Unit 2: Bacteriology
66
Lecture 4 - The Molecular Basis of
Bacterial Genetics
The Structure of Bacterial DNA
A bacterium’s genetic information is stored in its
chromosome and plasmids. Each of these structures is
made of a single DNA double helix twisted to the right,
then additionally twisted to the left about its helical axis
(supercoiled), Plasmids consisting of linear DNA also
occur, although this is rare.
Chromosome.
The chromosome corresponds to the nucleoid . The gene
sequence is colinear with the expressed genetic products.
The noncoding interposed sequences (introns) normally
seen in eukaryotic genes are very rare.
Plasmids
The plasmids are autonomous DNA molecules of varying
size (3 X 103 to 4.5 X 105 bp) localized in the cytoplasm.
Large plasmids are usually present in one to two copies
per cell, whereas small ones may be present in 10, 40, or
100 copies. Plasmids are not essential to a cell’s survival.
Many of them carry genes that code for certain
phenotypic characteristics of the host cell. The following
plasmid types are medically relevant:
▪
Virulence plasmids.
Carry determinants of bacterial
virulence, e.g., enterotoxin genes or hemolysin genes.
▪
Resistance plasmids. Carry genetic information bearing
on resistance to anti-infective agents. R plasmids may
carry several R genes at once . Plasmids have also been
described that carry both virulence and resistance genes.
DNA Replication
The identical duplication process of DNA is termed
semiconservative because the double strand of DNA is
opened up during replication, where upon each strand
serves as the matrix for synthesis of a complementary
strand. Thus each of the two new double strands
“conserves” one old strand. The doubling of each DNA
molecule (replicon) begins at a given starting point, the
so-called origin of replication. This process continues
throughout the entire fission cycle.
Transcription and Translation
Transcription
. Copying of the sense strand of the DNA
into mRNA. The continuous genetic nucleotide sequence
is transcribed “colinearly” into mRNA. This principle of
colinearity applies with very few exceptions. The
transcription process can be broken down into the three
phases promoter recognition, elongation, and
termination. The promoter region is the site where the
RNA polymerase begins reading the DNA sequence. A
sigma factor is required for binding to the promoter.
Sigma factors are proteins that associate temporarily with
the RNA polymerase (core enzyme) to form a
holoenzyme, then dissociate themselves once the
transcription process has begun, making them available to
associate once again. The mRNA synthesized by the
transcription of an operon is polycistronic, i.e., it contains
the information sequences of several genes.
Translation
. Transformation of the nucleotide sequence
carried by the mRNA into the polypeptide amino acid
sequence at the 70S ribosomes. In principle, bacterial and
eukaryotic translation is the same. The enzymes and other
factors involved do, however, differ structurally and can
therefore be selectively blocked by antibiotics
.
The Genetic Variability of Bacteria
Changes in bacterial DNA are the result of spontaneous
mutations in individual genes as well as recombination
processes resulting in new genes or genetic combinations.
Based on the molecular mechanisms involved, bacterial
recombinations are classified as homologous, site-
specific, and transpositional.
The latter two in particular reflect the high level of
mobility of many genes and have made essential
contributions to the evolution of bacteria. Although sexual
heredity is unknown in bacteria, they do make use of the
mechanisms of intercellular transfer of genomic material
known as parasexual processes.
Transformation designates transfer of DNA that is
essentially chemically pure from a donor into a receptor
cell. In transduction, bacteriophages serve as the vehicles
for DNA transport.
Unit 2: Bacteriology
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Conjugation is the transfer of DNA by means of cell-to-
cell contact. This process, made possible by conjugative
plasmids and transposons, can be a high-frequency one
and may even occur between partners of different species,
genera, or families. The transfer primarily involves the
conjugative elements themselves. Conjugative structures
carrying resistance or virulence genes are of
considerable medical significance.
The processes of restriction and modification are
important factors limiting genetic exchange among
different taxa. Restriction is based on the effects of
restriction endonucleases capable of specific excision of
foreign DNA sequences. These enzymes have become
invaluable tools in the field of genetic engineering.
Molecular Mechanisms of Genetic Variability
Spontaneous Mutation
In the year 1943, Luria and Delbru¨ ck used the so-
called fluctuation test to demonstrate that changes in the
characteristics of bacterial populations were the results of
rare, random mutations in the genes of individual cells,
which then were selected. Such mutations may involve
substitution of a single nucleotide, frame-shifts,
deletions, inversions, or insertions. The frequency of
mutations is expressed as the mutation rate, which is
defined as the probability of mutation per gene per cell
division. The rate varies depending on the gene involved
and is approximately 10-6 to 10-10. Mutation rates may
increase drastically due to mutagenic factors such as
radioactivity, UV radiation, alkylating chemicals, etc.
Recombination
The term recombination designates processes that lead
to the restructuring of DNA, formation of new genes or
genetic combinations.They may be Homologous
(generalized) recombination, Site-specific
recombination,or Transposition.
Intercellular Mechanisms of Genetic Variability
Although bacteria have no sexual heredity in the strict
sense, they do have mechanisms that allow for
intercellular DNA transfer. These mechanisms, which
involve a unilateral transfer of genetic information from a
donor cell to a receptor cell, are subsumed under the term
parasexuality.
Transformation.
Transfer of “naked” DNA.
Transduction.
Transfer of DNA from a donor to a
receptor with the help of transport bacteriophage
.Bacteriophages are viruses that infect bacteria.
Bacterial gene-transfer mechanism
T bacteriophage
Conjugation.
Conjugation is the transfer of DNA from a
donor to a receptor in a conjugal process involving cell-
to-cell contact. Conjugation is made possible by two
genetic elements: the conjugative plasmids and the
conjugative transposons.
Unit 2: Bacteriology
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Schematic drawing of bacterial conjugation.
Conjugation diagram 1-Donor cell produces
. 2-
Pilus attaches to recipient cell and brings the two cells
together. 3- The mobile plasmid is nicked and a single
strand of DNA is then transferred to the recipient cell. 4-
Both cells synthesize a complementary strand to produce
a double stranded circular plasmid and also reproduce
pili; both cells are now viable donors.
The F-factor in Escherichia coli
. This is the prototype of
a conjugative plasmid. This factor contains the so-called
tra (transfer) genes responsible both for Conjugative
resistance and virulence plasmids. Conjugative
plasmids that carry determinants coding for antibiotic
resistance and/or virulence in addition to the tra genes
and repA are of considerable medical importance.
Restriction, Modification, and Gene Cloning .
A number of control mechanisms limit these genetic
exchange processes. Among the most important are
restriction and modification. Restriction endonucleases
can destroy foreign DNA that bears no “fingerprint”
(modification) signifying “self.” These modifications take
the form of methylation of the DNA bases by
modification enzymes. Bacterial restriction endonucleases
are invaluable tools in modern gene cloning techniques.
The process is termed gene “cloning” because it involves
replication of DNA that has been manipulated in vitro in a
suitable host cell so as to produce identical copies of this
DNA: molecular clones or gene clones The technique
simplifies the replication of DNA, making experimental
manipulations easier. On the other hand, the bacteria can
also be used to synthesize gene products of the foreign
genes. Such foreign proteins are called recombinant
proteins. Bacterial plasmids often function in the role of
vectors into which the sequences to be cloned are
inserted.
Summary:
Bacteria possess two genetic structures: the chromosome
and the plasmid. Both of these structures consist of a
single circular DNA double helix twisted
counterclockwise about its helical axis. Replication of this
DNA molecule always starts at a certain point (the origin
of replication) and is “semiconservative,” that is, one
strand in each of the two resulting double strands is
conserved. Most bacterial genes code for proteins
(polypeptides). Noncoding
interposed sequences (introns), like those seen in
eukaryotes, are the exception. Certain bacterial genes
have a mosaic structure. The phases of transcription are
promoter recognition, elongation, and termination. Many
bacterial mRNAs are polycistronic, meaning they contain
the genetic information for several polypeptides.
Translation takes place on the 70S ribosomes. Special
mRNA codons mark the start and stop of polypeptide
synthesis. Many genes that code for functionally related
polypeptides are grouped together in chromosome or
plasmid segments known as operons. The most important
regulatory
mechanism is the positive or negative control of
transcription initiation. This control function may be
exercised by individual localized genes, the genes of an
operon or genes in a regulon.