Chapter 10 I. Mutation •

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Chapter 10
Genetics of Bacteria and Archaea
I. Mutation
• 10.1 Mutations and Mutants
• 10.2 Molecular Basis of Mutation
• 10.3 Reversions and Mutation Rates
• 10.4 Mutagenesis
10.1 Mutations and Mutants
• Mutation
•
•
Heritable change in DNA sequence that can lead to a
change in phenotype (observable properties of an
organism)
Mutant
• A strain of any cell or virus differing from parental strain in
genotype (nucleotide sequence of genome)
•
Wild-type strain
• Typically refers to strain isolated from nature
10.1 Mutations and Mutants
• Selectable mutations (Figure 10.1)
•
•
Those that give the mutant a growth advantage under
certain conditions
Useful in genetic research
•
Nonselectable mutations
• Those that usually have neither an advantage nor a
•
disadvantage over the parent
Detecting such mutations requires examining a large
number of colonies and looking for differences (screening)
10.1 Mutations and Mutants
• Screening is always more tedious than selection
•
•
Methods are available to facilitate screening (Figure 10.2)
• Example: replica plating
Replica plating is useful for identifying cells with a nutritional
requirement for growth (auxotroph)
10.2 Molecular Basis of Mutation
• Induced mutations
•
•
Those made environmentally or deliberately
Can result from exposure to natural radiation or oxygen
radicals
•
Spontaneous mutations
• Those that occur without external intervention
•
Point mutations (Figure 10.3)
• Mutations that change only one base pair
• Can lead to single amino acid change in a protein, an
incomplete protein, or no change at all
10.2 Molecular Basis of Mutation
•
Silent mutation
• Does not affect amino acid sequence
•
Missense mutation
• Amino acid changed; polypeptide altered
•
Nonsense mutation
• Codon becomes stop codon; polypeptide is incomplete
10.2 Molecular Basis of Mutation
• Deletions and insertions cause more dramatic changes
in DNA
•
Frameshift mutations (Figure 10.4)
• Deletions or insertions that result in a shift in the reading
•
frame
Often result in complete loss of gene function
10.3 Reversion and Mutation Rates
• Point mutations are typically reversible
• Reversion
•
Alteration in DNA that reverses the effects of a prior
mutation
10.3 Reversion and Mutation Rates
• Revertant
•
Strain in which original phenotype is restored
•
Two types
• Same-site revertant: mutation is at the same site as original
mutation
• Second-site revertant: mutation is at a different site in the DNA
• Suppressor: mutation that compensates for the effect of the
original mutation (Figure 10.5)
10.3 Reversion and Mutation Rates
• For most microorganisms, errors in DNA replication
occur at a frequency of 106 to107 per kilobase
•
•
DNA viruses have error rates 100–1000 greater
The mutation rate in RNA genomes is 1000-fold higher
than in DNA genomes
• Some RNA polymerases have proofreading capabilities
• However, RNA repair mechanisms similar to DNA repair
mechanisms do not exist
10.3 Reversion and Mutation Rates
• The Ames test makes practical use of bacterial
mutations to detect for potentially hazardous chemicals
(Figure 10.6)
• Looks for an increase in mutation of bacteria in the
•
presence of suspected mutagen
A wide variety of chemicals have been screened for toxicity
and carcinogenicity
10.4 Mutagenesis
•
Mutagens: chemical, physical, or biological agents that
increase mutation rates
•
Several classes of chemical mutagens exist:
• Nucleotide base analogs: resemble nucleotides (Figure
•
•
10.7)
Chemical mutagens that induce chemical modifications
• For example, alkylating agents such as nitrosoguanidine
Chemical mutagens that cause frameshift mutations
• For example, intercalating agents such as acridines
10.4 Mutagenesis
• Several forms of radiation (Figure 10.8)
• Two main categories of mutagenic electromagnetic
radiation
• Nonionizing (i.e., UV radiation)
•
• Purines and pyrimidines strongly absorb UV
• Pyrimidine dimer is one effect of UV radiation
Ionizing (i.e., X-rays, cosmic rays, and gamma rays)
• Ionize water and produce free radicals
• Free radicals damage macromolecules in the cell
10.4 Mutagenesis
• Three types of DNA repair systems
•
•
•
Direct reversal: mutated base is still recognizable and can
be repaired without referring to other strand
Repair of single-strand damage: damaged DNA is removed
and repaired using opposite strand as template
Repair of double-strand damage: a break in the DNA
• Requires more error-prone repair mechanisms
10.4 Mutagenesis
• When DNA damage is large scale, the cell may use a
different type of repair system (i.e., damage interferes
with DNA replication)
• Mechanism called the SOS regulatory system (Figure 10.9)
• This system is more error-prone
• Allows replication to proceed and cell to replicate, but errors are
more likely
•
Translesion synthesis allows DNA to be synthesized with no
template
10.4 Mutagenesis
• Perfect fidelity in organisms is counterproductive
because it prevents evolution
•
The mutation rate of an organism is subject to change
• Mutants can be isolated that are hyperaccurate or have
•
increased mutation rates
Mutator strains: bacteria that benefit from increased
mutation rates
II. Gene Transfer in Bacteria
• 10.5 Genetic Recombination
• 10.6 Transformation
• 10.7 Transduction
•
•
10.8 Conjugation
10.9 The Formation of Hfr Strains and Chromosome
Mobilization
10.5 Genetic Recombination
• Recombination
•
•
Physical exchange of DNA between genetic elements
Homologous recombination
• Process that results in genetic exchange between
homologous DNA from two different sources (Figure 10.11)
•
Selective medium can be used to detect rare genetic
recombinants (Figure 10.12)
10.6 Transformation
• Transformation
•
Genetic transfer process by which DNA is incorporated into
a recipient cell and brings about genetic change
10.6 Transformation
• Competent: cells are capable of taking up DNA and
being transformed
• In naturally transformable bacteria, competence is regulated
• In other strains, specific procedures are necessary to make
•
cells competent
Electricity can be used to force cells to take up DNA
(electroporation)
10.6 Transformation
• During natural transformation, integration of transforming
DNA is a highly regulated, multistep process (Figure
10.13)
10.6 Transformation
• Transfection
•
Transformation of bacteria with DNA extracted from a
bacterial virus
10.7 Transduction
• Transduction
•
•
Transfer of DNA from one cell to another by a
bacteriophage
Two modes
• Generalized transduction: DNA from any portion of the host
genome is packaged inside the virion
• Specialized transduction: DNA from a specific region of the host
chromosome is integrated directly into the virus genome
10.7 Transduction
• Generalized transduction: DNA derived from virtually any
portion of the host genome is packaged inside the
mature virion (Figure 10.14)
•
•
•
Defective virus particle incorporates fragment of the cell's
chromosome randomly
Virus can be temperate or virulent
Low efficiency
10.7 Transduction
• Specialized transduction: DNA from a specific region of
the host chromosome is integrated directly in the virus
genome
• DNA of temperate virus excises incorrectly and takes
•
adjacent host genes along with it (Figure 10.15)
Transducing efficiency can be high
10.7 Transduction
• Phage conversion: alteration of the phenotype of a host
cell by a lysogenization
• Nondefective temperate phage lysogenizes a cell and
•
•
becomes a prophage
Host cell becomes immune to further infection by same
phage
Other phenotypic changes can also occur
• Salmonella enterica serovar Anatum and bacteriophage ε
• Corynebacterium diphtheriae and bacteriophage β
10.8 Conjugation
• Bacterial conjugation (mating): mechanism of genetic
transfer that involves cell-to-cell contact
• Plasmid-encoded mechanism
• Donor cell: contains conjugative plasmid
• Recipient cell: does not contain plasmid
10.8 Conjugation
• F (fertility) plasmid (Figure 10.16)
•
•
•
•
Circular DNA molecule; ~100 kbp
Contains genes that regulate DNA replication
Contains several transposable elements that allow the
plasmid to integrate into the host chromosome
Contains tra genes that encode transfer functions
10.8 Conjugation
• Sex pilus is essential for conjugation (Figure 10.17)
•
Produced only by donor cell
10.8 Conjugation
• DNA synthesis is necessary for DNA transfer by
conjugation
• DNA synthesized by rolling circle replication (Figure 10.18)
• Mechanism also used by some viruses
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• F plasmid is an episome; can integrate into host
chromosome
•
•
Cells possessing a nonintegrated F plasmid are called F+
Cells possessing an integrated F plasmid are called Hfr
(high frequency of recombination)
• High rates of genetic recombination between genes on the
donor chromosome and those of the recipient
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• Presence of the F plasmid results in alterations in cell
properties
• Ability to synthesize F pilus
• Mobilization of DNA for transfer to another cell
• Alteration of surface receptors so that cell can no longer act
as a recipient in conjugation
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• Insertion sequences (mobile elements) are present in
both the F plasmid and E. coli chromosome
• Facilitate homologous recombination (Figure 10.19)
•
Plasmid is now part of chromosome
• Chromosomal genes transferred with plasmid (Figure 10.20)
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• Recipient cell does not become Hfr because only a
portion of the integrated F plasmid is transferred by the
donor (Figure 10.21)
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• Hfr strains that differ in the integration position of the F
plasmid in the chromosome transfer genes in different
orders (Figure 10.22)
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• Genetic crosses with Hfr strains can be used to map the
order of genes on the chromosome
10.9 The Formation of Hfr Strains and
Chromosome Mobilization
• F′ plasmids
•
Previously integrated F plasmids that have excised and
captured some chromosomal genes
III. Gene Transfer in Archaea and Other Genetic
Elements
• 10.10 Horizontal Gene Transfer in Archaea
• 10.11 Mobile DNA: Transposable Elements
• 10.12 Preserving Genome Integrity: CRISPR
Interference
10.10 Horizontal Gene Transfer in Archaea
• Archaea contain single circular chromosome (Figure
10.23)
•
Genetic manipulation of Archaea lags behind Bacteria
• Archaea need to be grown in extreme conditions
• Most antibiotics do not affect Archaea
• No single species is a model organism for Archaea
•
Examples of transformation, transduction, and
conjugation exist
•
Transformation works reasonably well in Archaea
10.11 Mobile DNA: Transposable Elements
• Discrete segments of DNA that move as a unit from one
location to another within other DNA molecules are
transposable elements
•
Transposable elements can be found in all three
domains of life
•
Move by a process called transposition
• Frequency of transposition is 1 in 1000 to 1 in 10,000,000
•
per generation
First observed by Barbara McClintock
10.11 Mobile DNA: Transposable Elements
• Two main types of transposable elements in Bacteria are
transposons and insertion sequences (Figure 10.24)
• Both carry genes encoding transposase
• Both have inverted repeats at their ends
10.11 Mobile DNA: Transposable Elements
• Insertion sequences are the simplest transposable
element
• ~1000 nucleotides long
• Inverted repeats are 10–50 base pairs
• Only gene is for the transposase
• Found in plasmids and chromosomes of Bacteria and
•
Archaea
Found in some bacteriophages
10.11 Mobile DNA: Transposable Elements
• Transposons are larger than insertion sequences
•
•
Transposase moves any DNA between inverted repeats
Insertion of a transposable element generates a duplicate
target sequence (Figure 10.25)
•
May include antibiotic resistance
• Examples are Tn5 and Tn10
10.11 Mobile DNA: Transposable Elements
• Mechanisms of transposition: two types
•
•
Conservative: transposon is excised from one location and
reinserted at a second location (e.g., Tn5; Figure 10.26)
• Number of transposons stays constant
Replicative: a new copy of transposon is produced and
inserted at a second location
• Number of transposons present doubles
10.11 Mobile DNA: Transposable Elements
• Using transposons to make mutants (Figure 10.27)
•
•
•
•
•
Transposons with antibiotic resistance are used
Transposon is on a plasmid that cannot be replicated in the
cell
Cells capable of growing on selective medium likely
acquired transposon
Most insertions will be in genes that encode proteins
Next step: screen for loss of function to determine insertion
site
10.12 Preserving Genome Integrity: CRISPR
Interference
• CRISPR: Clustered Regulatory Interspaced Short
Palindromic Repeats
•
Type of prokaryotic "immune system"
• Region of bacterial chromosome containing DNA
•
sequences similar to foreign DNA (spacers) alternating with
identical repeated sequences (Figure 10.28)
CRISPR-associated proteins (Cas proteins)
• Obtain and store segments of foreign DNA as spacers
• Recognize and destroy foreign DNA
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