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Chapter 8
Bacterial genetics
9.1 Mutations and Mutants
9.2 Genetic Recombination
9.3 Genetic Transformation
9.4 Transduction
9.5 Conjugation
9.6 Plasmids
9.7 Transposons and Insertion Sequences
9.8 Comparative Prokaryotic Genomics
9.9 Genetics in Eukaryotic Microorganisms
• Microorganisms provide relatively simple systems
for studying genetic phenomena and are thus
useful tools in attempts to decipher the
mechanisms underlying the genetics of all
organisms.
• Microorganisms are used for the isolation and
duplication of specific genes from other organisms,
a technique called molecular cloning. In molecular
cloning, genes are manipulated and placed in a
microorganism where they can be induced to
increase in number.
Section 1. Mutation and recombination
Mutation is an inherited change in the base sequence
of the nucleic acid comprising the genome of an
organism. Mutation usually brings about only a very
small amount of genetic change in a cell.
Genetic recombination is the process by which genetic
elements contained in two separate genomes are brought
together in one unit. This mechanism may enable the
organism to carry out some new function and result in
adaptation to changing environments. Genetic
recombination usually involves much larger changes.
Entire genes, sets of genes, or even whole chromosomes,
are transferred between organisms.
Section 2.
Techniques of bacterial genetics: in vivo
In vivo : manipulate the genetic material within the organism
Genetic Transformation
Transduction
Conjugation
Section 3. Techniques
of bacterial genetics: in vitro
In vitro: operate genetic material in the test tube
Restriction Enzymes
Molecular Cloning
Amplifying DNA: PCR
WORKING GLOSSARY
Auxotroph an organism that has developed a nutritional
requirement through mutation
Cloning vector genetic element into which genes can be
recombined and replicated
Conjugation transfer of genes from one prokaryotic cell to another
by a mechanism involving cell-to-cell contact and a plasmid
Diploid a eukaryotic cell or organism containing two sets of
chromosomes
Electroporation the use of an electric pulse to induce cells to take
up free DNA
Gene disruption use of genetic techniques to inactivate a gene by
inserting within it a DNA fragment containing an easily selectable
marker. The inserted fragment is called a cassette, and the process of
insertion, cassette mutagenesis
Genetic map the arrangement of genes on a chromosome
Genome the total complement of genes of a cell or a virus
Genotype the precise genetic makeup of an organism
Hybridization formation of a duplex nucleic acid molecule
with strands derived from different sources by
complementary base pairing
Molecular cloning isolation and incorporation of a
fragment of DNA into a vector where it can be replicated
Haploid a cell or organism that has only one set of
chromosomes
Mutagens agents that cause mutation
Mutant an organism whose genome carries a mutation
Mutation an inheritable change in the base sequence of the
genome of an organism
Nucleic acid probe a strand of nucleic acid that can be
labeled and used to hybridize to a complementary molecule
from mixture of other nucleic acids
Phenotype the observable characteristics of an organism
Plasmid an extrachromosomal genetic element that has no
extracellular form
Point mutation a mutation that involves one or only a very
few base pairs
Polymerase chain reaction (PCR) a method used to
amplify a specific DNA sequence in vitro by repeated cycles
of synthesis using specific primers and DNA polymerase
Recombination the process by which parts or all of the
DNA molecules from two separate sources are exchanged or
brought together into a single unit.
Restriction enzyme an enzyme that recognizes and makes
double-stranded breaks at specific DNA sequences
Shotgun cloning making a gene library by closing random
DNA fragments
Site-directed mutagenesis a technique whereby a gene
with a specific mutation can be constructed in vitro
Synthetic DNA a DNA molecule made by a chemical
process in a laboratory
Transduction transfer of host genes from one cell to
another by a virus
Transformation transfer of bacterial genes involving free
DNA
Transposon a type of transposable element that carries
genes in addition to those involved in transposition
Genetic Recombination
Genetic recombination involves the physical exchange
of genetic material between genetic elements.
Homologous recombination results in genetic exchange
between homologous DNA sequences from two different
sources. This type of recombination is extremely
important to all organisms. However, it is also very
complex. Even in the bacterium Escherichia coli there
are at least 25 genes involved.
A simplified version of one molecular mechanism
of recombination. Homologous DNA molecules
pair and exchange DNA segments.
The mechanism involves breakage and
reunion of paired segments. Two of the
proteins involved, a single-stranded
binding (SSB) protein and the RecA
protein.
Note that there are two possible
outcomes, depending on which strands
are cut during the resolution process. In
one outcome the recombinant molecules
have patches, whereas in the other the
two parental molecules appear to have
been cut and then spliced together.
Detection of Recombination
In order to detect physical exchange of DNA segments, the
cells resulting from recombination must be phenotypically
different from the parents.
Strains that lack some selectable characteristic that the
recombinants will possess. For instance, the recipient may
not be able to grow on a particular medium, and genetic
recombinants are selected that can.
Various kinds of selectable and nonselectable markers (such
as drug resistance, nutritional requirements, and so on) may
be used.
Complementation
This can be determined by a type of experiment called a
complementation test.
Complementation was first used in diploid eukaryotic
organisms
Genetic Transformation
In prokaryotes genetic recombination is observed because
fragments of homologous DNA from a donor chromosome
are transferred to a recipient cell by one of three processes:
(1) transformation, which involves donor DNA free in the
environment
(2) transduction, in which the donor DNA transfer is
mediated by a virus
(3) conjugation, in which the transfer involves cell-to-cell
contact
Three main processes of genetic recombination in prokaryotes
fragments of homologous DNA from a donor chromosome are
transferred to a recipient cell
(1) Transformation, which involves donor DNA free
in the environment
(2) Transduction, in which the donor DNA transfer
is mediated by a virus
(3) Conjugation, in which the transfer involves cellto-cell contact and a conjugative plasmid in the donor
cell
Transformation
Transduction
Conjugation
Transformation
Competence
A cell that is able to take up a molecule of DNA and be
transformed is said to be competent.
The introduction of DNA into cells
by mixing the DNA and the cell
(a) Binding of free DNA by a membranebound DNA binding protein.
(b) Passage of one of the two strands into
the cell while nuclease activity
degrades the other strand.
(c) The single strand in the cell is bound
by specific proteins, and
recombination with homologous
regions of the bacterial chromosome
mediated by RecA protein occurs.
Transformed cell
The mechanism of bacterial transformation
Transduction
Concept
Transduction involves transfer of host genes from one
bacterium to another by viruses. In generalized
transduction, defective virus particles randomly
incorporate fragments of the cell's chromosomal DNA;
virtually any gene of the donor can be transferred, but
the efficiency is low. In specialized transduction, the
DNA of a temperate virus excises incorrectly and
brings adjacent host genes along with it; only genes
close to the integration point of the virus are
transferred, but the efficiency may be high.
In transduction, DNA is transferred from cell
to cell through the agency of viruses. Genetic
transfer of host genes by viruses can occur in
two ways.
Generalized transduction
And
Specialized transduction
Generalized transduction: host DNA derived from
virtually any portion of the host genome becomes a
part of the DNA of the mature virus particle in
place of the virus genome.
Specialized transduction: occurs only in some
temperate viruses; DNA from a specific region of the
host chromosome is integrated directly into the virus
genome - usually replacing some of the virus genes.
Generalized transduction
In generalized transduction, virtually any genetic marker
can be transferred from donor to recipient
During a lytic infection, the
enzymes responsible for
packaging viral DNA into the
bacteriophage sometimes
accidentally package host DNA.
This DNA cannot replicate, it
can undergo genetic
recombination with the DNA of
the new host.
Specialized Transduction
the DNA of lambda is inserted into the host DNA
at the site adjacent to the galactose genes
On induction, Under rare conditions, the phage
genome is excised incorrectly
A portion of host DNA is exchanged for phage
DNA, called lambda dgal ( dgal means
"defective galactose“ )
Phage synthesis is completed
Cell lyses and releases defective phage
capable of transducing galactose genes
Genetic
Recombination
Genetic recombination involves the physical exchange of
genetic material between genetic elements. It results in
genetic exchange between homologous DNA sequences
from two different sources.
Homologous recombination is extremely important to all
organisms. However, it is also very complex .
conjugation
Bacterial conjugation (mating) is a process of genetic transfer
that involves cell-to-cell contact.
Direct contact between two conjugating bacteria is first made via a
pilus. The cells are then drawn together for the actual transfer of DNA.
Note the F-specific bacteriophages on the pilus
Conjugation involves a donor cell, which contains a particular
type of conjugative plasmid, and a recipient cell, which does not.
The genes that control conjugation are contained in the tra region
of the plasmid (see Section 9.8 in your text ). Many genes in the
tra region have to do with the synthesis of a surface structure, the
sex pilus . Only donor cells have these pili,
The pili make specific contact with a receptor on the recipient
and then retract, pulling the two cells together. The contacts
between the donor and recipient cells then become stabilized,
probably from fusion of the outer membranes, and the DNA is then
transferred from one cell to another.
Mechanism of DNA Transfer During Conjugation
A mechanism of DNA synthesis in
certain bacteriophages, called rolling
circle replication, was presented here
to explains DNA transfer during
conjugation .
if the DNA of the donor is labeled, some
labeled DNA is transferred to the
recipient but only a single labeled strand
is transferred. Therefore, at the end of
the process, both donor and recipient
possess completely formed plasmids.
DNA Transfer in Bacteria
transformation
transduction
conjugation
Three main processes of genetic recombination in prokaryotes
fragments of homologous DNA from a donor chromosome are
transferred to a recipient cell
(1) Transformation, which involves donor DNA free
in the environment
(2) Transduction, in which the donor DNA transfer
is mediated by a virus
(3) Conjugation, in which the transfer involves cellto-cell contact and a conjugative plasmid in the donor
cell
Transformation
Transduction
Conjugation
Transformation
A number of prokaryotes have been found to be
naturally transformable, including certain species of
both gram-negative and gram-positive Bacteria and
some species of Archaea. However, even within
transformable genera, only certain strains or species
are transformable
Competence
A cell that is able to take up a molecule of DNA and be
transformed is said to be competent. Competence in
most naturally transformable bacteria is regulated,
and special proteins play a role in the uptake and
processing of DNA. These competence-specific
proteins may include a membrane-associated DNA
binding protein, a cell wall autolysin, and various
nucleases.
Competent cells bind much more DNA than do
noncompetent cells as much as 1000 times more
The introduction of DNA into cells
by mixing the DNA and the cell
(a) Binding of free DNA by a membranebound DNA binding protein.
(b) Passage of one of the two strands into
the cell while nuclease activity
degrades the other strand.
(c) The single strand in the cell is bound
by specific proteins, and
recombination with homologous
regions of the bacterial chromosome
mediated by RecA protein occurs.
Transformed cell
The mechanism of bacterial transformation
Artificially Induced Competence
High efficiency natural transformation is found only in a few
bacteria; Azotobacter, Bacillus, Streptococcus,, for example,
are easily transformed. Many prokaryotes are transformed
only poorly or not at all under natural conditions.
Determination of how to induce competence in such bacteria
may involve considerable empirical study, with variation in
culture medium, temperature, and other factors
when E. coli is treated with high concentrations of calcium ions
and then stored in the cold, the transformation by plasmid DNA
is relatively efficient.
DNA Transfer by Electroporation
for artificial induction of competence are being
supplanted by a new method termed electroporation.
Small pores are produced in the membranes of cells
exposed to pulsed electric fields. When DNA
molecules are
present outside the cells during the electric pulse,
they can then enter the cells through these pores.
This process is called electroporation.
Transfection
Bacteria can be transformed with DNA extracted
from a bacterial virus rather than from another
bacterium, a process known as transfection.
Transduction
In transduction, DNA is transferred from cell
to cell through the agency of viruses. Genetic
transfer of host genes by viruses can occur in
two ways.
Generalized transduction
And
Specialized transduction
Concept:
Transduction involves transfer of host genes from one
bacterium to another by viruses. In generalized
transduction, defective virus particles randomly
incorporate fragments of the cell's chromosomal DNA;
virtually any gene of the donor can be transferred, but
the efficiency is low. In specialized transduction, the
DNA of a temperate virus excises incorrectly and
brings adjacent host genes along with it; only genes
close to the integration point of the virus are
transferred, but the efficiency may be high.
Generalized transduction: host DNA derived from
virtually any portion of the host genome becomes a
part of the DNA of the mature virus particle in
place of the virus genome.
Specialized transduction: occurs only in some
temperate viruses; DNA from a specific region of the
host chromosome is integrated directly into the virus
genome - usually replacing some of the virus genes.
Transduction has been found to occur in a variety of
prokaryotes, including certain species of the
Bacteria: Desulfovibrio, Escherichia, Pseudomonas,
Rhodococcus, Rhodobacter, Salmonella,
Staphylococcus, and Xanthobacter, as well as the
archaean Methanobacterium thermoautotrophicum.
Not all phages can be transducer and not all bacteria are transducible
Generalized transduction
In generalized transduction, virtually any genetic marker
can be transferred from donor to recipient
During a lytic infection,
the enzymes responsible
for packaging viral DNA
into the bacteriophage
sometimes accidentally
package host DNA. This
DNA cannot replicate, it
can undergo genetic
recombination with the
DNA of the new host.
Generalized transduction
Specialized Transduction
the DNA of lambda is inserted into the host DNA
at the site adjacent to the galactose genes
On induction, Under rare conditions, the phage
genome is excised incorrectly
A portion of host DNA is exchanged for phage
DNA, called lambda dgal ( dgal means
"defective galactose“ )
Phage synthesis is completed
Cell lyses and releases defective phage
capable of transducing galactose genes
Conjugation
Bacterial conjugation (mating) is a process of genetic transfer
that involves cell-to-cell contact.
Direct contact between two conjugating bacteria is first made via a
pilus. The cells are then drawn together for the actual transfer of DNA.
Conjugation involves a donor cell, which contains a particular
type of conjugative plasmid, and a recipient cell, which does not.
The genes that control conjugation are contained in the tra region
of the plasmid (see Section 9.8 in your text ). Many genes in the
tra region have to do with the synthesis of a surface structure, the
sex pilus . Only donor cells have these pili,
The pili make specific contact with a receptor on the recipient
and then retract, pulling the two cells together. The contacts
between the donor and recipient cells then become stabilized,
probably from fusion of the outer membranes, and the DNA is then
transferred from one cell to another.
Result of selected conjugation
Donor Recipient Molecules transferred
Product
F+
F-
F plasmid
F+ Cell
Hfr
F-
F- with variable
quantity of
chromosomal DNA
F+
F-
Initiating segment of F
plasmid and variable
quantity of
chromosomal DNA
F+ plasmid and some
chromosomal genes it
carries with it
F+ Cell with some
duplicate gene pairs:
one on chromosom,
one on plasmid
F
+
F
Hrf
F’
Cells possessing an
unintegrated F plasmid are
called F', and strains that
F'
can act as recipients for
(or Hfr, see later in this
section) are called F-. F- cells
lack the F plasmid;
F
F’
F+ x F-
+
F
Hrf
Mechanism of DNA Transfer During Conjugation
A mechanism of DNA synthesis in
certain bacteriophages, called rolling
circle replication, was presented here
to explains DNA transfer during
conjugation .
if the DNA of the donor is labeled, some
labeled DNA is transferred to the
recipient but only a single labeled strand
is transferred. Therefore, at the end of
the process, both donor and recipient
possess completely formed plasmids.