MLS 321: BASIC MICROBIOLOGY

BACTERIAL GENETICS by Dr. Ezeumeh E.N

INTRODUCTION
Bacterial genetics is the subfield of genetics that studies the gene and genetic materials, that is
information stored in the gene; how the information is expressed and transferred, either from a
particular bacterium to its offspring or between interbreeding lines of bacteria, and how the
genetic information (genotype) determines the physiology of the bacterium (phenotype)
otherwise known as heredity and variation. Occasionally genetic variation or the transfer of
genetic information between bacteria gives rise to mutations of the original DNA. The large sizes
of bacterial populations ensure that even extremely rare genetic events are likely to occur. This
genetic variation makes it possible for individual members of huge populations of bacteria to
quickly evolve new traits. In the laboratory, genetic variation is exploited to study the properties
of bacteria, to explore the fundamental characteristics of gene transfer and gene expression, and
to construct mutants with desired characteristics. A hallmark of bacterial genetics is the ability to
analyze very large populations of cells to identify rare genetic events. The essential techniques
of bacterial genetics include the isolation of mutations, the ability to transfer genes
between bacterial strains, the ability to isolate recombinants, and the ability to do
complementation tests. These tools or techniques have been finely tabled for a select group of
model bacteria, including E. coli, Salmonella enterica and Bacillus subtilis. The concepts
developed for these model bacteria are readily applicable to other bacteria as well, although the
experimental details typically require adaptation for each particular species of bacteria

Fundamentals of Genetic Study

Nuclei acid is the blue print of genetics. It comprises of deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) molecules. A gene is defined as the unit of inheritance and a subset of
chromosome (usually circular in bacteria). The total chromosomal structure or gene in an
organism is referred to as the genome. DNA plays crucial roles in cell division as well as
reproduction. The central dogma of molecular biology (in which genetics is a branch of study),
is the process which explains how genetic information flows from DNA to RNA to protein. The
theory (central dogma) emphases the unidirectional flow of information from DNA to protein
synthesis. This involves replication (duplication) of the existing DNA double strands to form
two new but conserved daughter DNA double strands facilitated by the activities of the enzymes,
DNA polynerases. One of the two DNA strands serves as the template strand while the other
strand serves as the coding strand. The template strand synthesizes RNA in the process known
as RNA ‘transcription’. This process is facilitated by the enzyme, RNA polymerase. The
arrangement of the four nitrogenous bases, adenine (A), cytosine (C), guanine (G), and thymine
(T) within the DNA structure determines basis of genetic information known as genetic code.
During RNA transcription one of the bases, Thymine is substituted with Uracil (U). The reading
and transcribing of the DNA template strand gives rise to a single strand known as RNA
transcript. Finally, the RNA transcript is used as a precursor during gene expression to synthesize

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protein in a process known as RNA translation. In a nutshell, DNA performs a wide range of
roles which include; replication, gene expression, control and regulation of gene expressions. On
the other hand, RNA performs the tasks of protein synthesis, gene expression, and cell signaling.

The Central Dogma

The central dogma was first proposed in 1957 by Francis Crick and James Watson. It states that :
DNA to RNA: DNA is copied into a messenger RNA (mRNA) molecule in the nucleus of a cell
RNA to protein: The mRNA is translated into a protein in the cytoplasm by ribosomes
Structure of DNA
The structure of DNA molecule is composed of two chains of nucleotides wound around each
other in the form of “double helix” and in antiparallel manner. Chains of different arrangements
of these DNA units give rise to chromosomes. The backbone of each strand of DNA comprises
of repeating units of deoxyribose and phosphate residue. Attached to the deoxyribose is purine
(AG) or pyrimidine (CT) base. Nucleic acids are large polymers consisting of repeating
nucleotide units
Each nucleotide contains one phosphate group, one deoxyribose sugar, and one purine or
pyrimidine base. In DNA the sugar is deoxyribose; in RNA the sugar is ribose. The double helix
is stabilized by hydrogen bonds between purine and pyrimidine bases on the opposite strands. A
on one strand pairs by two hydrogen bonds with T on the opposite strand, or G pairs by three
hydrogen bonds with C. The two strands of double-helical DNA are, therefore complementary.
Because of complementarity, double-stranded DNA contains equimolar amounts of purines (A +
G) and pyrimidines (T + C), with A equal to T and G equal to C, but the mole fraction of G + C
in DNA varies widely among different bacteria. Another difference between DNA and RNA is
that RNA contains uracil instead of the base thymine.

a. Deoxyribonucleic acid (DNA) b. Ribonucleic acid (RNA)

Fig 1: Structure of DNA and RNA

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Important Features of the Nucleic Acid
Sugar
Both DNA and RNA are built with a sugar backbone, but whereas the sugar in DNA is called
deoxyribose (left in image), the sugar in RNA is called simply ribose (right in image). The
‘deoxy’ prefix denotes that, whilst RNA has two hydroxyl (-OH) groups attached to its carbon
backbone, DNA has only one, and has a lone hydrogen atom attached instead. RNA’s extra
hydroxyl group proves useful in the process of converting genetic code into mRNAs that can be
made into proteins,whilst the deoxyribose sugar gives DNA more stability
Bases
The nitrogen bases in DNA are the basic units of genetic code, and their correct ordering and
pairing is essential to biological function. The four bases that make up this code are adenine (A),
thymine (T), guanine (G) and cytosine (C). Bases pair off together in a double helix structure,
these pairs being A and T, and C and G. RNA doesn’t contain thymine bases, replacing them
with uracil bases (U), which pair to adenine
Phosphate group: forms phosphodiester bond with OH of the adjacent nucleotide to form a rigid
backbone of the nucleotide strands.

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 Fig2: Nucleic acid in a chromosome strands

Differences between DNA and RNA

1. Function
DNA encodes all genetic information, and is the blueprint from which all biological life is
created. And that’s only in the short-term. In the long-term, DNA is a storage device, a biological
flash drive that allows the blueprint of life to be passed between generations. RNA functions as
the reader that decodes this flash drive. This reading process is multi-step and there are
specialized RNAs for each of these steps. Below, we look in more detail at the three most
important types of RNA.
2. Structure
DNA double helix means that the two-stranded structure of DNA structure is common
knowledge whereas RNA is in single-stranded format. The DNA double helix was promulgated
by two scientists, Francis Crick and James Watson. RNA can form into double-stranded
structures, such as during translation, when mRNA and tRNA molecules pair. DNA polymers are
also much longer than RNA polymers

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 Fig 3: Differences between DNA and RNA base pairing

3. Bases
The nitrogen bases in DNA are the basic units of genetic code, and their correct ordering and
pairing is essential to biological function. The four bases that make up this code are adenine (A),
thymine (T), guanine (G) and cytosine (C). Bases pair off together in a double helix structure,
these pairs being A and T, and C and G. RNA doesn’t contain thymine bases, replacing them
with uracil bases (U), which pair to adenine1.

DNA Replication
DNA replication is the process by which DNA makes a copy of itself. This happens during cell
division, making sure that each new cell receives the same genetic information as the parent
cellEach time a cell divides, its DNA is carefully copied, creating a new DNA molecule that is
passed to the new daughter cell. This is the process of DNA replication.
DNA replication involves three main steps: initiation, elongation and termination.

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1. Preparing for replication (known as initiation)
 The first step is to unwind the double helix structure of the DNA molecule, and ‘unzip’ the
strands. The separated strands will act as templates for making the new DNA.
 This is carried out by an enzyme called helicase, which breaks the hydrogen bonds holding the
base pairs of the 2 strands together.
 Separating the strands creates a ‘Y’ shape called a ‘replication fork’

2. DNA replication (known as elongation)

 The two strands are replicated in different ways, because they run in opposite directions to each
other.
 One is oriented in the 3’ to 5’ direction, towards the replication fork. This is the leading strand.
 The other is oriented away from the replication fork. This is the lagging strand.

3. Finishing the process (known as termination)

 Once all the bases are matched up (A with T, C with G), an enzyme called exonuclease strips
away the primers. The gaps where the primers were are then filled by more complementary
bases.
 A DNA polymerase proofreads the new strands to make sure there are no mistakes in the new
DNA sequence.
 Finally, an enzyme called DNA ligase seals the DNA back into two continuous double strands.
The DNA automatically winds itself back into a double helix.
 The result is 2 DNA molecules that each consist of one new and one old strand of DNA. This is
why DNA replication is described as semi-conservative – half of the chain is part of the original
DNA molecule, and half is brand new

Continuous replication of the leading strand

 To replicate the leading strand, a short piece of RNA called a primer (produced by an enzyme
called primase) binds to its 3’ end, marking the starting point for DNA synthesis.
 An enzyme called DNA polymerase binds to and ‘walks’ along the strand, towards the
replication fork.
 As it reads the DNA sequence, it adds complementary bases, pairing A with T, and C with G.
 This type of replication is called continuous.

Discontinuous replication of the lagging strand

Because the lagging strand runs in the opposite direction, the DNA polymerase can only copy
small lengths of it at one time.

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 Many RNA primers bind at various points along the lagging strand. The DNA polymerase reads
the DNA from these points and adds complementary base pairs – creating chunks of DNA called
Okazaki fragments. This is called discontinuous replication.
 The Okazaki fragments are then joined up to make one continuous sequence.

Gene Expression
Genetic information encoded in DNA is expressed by synthesis of specific RNAs and proteins,
and information flows from DNA to RNA to protein. The DNA-directed synthesis of RNA is
called transcription. Because the strands of double-helical DNA are antiparallel and
complementary, only one of the two DNA strands can serve as template for synthesis of a
specific mRNA molecule. Messenger RNAs (mRNAs) transmit information from DNA, and
each mRNA in bacteria functions as the template for synthesis of one or more specific proteins.
The process by which the nucleotide sequence of an mRNA molecule determines the primary
amino acid sequence of a protein is called translation

Genome Organization
The bacterial chromosome is a circular molecule of DNA that functions as a self-replicating
genetic element (replicon). Extrachromosomal genetic elements such as plasmids and
bacteriophages are nonessential replicons which often determine resistance to antimicrobial
agents, production of virulence factors, or other functions. The chromosome replicates
semiconservatively; each DNA strand serves as template for synthesis of its complementary
strand

Mutations/ Variations
The term “mutation” was coined by Hugo de Vries, which is derived from Latin word meaning
“to change”. Mutations are heritable changes in genotype that can occur spontaneously or be
induced by chemical or physical treatments. (Organisms selected as reference strains are called
wild type, and their progeny with mutations are called mutants.) The process of mutation is
called mutagenesis and the agent inducing mutations is called mutagen. Changes in the sequence
of template DNA (mutations) can drastically affect the type of protein end product produced. For
a particular bacterial strain under defined growth conditions, the mutation rate for any specific
gene is constant and is expressed as the probability of mutation per cell division. Spontaneous
mutation occurs naturally about one in every million to one in every billion divisions. Mutation
rates of individual genes in bacteria range from 10-2 to 10-10 per bacterium per division. Most
spontaneous mutations occur during DNA replication. Mechanisms of mutation a. Substitution of
a nucleotide: Base substitution, also called point mutation, involves the changing of single base
in the DNA sequence. This mistake is copied during replication to produce a permanent change.
If one purine [A or G] or pyrimidine [C or T] is replaced by the other, the substitution is called a
transition. If a purine is replaced by a pyrimidine or vice-versa, the substitution is called a
transversion. This is the most common mechanism of mutation. b. Deletion or addition of a

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nucleotide: deletion or addition of a nucleotide during DNA replication. When a transposon
(jumping gene) inserts itself into a gene, it leads to disruption of gene and is called insertional
mutation.

Transfer of Genetic Material

Sometimes when two pieces of DNA come into contact with each other, sections of each DNA
strand will be exchanged. This is usually done through a process called crossing over in which
the DNA breaks and is attached on the other DNA strand leading to the transfer of genes and
possibly the formation of new genes. Genetic recombination is the transfer of DNA from one
organism to another. The transferred donor DNA may then be integrated into the recipient's
nucleoid by various mechanisms. In the case of homologous recombination, homologous DNA
sequences having nearly the same nucleotide sequences are exchanged by means of breakage and
reunion of paired DNA segments. Genetic information can be transferred from organism to
organism through vertical transfer (from a parent to offspring) or through horizontal transfer
methods such as conjugation, transformation or transduction. Bacterial genes are usually
transferred to members of the same species but occasionally transfer to other species can also
occur. The mutations are transferred from one bacteria to another through horizontal
transmission. There are three different types of horizontal transmission for the transfer of genetic
information.

 Conjugation
 Transduction
 Transformation
Bacterial Conjugation
Conjugation is the method of transfer of genetic material from one bacteria to another placed in
contact. This method was proposed by Lederberg and Tatum. They discovered that the F-factor
can move between E.coli cells and proposed the concept of conjugation.

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 Fig 4: fig

Fig 4: Conjugation mechanism of gene transfer

There are various conjugal plasmids carried by various bacterial species. Conjugation is carried
out in several steps:

 Mating pair formation

 Conjugal DNA synthesis
 DNA transfer
 Maturation
Mechanism of Bacterial Conjugation
Bacterial conjugation involves the following steps:

Pilus Formation
The donor cells (F+ cells) form a sex pilus and begin contact with an F- recipient cell.

Physical Contact between Donor and Recipient Cell

The pilus forms a conjugation tube and enables direct contact between the donor and the
recipient cells.

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Transfer of F-Plasmid
The F-factor opens at the origin of replication. One strand is cut at the origin of replication, and
the 5’ end enters the recipient cell.

Synthesis of Complementary Strand

The donor and the recipient strand both contain a single strand of the F-plasmid. Thus, a
complementary strand is synthesized in both the recipient and the donor. The recipient cell now
contains a copy of F plasmid and becomes a donor cell.

Bacterial Transduction
Transduction is the process of transfer of genes from the recipient to the donor through
bacteriophage.

Fig5: Transfer by Transduction

Transduction is of two types:

 Generalized Transduction
 Specialized Transduction

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Generalized Transduction
In this type, the bacteriophage first infects the donor cells and begins the lytic cycle. The virus
then develops its components using the host cell machinery. The host cell DNA is hydrolyzed
into small fragments by the viral enzymes.

Small pieces of bacteria DNA is now integrated into viral genome. When the virus infects
another bacteria the DNA is transferred into it.

Specialized Transduction
In this, only a few restricted bacteria are transferred from donor to recipient bacteria. This is
carried out by temperate bacteriophage which undergoes the lysogenic cycle.

The virus enters the bacteria and integrates its genome within the host cell DNA. It remains
dormant and passes on from generation to generation. When the lysogenic cell is exposed to
some external stimulus, the lytic cycle begins.

The viral genome is induced in the host cell genome. Due to this, the phage genome sometimes
carries the bacterial genome with it and integrates it into the genome of the recipient cell. Here,
only the restricted genome has the possibility of entering the recipient cells.

Bacterial Transformation
Transformation is the process of DNA uptake by the bacteria from the surrounding environment.
The cells that have the ability to uptake DNA are known as competent cells. This process was
first reported in Streptococcus pneumonia by Griffith.

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Fig 6: Bacterial gene transformation

Bacterial Competence
Not all bacteria are capable of taking up DNA from the surrounding environment. Such bacteria
are made artificially competent. This is achieved by using chemicals and electrical pulses.

 Chemicals- The cells are chilled and made permeable in the presence of calcium
phosphate. They are then incubated with the DNA and provided with a heat shock
treatment that causes the DNA to enter the cells.
 Electroporation- The bacterial cells are subjected to electrical pulses to make them
permeable and cause the DNA to enter into cells.

How to Identify Transformed Cells?

The bacteria are grown on an agar medium with antibiotics to check for transformed cells. Only
the bacteria containing the antibiotic resistance gene will grow in the presence of antibiotics. The
cells that survive and grow are transformed cells. The others are non-transformed

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