BACTERIAL GENETICS

Lecture # 01: Introduction

Genetics
• Genetics is the branch of science concerned with genes, heredity, and
variation in living organisms.

• Genetics can be simply defined as the manipulation of DNA to study

cellular and organismal functions.

• Since DNA encodes all of the information needed to make the cell and
the complete organism, the effects of changing this molecule can give
clues to the normal functions of the cell and organism.
Genetics (contd.)
• Before the advent of methods for manipulating DNA in the test tube,
the only genetic approaches available for studying cellular and
organismal functions were those of classical genetics.

• In this type of analysis, mutants (i.e., individuals that differ from the
normal, or wild-type, members of the species by a certain observable
attribute, or phenotype) that have alterations in the function being
studied are isolated.
Genetics (contd.)

• The changes in the DNA, or mutations, responsible for the altered

function are then localized in the chromosome by genetic crosses.

• The development of molecular genetic techniques has greatly

expanded the range of methods available for studying genes and their
functions.
Genetics (contd.)
• These techniques include

• methods for isolating DNA and

• identifying the regions of DNA that encode particular functions,

• as well as methods for

• altering or mutating DNA in the test tube and

• then returning the mutated DNA to cells to determine the effect of the
mutation on the organism.
Genetics (contd.)

• The approach of first cloning a gene and then altering it in the test
tube before reintroducing it into the cells to determine the effects of
the alterations is sometimes called reverse genetics and is essentially
the reverse of a classical genetic analysis.

• In classical genetics, a gene is known to exist only because a mutation

in it has caused an observable change in the organism.
Genetics (contd.)
• 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.
Bacterial Genetics
• Applying genetic analysis to bacteria is no 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.
Bacterial Genetics: 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.
Bacterial Genetics: Bacteria are Haploid
(contd.)
• 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.
Bacterial Genetics: 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.

• Some strains of the bacterium E. coli can reproduce every 20 minutes

under ideal conditions.
Bacterial Genetics: 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.
Bacterial Genetics: Asexual Reproduction
(contd.)
• 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.

Bacterial Genetics: Colony Growth

• 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, millions, or even billions of

individuals can be screened on a single agar-containing petri plate.
Bacterial Genetics: Colony Growth (contd.)

• 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 the

original bacterium and hence all clones of the original bacterium.
Bacterial Genetics: Colony Purification

• 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.
Bacterial Genetics: Colony Purification
(contd.)
• If the colonies are picked and the bacteria are diluted before re-
plating, 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.
Bacterial Genetics: 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.
Bacterial Genetics: Serial Dilutions (contd.)

• 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.
Bacterial Genetics: Serial Dilutions (contd.)

• For example, if a solution is diluted in three steps

• by adding 1 ml of the solution to 99 ml of water, 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 × 10−2 × 10−2 = 10−6, or one in a million.

Bacterial Genetics: Serial Dilutions (contd.)

• To achieve the same dilution in a single step, 1 ml of the original

solution would have to be added to 1,000 liters of water.

• Obviously, it is more convenient to handle three solutions of 100 ml

each than to handle a solution of 1,000 L, which weighs about 2,000
lb.
Bacterial Genetics: 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.
Bacterial Genetics: Selections (contd.)

• 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.
Bacterial Genetics: Selections (contd.)

• 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.
Bacterial Genetics: Storing Stocks

• Most types of organisms must be continuously propagated;

otherwise, they age and die.

• Propagating organisms 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.
Bacterial Genetics: Storing Stocks (contd.)

• 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.

• The strains remain dormant until the cells are needed, at which time
they can be revived.
Bacterial Genetics: 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.
Bacterial Genetics: Genetic Exchange (contd.)
• Exchange of DNA from one bacterium to another can occur in one of
three ways.

• In transformation, DNA released from one cell (‘free’ in the

environment) 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.
Bacterial Genetics: Genetic Exchange (contd.)

• 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, as well as the tests essential to genetic
analysis.