higher organisms follows Darwinian principles. According to Charles Darwin, changes in the hereditary properties of organisms occur randomly and are passed on to the progeny. In general, the changes that happen to be beneficial to the organism are more apt to be passed on to subsequent generations.
With the discovery of the molecular basis for heredity, Darwinian evolution now has a strong theoretical foundation. The properties of organisms are determined by the sequence of their DNA, and as the organisms multiply, changes in this sequence sometimes occur randomly and without regard to the organism’s environment. However, if a random change in the DNA happens to be beneficial in the situation in which the organism finds itself, the organism has an improved chance of surviving and reproducing.
As late as the 1940s, many bacteriologists thought that inheritance in bacteria was different from inheritance in other organisms. It was thought that rather than enduring random changes, bacteria could adapt to their environment by some sort of “directed” change and that the adapted organisms could then somehow pass on the change to their offspring. Such opinions were encouraged by the observations of bacteria growing under selective conditions. For example, in the presence of an antibiotic, all the bacteria in the culture soon become resistant to the antibiotic. It seemed as though the resistant bacterial mutants appeared in response to the antibiotic.
One of the first convincing demonstrations that inheritance in bacteria follows Darwinian principles was made in 1943 by Salvador Luria and Max Delbrück (see chapter 3 and Suggested Reading). Their work demonstrated that particular phenotypes, in their case resistance to a virus, occur randomly in a growlng population, even in the absence of the virus. By the directed-change or adaptive-mutation hypothesis, the resistant mutants should have appeared only in the presence of the virus.
The demonstration that inheritance in bacteria follows the same principles as inheritance in eukaryotic organisms set the stage for the use of bacteria in studies of basic genetic principles common to all organisms.