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BACTERIAL GENETIC
MLS 315
ABUAD DEPARTMENT OF MEDICAL
LABORATORY SCIENCES
Overview
• Because a single type of molecule, DNA, is the
genetic material of all cellular organisms from
bacteria to humans, basic genetic phenomena
gene mutation, gene replication, and gene
recombination are much the same for all life forms.
• The prototypic organism used in microbial genetic
studies for the past fifty years is the enteric, gramnegative Escherichia coli
• An aspect of microbial genetics of great clinical
importance is the ability of bacteria to transfer
genes, especially genes for antibiotic resistance,
to other bacteria both within and between
species.
• Such transfer allows the flow of antibiotic
resistance genes from nonpathogenic bacterial
populations to pathogenic populations, with
potentially dire consequences for public health.
The Bacterial Genome
• The genome of an organism is defined as the
totality of its genetic material.
• For bacteria, the genome consists of a single
chromosome that carries all of the essential genes
and one or more varieties of plasmid that generally
carry nonessential genes.
– Let’s consider some important terms
• Chromosomes
• Plasmids
• Transposons
A. The chromosome
• All of the essential genes and many nonessential
genes of the bacterium are carried on a single,
long piece of circular, double-stranded DNA.
• This molecular structure is called the
chromosome•by analogy with the hereditycarriers of eukaryotic cells. Most bacteria have
chromosomes that contain 2000 to 4000 genes.
– Note that in bacteria they are
•
•
•
•
Circular
Contains and average of 3000 genes (3000kb)
One copy contained in each cell
Are highly folded in cells
B. Plasmids
• Is an extrachromosomal genetic element that occurs in many
bacterial strains.
• Plasmids are circular deoxyribonucleic acid (DNA) molecules
that replicate independently of the bacterial chromosome.
• They are not essential for the bacterium but may confer a
selective advantage.
• Two common classes of plasmids,
– colicinogenic (or Col ) factors, determines the production of
proteins called colicins, which have antibiotic activity and can kill
other bacteria.
– R factors, confers upon bacteria resistance to antibiotics.
• Some Col factors and R factors can transfer themselves from
one cell to another and thus are capable of spreading rapidly
through a bacterial population.
• A plasmid that is attached to the cell membrane or integrated
into the bacterial chromosome is called an episome.
• Plasmids are extremely valuable tools in the fields of molecular
biology and genetics, specifically in the area of genetic
engineering.
• They play a critical role in such procedures as gene cloning,
recombinant protein production (e.g., of human insulin), and
gene therapy research.
• Typically, bacteria contain small DNA circles (plasmids), which
range in size from 1.5 kilobase (kb) pairs to 120 kb pairs (less
than one-tenth the size of the bacterial chromosome).
• Plasmids replicate independently of the chromosome, and can
exist in the cell as one copy or as many copies.
• Plasmids can carry genes for toxins and for proteins that
promote the transfer of the plasmid to other cells, but usually
do not include genes that are essential for cell growth or
replication.
• Many plasmids contain mobile DNA sequences (transposons)
that can move between plasmids, and between plasmids and
the chromosome
• NB.
– The are circular in nature.
– Contains 5-100 genes (5 – 100 kB).
– There are 1 -20 pairs/cell.
C. Transposons
• These are mobile DNA sequences contained
in many plasmids that can move between
plasmids, and between plasmids and the
chromosome
• They are repository for many antibiotic
resistance genes, thus responsible for the
ability of some plasmids to integrate into the
chromosome.
Bacteriophage
• A bacteriophage (phage) is a virus that replicates
inside a bacterial cell;
• it consists of nothing more than a piece of nucleic
acid encapsulated in a protective protein coat.
• Depending on the phage, the nucleic acid can be
DNA or RNA, double-stranded or single-stranded,
and range in size from about 3000 bases (3 genes)
to about 200,000 bases (200 genes).
Bacteriophage
• The typical replicative cycle begins with
– attachment of the phage to receptors on the cell surface,
followed by
– injection of the nucleic acid into the bacterial cell, leaving
all or most of the protein outside the cell.
• [Note: This is in contrast to viral infection of vertebrate cells, in
which the entire virus is taken up by the cell, and its nucleic acid
released intracellularly.
– The phage nucleic acid takes over the cell's biosynthetic
machinery to replicate its own genetic material, and to
synthesize phage-specific proteins.
– When sufficient coat proteins and new phage DNA have
accumulated, these components self-assemble into mature
phage particles, with the DNA encapsulated by the phage
coat.
– Release of new phage particles is accomplished by a phagespecific enzyme (lysozyme) that dissolves the bacterial cell
wall.
• The number of phage particles in a sample can
be determined by a simple and rapid plaque
assay. If a single phage particle is immobilized in
a confluent bacterial lawn growing on a
nutrient agar surface, this phage, within a few
hours, will produce millions of progeny at the
expense of neighboring bacterial cells, leaving a
visible hole or plaque in the otherwise opaque
lawn
Classes of phage.
• A phage could classified as
– Virulent phage or
– Temperate phage.
• This classification as virulent or temperate
depending on the nature of their relationship to
the host bacterium.
• A. Virulent phage (results in a lytic cycle)
– Infection of a bacterium with a virulent phage inevitably results
in the death of the cell by lysis, with release of newly replicated
phage particles.
– Under optimal conditions, a bacterial cell infected with only one
phage particle can produce hundreds of progeny in twenty
minutes.
– [Note: Generally, phage that attack one bactrial species do not
attack other bacterial species.]
• B. Temperate phage (results in a lysogenic cycle)
– A bacterium infected with a temperate phage can have the same
fate as a bacterium infected with a virulent phage (lysis rapidly
following infection).
– However, an alternative outcome is also possible, namely,
• after entering the cell, the phage DNA, rather than replicating
autonomously, can fuse or integrate with the chromosome of the host
cell. In this state (prophage), the expression of phage genes is repressed
indefinitely by a protein (repressor) encoded within the phage genome.
No new phage particles are produced, the host cell survives, and the
phage DNA replicates as part of the host chromosome.
Lytic cycle
Lysogenic bacteria
• Lysogenic bacteria carry a prophage; the phenomenon is termed
lysogeny, and the bacterial cell is said to be lysogenized.
• Nonlysogenic bacteria can be made lysogenic by infection with a
temperate phage.
• The association of prophage and bacterial cell is highly stable,
but can be destabilized by various treatments, such as exposure
to ultraviolet light, that damage the host DNA.
• When DNA damage occurs,
–
–
–
–
–
repression of phage genes is lifted,
the prophage excises from the host chromosome,
replicates autonomously, and
produces progeny phage particles.
The host cell is lysed just as with a virulent phage.
• The emergence of the virus from its latent prophage state is
called induction.
• The acquisition by bacteria of properties due to the presence of
a prophage is called lysogenic conversion.
PLAQUE ASSAY
GENE TRANSFER
• Genes can be transferred from one bacterial cell to
another by three distinct mechanisms:
– conjugation,
– transduction, and
– transformation.
• Because the transferred DNA usually does not
contain an origin of replication, these genes will be
passed on to succeeding generations only if the
transferred DNA becomes incorporated into the
recipient chromosome, which has an origin of
replication.
A. Conjugation
• Conjugation is the process by which bacteria
transfer genes from one cell to another by cell-tocell contact.
• The donor (male) and recipient (female) cells must
have the proper genetic constitution to adhere to
each other, and form a cytoplasmic bridge
between the cells through which DNA can pass.
• Specifically, the process requires the presence on
the donor cell of hairlike projections called sex pili
that make contact with specific receptor sites on
the surface of the recipient cell.
• This contact results in the formation of a relatively
stable cell pair, and the initiation of DNA transfer.
• Conjugation in bacteria is a one way transfer of
genetic information.
• F Factor is a fertility factor possessed by the donor
strain. It is necessary to have this in order to transfer
genetic material.
• In its simplest form, the F factor is a plasmid, a small
circular DNA molecule, separate from the large
circular chromosome containing most genes.
Plasmids are autonomously replicating genetic
elements.
• F Factors are also episomes. Episomes can on
occasion integrate into the main chromosome.
• When normal F+ is crossed to F-, the only genetic
material usually transferred is the F factor itself.
• F+ types are able to synthesize a special "sex" pilus.
• Special F+ strains were found that transferred
chromosomal DNA from the F+ to the F-, but
only rarely transferred the F factor itself.
• It was found that the F factor plasmid had
integrated into the chromosome of the F+
strain forming what was called a Hfr for high
frequency type.
B. Transduction
• Transduction refers to transfer of genes from
one cell to another via a phage vector without
cell-to-cell contact.
• There are two ways in which this can occur:
– generalized transduction and
– specialized transduction.
• In each case, the transducing phage is a
temperate phage, so that the recipient cell
survives the phage infection.
• Generalized transduction:
– In generalized transduction, a random fragment of
bacterial DNA, resulting from phage-induced cleavage of
the bacterial chromosome, is accidentally encapsulated
in a phage protein coat in place of the phage DNA .
– When this rare phage particle infects a cell, it injects the
bacterial DNA fragment into the cell.
– If this fragment becomes integrated into the recipient
chromosome by recombination, the recipient cell will be
stably transduced.
– NB. Any gene from the bacterial genome can be
transferred due to phage induced cleavage.
• Specialized transduction:
– In specialized transduction, only certain bacterial genes,
located on the bacterial chromosome in close proximity to
the prophage insertion site of the transducing phage, are
transduced.
– The phage acquires the bacterial genes by a rare, abnormal
excision from the bacterial chromosome.
– In general, a specialized transducing phage particle contains
both phage and bacterial DNA joined together as a single
molecule.
– After infecting another cell, this joint molecule integrates
into the recipient chromosome just as phage DNA normally
does in the process of becoming a prophage.
– NB only certain bacterial gene close to the site of insertion
of phage can be transfered to other bacteria cells
• C. Transformation
– Transformation is the transfer of genes from one cell to another by
means of naked DNA.
– The discovery of transformation in 1928, one of the most important in
all of biology, led eventually to the identification of DNA as the genetic
material.
– Transformation process: Studies of the transformation phenomenon
itself revealed that the ability of a cell to be transformed (called
competence) depends on a transitory physiologic state of the cell that
allows DNA to cross the cell membrane.
– As free, double-stranded DNA enters the recipient cell, one of the two
strands is destroyed by nucleases.
– The remaining single strand invades the resident chromosome, seeking
a region of sequence homology.
– If such a sequence is found, the invading strand replaces one of the two
resident strands by a complex cut-and-paste process.
– Transformation probably has only a minor effect on gene flow in natural
bacterial populations, but it is useful experimentally for introducing a
cloned gene (for example, the human gene for insulin) into bacterial
cells.
• Thus, for transformation to occur, 2 things must exist.
1. the DNA must be competent.
2. the cell must be competent.
– Competent DNA implies it is large enough and it is doublestranded.
– A competent cell is one that has a competence factor, a cellsurface protein that is used to transport the DNA into the cell.
• How does it occur?
– Extracellular DNA is bound by CF protein.
– One of the strands is hydrolyzed to provide energy for transport.
– The other single-stranded DNA molecule is transported into the
cell.
– Crossing over between homologous regions occurs. It is not
reciprocal and only involves replacement of DNA on the existing
chromosomal strand.
– Of the bacteria that take up the DNA, only some will be
transformed.
Genetic Variation
• Although all of the cells in pure•bacterial culture
are derived from a single original cell, the
culture typically contains rare cells that differ
from the originating cell.
• The majority, if not all, of such variants
(mutants) are due to changes (mutations) in
their DNA.
Mutation
• Strictly speaking, any change in the structure of genetic
material or, more specifically, any change in the base
sequence of DNA, is called a mutation.
• Some mutations are unstable (that is, they frequently
revert back to their original state), and others do not
noticeably affect the organism.
• Mutations that come under study are usually those that
are stable, and that cause some change in the
characteristics of the organism.
• Mutations can be classified according to the kind of
chemical change that occurs in the DNA or, when the
mutation affects a protein-coding gene, by the effect
the mutation has on the translation of the message.
B. Mobile genetic elements
• In recent years, it has been recognized that the arrangement
of genes in the genome of bacteria and probably all
organisms is
not entirely static.
• Certain DNA segments, called transposons, have the ability
to move from place to place on the chromosome and into
and out of plasmids.
• Transposons do not exist as segments free of the genome
but only as segments within the genome.
• There are two general types of transposons:
• Replicative and
• Nonreplicative.
– A replicative transposon leaves a copy of itself at the original
location. Thus, the transposition process doubles the number of
copies of the transposon.
– A nonreplicative transposon does not leave a copy of itself at
the original location.
• If transposition inserts a transposon into a
functional gene, the function of the gene is
generally destroyed; this was the original basis by
which transposons were discovered.
• Transposons can thus be viewed as internal
mutagenic agents. The transposition process and
the structure of a typical replicative transposon are
shown in.
• Mobile genetic elements are probably responsible
for most of the genetic variability in natural
bacterial populations, and for the spread of
antibiotic resistance genes.
Mechanisms of acquired antibiotic resistance
• Acquired antibiotic resistance requires a temporary
or permanent gain or alteration of bacterial genetic
information.
• Most resistance genes are plasmid-mediated;
however, plasmid-mediated traits can interchange
with chromosomal elements.
• Transfer of genetic material from plasmid to
chromosome can occur by simple recombinational
events, but the process is greatly facilitated by
transposons.
• Many resistance genes, such as plasmid-mediated
β-lactamases, tetracycline-resistance genes, and
aminoglycoside-modifying enzymes, are organized
on transposons.
Resistance to antibiotics is accomplished by three principal mechanisms
• Decreased uptake (or increased efflux) of antibiotic: For
example, gram-negative organisms can limit the penetration of
certain agents, including β-lactam antibiotics, tetracyclines, and
chloramphenicol, as a result of alteration in the number and
structure of porins (proteins that form channels) in the outer
membrane.
• Alteration of the target site for antibiotic: For example,
Staphylococcus pneumoniae resistance to β-lactam antibiotics
involves alterations in one or more of the major bacterial
penicillin-binding proteins, which results in decreased binding of
the antibiotic to its target.
• Acquisition of the ability to destroy or modify the antibiotic:
Examples of antibiotic inactivating enzymes include:
– 1) β-lactamases that hydrolytically inactivate the β-lactam ring of
penicillins, cephalosporins, and related drugs;
– 2) acetyltransferases that transfer an acetyl group to the antibiotic,
inactivating chloramphenicol or aminoglycosides;
– 3) esterases that hydrolyze the lactone ring of macrolides.
MUTATION
• 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 nucleotides:
– This involves 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.
Results of mutation
• a. Missense mutation:
– Missense mutations are DNA mutations which lead to changes
in the amino acid sequence (one wrong codon and one wrong
amino acid) of the protein product.
– This could be caused by a single point mutation or a series of
mutations.
• b. Nonsense mutation:
– A mutation that leads to the formation of a stop codon is called
a nonsense mutation.
– Since these codon cause the termination of protein synthesis, a
nonsense mutation leads to incomplete protein products.
• c. Silent mutation:
– Sometimes a single substitution mutation change in the DNA
base sequence results in a new codon still coding for the same
amino acid.
– Since there is no change in the product, such mutations are
called silent.
• d. Frameshift mutation:
– Frameshift mutations involve the addition or deletion of base
pairs causing a shift in the “reading frame” of the gene.
– This causes a reading frame shift and all of the codons and all of
the amino acids after that mutation are usually wrong.
– Since the addition of amino acids to the protein chain is
determined by the three base codons, when the overall
sequence of the gene is altered, the amino acid sequence may
be altered as well.
e. Lethal mutation: Sometimes some mutations affect vital
functions and the bacterial cell become nonviable. Hence those
mutations that can kill the cell are called lethal mutation.
f. Suppressor mutation: It is a reversal of a mutant phenotype
by another mutation at a position on the DNA distinct from that
of original mutation. True reversion or back mutation results in
reversion of a mutant to original form, which occurs as a result
of mutation occurring at the same spot once again.
Example of frame shift.
•
•
Mutations can also occur in which nucleotide base pairs are inserted into or deleted
from the original gene sequence. This type of gene mutation is dangerous because it
alters the template from which amino acids are read. Insertions and deletions can
cause frame shift mutations when base pairs that are not a multiple of three are
added to or deleted from the sequence. Since the nucleotide sequences are read in
groupings of three, this will cause a shift in the reading frame. For example, if the
original transcribed DNA sequence is CGA CCA ACG GCG ..., and two base pairs (GA)
are inserted between the second and third groupings, the reading frame will be
shifted.
Original Sequence:
CGA-CCA-ACG-GCG...
•
Amino Acids Produced: Arginine - Proline - Threonine - Alanine ...
•
Inserted Base Pairs (GA): CGA-CCA-GAA-CGG-CG...
•
•
Amino Acids Produced: Arginine - Proline - Glutamic Acid - Arginine ...
The insertion shifts the reading frame by two and changes the amino acids that are
produced after the insertion. The insertion can code for a stop codon too soon or too
late in the translation process. The resulting proteins will be either too short or too
long. These proteins are for the most part defunct.
• g. Conditional lethal mutation: Sometimes a
mutation may affect an organism in such a way
that the mutant can survive only in certain
environmental condition. Example; a temperature
sensitive mutant can survive at permissive
temperature of 35oC but not at restrictive
temperature of 39oC.
h. Inversion mutation: If a segment of DNA is
removed and reinserted in a reverse direction, it
is called inversion mutation.
• Based on extent of base pair changes,
mutations can be of two types; microlesion
and macrolesion. Microlesions are basically
point mutations (affecting single base pairs)
whereas macrolesions involve addition,
deletion, inversion or duplication of several
base pairs.
• The mutations in DNA can occur spontaneously or can be
caused by an external force or substance called a mutagen.
Mutagens can be chemicals such as nitrous acid, which
alters adenine to pair with cytosine instead of thymine.
Other chemical mutagens include acridine dyes, nucleoside
analogs that are similar in structure to nitrogenous bases,
benzpyrene (from smoke and soot) and aflatoxin. Radiation
can also be a cause of DNA mutations. High energy light
waves such as X-rays, gamma rays, and ultraviolet light have
been shown to damage DNA. UV light is responsible for the
formation of thymine dimers in which covalent links are
established between the thymine molecules. These links
change the physical shape of the DNA preventing
transcription and replication.
Polycytic kidney due to mutation
Replica plate
Replica plate
Mutagens
• A mutation can be the result of different events.
• Errors made during replication, repair, or
recombination can all lead to point or frameshift
mutations. Mutations resulting from such errors
are spontaneous mutations.
• A mutation can also result from the action of
physical and chemical agents known as
mutagens. We will now explore three mutagens:
nitrous acid, base analogs, and UV light.
• Nitrous Acid:
– Nitrous Acid affects DNA complementation.
– The acid randomly modifies the base adenine so that it will pair with cytosine instead
of thymine.
– This change is made evident during DNA replication when a new base pair appears in
daughter cells in a later generation.
• A Base Analog:
– A base analog is a compound sufficiently similar to one of the four DNA bases but
have different pairing properties.
– For example, 5-bromouracil is the analog of thymine but sometimes pairs with
guanine and 2-aminopurine is the analog of adanine but sometimes pairs with
cytosine.
– The incorporation of a base analog will to a base pair substitution in that appears in
daughter cells in a later generation.
• UV Light:
– Exposure to direct UV light induces covalent linking between adjacent thymine
nucleotides on a DNA strand forming a thymine dimer.
– These dimers cause the strand to buckle, disrupting normal base pairing. This
prevents proper replication and transcription.
– Bacteria have enzymes to fix the damage created by UV light.
– An enzyme cuts the DNA at two point and removes the damaged portion.
– DNA polymerase synthesizes a new DNA segment using the healthly strand as a
template.
– DNA ligase joins the new fragment to the old strand.
• Significance of mutation:
• Discovery of a mutation in a gene can help in
identifying the function of that gene.
• Mutations can be induced at a desired region to
create a suitable mutant, especially to produce
vaccines.
• Spontaneous mutations can result in emergence
of antibiotic resistance in bacteria.
• Mutations can result in change in phenotype such
as appearance of novel surface antigen, alternation
in physiological properties, change in colony
morphology, nutritional requirements, biochemical
reactions, growth characteristics, virulence and
host range.
Mechanisms of acquired antibiotic resistance
• Acquired antibiotic resistance requires a temporary
or permanent gain or alteration of bacterial genetic
information.
• Most resistance genes are plasmid-mediated;
however, plasmid-mediated traits can interchange
with chromosomal elements.
• Transfer of genetic material from plasmid to
chromosome can occur by simple recombinational
events, but the process is greatly facilitated by
transposons.
• Many resistance genes, such as plasmid-mediated
β-lactamases, tetracycline-resistance genes, and
aminoglycoside-modifying enzymes, are organized
on transposons.
Resistance to antibiotics is accomplished by three principal mechanisms
• Decreased uptake (or increased efflux) of antibiotic: For
example, gram-negative organisms can limit the penetration of
certain agents, including β-lactam antibiotics, tetracyclines, and
chloramphenicol, as a result of alteration in the number and
structure of porins (proteins that form channels) in the outer
membrane.
• Alteration of the target site for antibiotic: For example,
Staphylococcus pneumoniae resistance to β-lactam antibiotics
involves alterations in one or more of the major bacterial
penicillin-binding proteins, which results in decreased binding of
the antibiotic to its target.
• Acquisition of the ability to destroy or modify the antibiotic:
Examples of antibiotic inactivating enzymes include:
– 1) β-lactamases that hydrolytically inactivate the β-lactam ring of
penicillins, cephalosporins, and related drugs;
– 2) acetyltransferases that transfer an acetyl group to the antibiotic,
inactivating chloramphenicol or aminoglycosides;
– 3) esterases that hydrolyze the lactone ring of macrolides.
• Significance of mutation:
• Discovery of a mutation in a gene can help in
identifying the function of that gene.
• Mutations can be induced at a desired region to
create a suitable mutant, especially to produce
vaccines.
• Spontaneous mutations can result in emergence of
antibiotic resistance in bacteria.
• Mutations can result in change in phenotype such as
appearance of novel surface antigen, alternation in
physiological properties, change in colony morphology,
nutritional requirements, biochemical reactions,
growth characteristics, virulence and host range.