Transcript Freeman 1e: How we got there

CHAPTER 10
Bacterial Genetics
Mutation and Recombination,
Mutations and Mutants
• Mutation is a heritable change in DNA sequence
that can lead to a change in phenotype.
•By definition, a mutant differs from its parental
strain in genotype, the nucleotide sequence of the
genome.
•hisC (1,2,3,…) mutants of E. coli
•Auxotroph – nutritional mutant rewiring a growth
factors, amino acids (e.g. His-).
•Prototroph – wild type
• Selectable mutations are those that give the
mutant a growth advantage under certain
environmental conditions and are especially
useful in genetic research.
•If selection is not possible, mutants must be
identified by screening.
• Although screening is always more tedious
than selection, methods are available for
screening large numbers of colonies in certain
types of mutations.
•Penicillin selection – pen kills growing but
not mutant (non growing) cells in minimal
medium
Nutritionally defective mutants can also be
detected by the technique of replica plating
(Figure 10.2).
Replica Plating
Molecular Basis of Mutation
• Mutations, which can be either spontaneous
or induced, arise because of changes in the
base sequence of the nucleic acid of an
organism's genome.
• A point mutation, which results from a
change in a single base pair, can lead to a
single amino acid change in a polypeptide or
to no change at all, depending on the
particular codon involved (Figure 10.3).
Possible effects of bp
substitution
• In a nonsense mutation, the codon becomes
a stop codon and an incomplete polypeptide is
made.
•In a missense mutation, the sequence of
amino acids in the ensuing polypeptide is
changed, resulting in an inactive protein or
one with reduced activity.
•Temperature-sensitive mutations/
conditionally lethal mutations
• Deletions and insertions cause more
dramatic changes in the DNA, including
frameshift mutations, and often result in
complete loss of gene function (Figure 10.4).
Table 10.1 lists various kinds of mutants.
Mutation Rates
• Different types of mutations can occur at
different frequencies. For a typical bacterium,
mutation rates of 10–7 to 10–11 per base pair
are generally seen.
• Although RNA and DNA polymerases make
errors at about the same rate, RNA genomes
typically accumulate mutations at much
higher frequencies than DNA genomes.
Mutagenesis
• Mutagens are chemical, physical, or
biological agents that increase the mutation
rate. Mutagens can alter DNA in many
different ways, but such alterations are not
mutations unless they can be inherited.
•Table 10.2 gives an overview of some of the major chemical and
physical mutagens and their modes of action.
• There are several classes of chemical
mutagens, one being the nucleotide base
analogs (Figure 10.5).
• Several forms of radiation are highly
mutagenic (Figure 10.6).
• Some DNA damage can lead to cell death if
not repaired. A complex cellular mechanism
called the SOS regulatory system is activated
as a result of some types of DNA damage and
initiates a number of DNA repair processes,
both error-prone and high-fidelity (Figure
10.7).
Mutagenesis and
Carcinogenesis: The Ames Test
• The Ames test employs a sensitive bacterial
assay system for detecting chemical mutagens
in the environment.
Genetic Recombination
• Homologous recombination arises when
closely related DNA sequences from two
distinct genetic elements are combined in a
single element (Figure 10.9).
• Recombination is an important evolutionary
process, and cells have specific mechanisms
for ensuring that recombination takes place.
• Mechanisms of recombination that occur in
prokaryotes involve DNA transfer during the
processes of transformation, transduction, and
conjugation (Figure 10.11).
Genetic Exchange in
Prokaryotes
Transformation
• The discovery of transformation was one
of the seminal events in biology because it led
to experiments demonstrating that DNA is the
genetic material (Figure 10.13).
• Certain prokaryotes exhibit competence, a
state in which cells are able to take up free
DNA released by other bacteria.
• Incorporation of donor DNA into a recipient
cell requires the activity of single-stranded
binding protein, RecA protein, and several
other enzymes. Only competent cells are
transformable (Figure 10.14).
Transduction
• Transduction involves the transfer of host
genes from one bacterium to another by
bacterial viruses.
• In generalized transduction (Figure 10.15),
defective virus particles incorporate fragments
of the cell's chromosomal DNA randomly, but
the efficiency is low.
• In specialized transduction (Figure 10.16),
the DNA of a temperate virus excises
incorrectly and takes adjacent host genes
along with it; transducing efficiency in this
case may be very high.
Plasmids: General Principles
• Plasmids are small circular or linear DNA
molecules that carry any of a variety of
unessential genes. Although a cell can contain
more than one plasmid, they cannot be
closely related genetically.
• Figure 10.18 shows a genetic map of the F
(fertility) plasmid, a very well characterized
plasmid of Escherichia coli.
• Lateral transfer in the process of conjugation
can transfer plasmids (Figure 10.19).
Types of Plasmids and Their
Biological Significance
• The genetic information that plasmids carry
is not essential for cell function under all
conditions but may confer a selective growth
advantage under certain conditions.
• Examples include antibiotic resistance
(Figure 10.20), enzymes for degradation of
unusual organic compounds, and special
metabolic pathways. Virulence factors of
many pathogenic bacteria are often plasmidencoded.
• Table 10.3 lists some phenotypes that
plasmids confer on prokaryotes.
Conjugation: Essential Features
• Conjugation is a mechanism of DNA
transfer in prokaryotes that requires cell-tocell contact.
• Genes carried by certain plasmids (such as
the F plasmid) control conjugation, and the
process involves transfer of the plasmid from
a donor cell to a recipient cell (Figure 10.22).
Plasmid DNA transfer involves replication via
the rolling circle mechanism.
10.12 The Formation of Hfr
Strains and Chromosome
Mobilization, p. 279
• The donor cell chromosome can be
mobilized for transfer to a recipient cell. This
requires that the F plasmid integrate into the
chromosome to form the Hfr phenotype.
Transfer of the host chromosome is rarely
complete but can be used to map the order of
the genes on the chromosome.
• F' plasmids are previously integrated F
plasmids that have deintegrated and excised
some chromosomal genes.
• Integration of the F plasmid into the host
chromosome can occur at several specific
sites, called IS (for insertion sequence) sites
(Figure 10.23). These sites are regions of
DNA sequence homology between
chromosomal and F plasmid DNA.
Complementation
• If a cell is treated so that it contains two
copies of a region of its genome,
complementation tests can determine if two
mutations are in the same or different genes
(Figure 10.28).
• This is often necessary because mutations in
different genes in the same pathway may give
the same phenotype. Complementation tests
do not involve recombination.
Transposons and Insertion
Sequences
• Transposons and insertion sequences are
genetic elements that can move from one
location on a chromosome to another by a
process called transposition, a type of sitespecific recombination (Figure 10.30).
• Transposition is linked to the presence of
special genetic elements called transposable
elements. Transposition can be either
replicative or conservative (Figures 10.31,
10.32).
• Transposons often carry genes encoding
antibiotic resistance, and they can be used as
biological mutagens (Figure 10.33).
Bacterial Genetics and Gene
Cloning
Essentials of Molecular Cloning
• A plasmid or virus is used as the cloning
vector to isolate a specific gene or region of a
chromosome by molecular cloning (Figure
10.35).
• An in vitro recombination procedure uses
restriction enzymes and DNA ligase to
produce the hybrid DNA molecule. Once
introduced into a suitable host, the cloning
vector can control production of large
amounts of the target DNA.
• Making a gene library by cloning random
fragments of a genome is called shotgun
cloning, and it is a widely practiced technique
in gene cloning and genomic analyses.
Plasmids as Cloning Vectors
• Plasmids are useful cloning vectors (Figure
10.36) because they are easy to isolate and
purify and can multiply to high copy numbers
in bacterial cells.
• Antibiotic resistance genes of the plasmid
are used to identify bacterial cells containing
the plasmid (Figure 10.37).
Bacteriophage Lambda as a
Cloning Vector
• Bacteriophages such as lambda have been
modified to make useful cloning vectors
(Figures 10.38, 10.39).
• Larger amounts of foreign DNA can be
cloned with lambda than with many other
plasmids. In addition, the recombinant DNA
can be packaged in vitro for efficient transfer
to a host cell. Plasmid vectors containing the
lambda cos sites are called cosmids, and they
can carry a large fragment of foreign DNA.
In Vitro and Site-Directed
Mutagenesis
• Site-directed mutagenesis allows synthetic
DNA molecules of desired sequence to be
made in vitro and used to construct a mutated
gene directly or to change specific base pairs
within a gene (Figure 10.40).
• Inserting DNA fragments, called cassettes,
into genes can also cause gene disruption
(Figure 10.41). The inserted cassette
eliminates the function of the wild-type gene
while conferring a new, and usually
selectable, phenotype on the cell.
The Bacterial Chromosome
Genetic Map of the Escherichia
coli Chromosome
• The Escherichia coli chromosome has been
mapped using conjugation, transduction,
molecular cloning, and sequencing (Figure
10.42).
• E. coli has been a useful model organism,
and a considerable amount of information has
been obtained from it, not only about gene
structure but also about gene function and
regulation.