In
bacterial genetics, genetic techniques are used to study bacteria. Applying
genetic analysis to bacteria is no dif-ferent 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.
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 homolo- gous 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 some 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 or- ganism 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 lower 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 trans-
planting a somatic cell into the ovary, bacteria form clones of themselves
every time they divide.
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 multi- plication 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 smaller dilutions are repeated in succession,
they can be multiplied to produce the total dilution.
For ex- ample, if a solution is diluted in three steps
by adding 1 ml of the solution to 99 ml of water, followed
by add- ing 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 102
x 102 x 102 = 106,
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 solu- tions 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
where 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 be-
ing 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 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 billions 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 organ- isms requires continuous 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 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 un- til the cells are needed, at which time they can be revived.
Genetic Exchange
Genetic
experiments with an organism usually require some form
of exchange of DNA or genes between mem- bers 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 re- leased from one cell enters another cell of the same species. In conjugation, plasmids, which are small autonomously replicating DNA molecules in bacterial cells, transfer DNA from one cell to another. Finally, in transduction, a bacterial virus accidentally picks up DNA from a cell it has infected and injects this DNA into another cell. 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.
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