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Prescott’s Microbiology, 9th Edition
16
Mechanisms of Genetic Variation
CHAPTER OVERVIEW
This chapter begins with a discussion of mutation and genetic variation and includes molecular mechanisms of
mutation and repair. A general discussion of bacterial recombination, plasmids, and transposable elements follows,
with examination of the acquisition of genetic information by conjugation, transformation, and transduction.
LEARNING OUTCOMES
After reading this chapter you should be able to:
•
distinguish spontaneous from induced mutations, and list the most common ways each arises
•
construct a table, concept map, or picture to summarize how base analogoues, DNA-modifying agents, and
intercalating agents cause mutations
•
discuss the possible effects of mutations
•
differentiate mutant detections from mutant selection
•
design an experiment to isolate mutant bacteria that are threonine auxotrophs
•
propose an experiment to isolate revertants of a threonine auxotroph and predict the types of mutations that
might lead to the revertant phenotype
•
explain how the Ames test is used to screen for potential carcinogens and evaluate its effectiveness
•
compare and contrast excision repair, direct repair, mismatch repair, and recombinational repair
•
propose a scenario that would elicit SOS response and describe the response to those conditions
•
describe in general terms how recombinant eukaryotic organisms arise
•
distinguish vertical gene transfer from horizontal gene transfer
•
summarize the four possible outcomes of horizontal gene transfer
•
compare and contrast homologous recombination and site-specific recombination
•
differentiate insertion sequences from transposons
•
distinguish simple transposition from replicative transposition
•
defend this statement “Transposable elements are important factors in the evolution of bacteria and archaea.”
•
identify the type of plasmids that are important creators of genetic variation
•
describe the features of the F factor that allow it to (1) transfer itself to a new host and (2) integrate into a host
cell’s chromosome
•
outline the events that occur when an F+ cell encounters an F- cell
•
distinguish F+, Hfr and F’ cells from each other
•
explain how Hfr cells arise
•
outline the events that occur when an Hfr cell encounters an F- cell
•
describe the factors that contribute to a bacterium being naturally transformation competent
•
predict the outcomes of transformation using a DNA fragment versus using a plasmid
•
design an experiment to transform bacteria that carries genes encoding ampicillin resistance and the protein that
generates green fluorescence
•
differentiate generalized transduction from specialized transduction
•
create a phage’s life cycle to its capacity to mediate generalized or specialized transduction
•
draw a figure, create a concept map, or construct a table that distinguishes conjugation, transformation, and
transduction
1
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Prescott’s Microbiology, 9th Edition
CHAPTER OUTLINE
I.
II.
Mutations
A. Mutation overview
1. A mutation is a stable, heritable change in the genomic nucleotide sequence; this can be a single base
change (point mutation), changes of several bases, or larger insertions, deletions, inversions,
duplications, and translocations
2. Mutations can arise in two ways:
a. Spontaneous mutations arise occasionally in the absence of any added agent
b. Induced mutations are the result of exposure to a mutagen (physical or
chemical agent)
B. Spontaneous mutations
1. Arise occasionally in all cells without exposure to external agents; they are often the result of errors
in replication or lesions to the DNA
2. Errors in replication can be due to tautomeric shifts, which cause base substitutions
a. Transition mutation—substitution of one purine for another, or of one pyrimidine for another
b. Transversion mutation—substitution of a purine for a pyrimidine or vice versa
3. Lesions in the structure of DNA; the loss of a nitrogenous base creating an apurinic or apyrimidinic
site can cause spontaneous mutations
C. Induced mutations
1. Mutations can be induced by agents that damage DNA, alter its chemistry, or interfere with its
functioning
2. Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into DNA
during replication, but exhibit base-pairing properties different from the bases they replace
3. Specific mispairing occurs when a mutagen is a DNA-modifying agents that changes a base’s
structure and thereby alters its pairing characteristics (e.g., alkylating agents)
4. Intercalating agents, which become inserted between the stacked bases of the helix, distort the DNA
and thus induce single nucleotide pair insertions or deletions
D. Effects of mutations
1. Forward mutation—a conversion from the most prevalent gene form (wild type) to a mutant form
2. Reversion mutation—a second mutation event that makes the mutant appear to be a wild type again
a. Back mutation (true reversion)—conversion of the mutant nucleotide sequence back to the
wild-type sequence
b. Suppressor mutation—a reestablishment of the wild-type phenotype by a second mutation that
overcomes the effect of the first mutation; can be in the same gene or a different gene, but does
not restore the original sequence
3. Mutations in protein-coding genes
a. Silent mutations are alterations of the base sequence that do not alter the amino acid sequence
of the protein because of code degeneracy
b. Missense mutations are alterations of the base sequence that result in the incorporation of a
different amino acid in the protein; at the level of protein function, the effect may range from
complete loss of activity to no change in activity
c. Nonsense mutations are alterations that produce a translation termination codon; this results in
premature termination of protein synthesis; location of the mutation within the protein will
determine the extent of change in function
d. Frameshift mutations are insertions or deletions of one or two base pairs that thereby alter the
reading frame of the codons
e. Conditional mutations are expressed only under certain environmental conditions
f.
Biochemical mutations result in changes in the metabolic capabilities of a cell; auxotrophs
cannot grow on minimal media because they have lost a biosynthetic capability and require
supplements; prototrophs are wild-type organisms that can grow on minimal media
g. Resistance mutations result in acquired resistance to some pathogen, chemical, or antibiotic
4. Mutations in regulatory sequences
Detection and Isolation of Mutants
A. Mutant detection
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Prescott’s Microbiology, 9th Edition
1.
2.
Visual observation of changes in colony characteristics
Auxotrophic mutants can be detected by replica plating on media with and without the growth factor
required; mutants are those growing with the factor but not without it
B. Mutant selection is achieved by finding the environmental condition under which the mutant will grow but
the wild type will not (useful for isolating auxotrophic revertants, resistance mutants, and substrate
utilization mutations)
C. Mutagens and carcinogens
1. Many cancer-causing agents (carcinogens) are also mutagens, therefore tests for mutagenicity can be
used as a screen for carcinogenic potential
2. The Ames test is a widely used mutagenicity test; it detects an increase in reversion of special strains
of Salmonella typhimurium from histidine auxotrophy to prototrophy after exposure to a potential
carcinogen
III. DNA Repair
A. Proofreading: The first line of defense
1. replicative DNA polymerases sometimes insert incorrect nucleotide sequences during DNA replication
2. DNA polymerases can evaluate and correct any errors
B. Mismatch repair
1. The mismatch repair system corrects replication errors that result in mismatched base pairs; newly
replicated DNA is detected by a lack of DNA methylation
2. The mismatch is detected by MutS and repaired through excision by MutH
C. Excision repair
1. Corrects damage that causes distortions of DNA (e.g., thymine dimers, apurinic or apyrimidinic
sites, damaged or unnatural DNA)
2. For nucleotide excision repair, the damaged area is excised, producing a single-stranded gap, and
then the gap is filled in by DNA polymerase I, and DNA ligase joins the new fragment into the
existing DNA strand
3. For base excision repair, DNA glycosylases remove the damaged base, and this signals AP nucleases
to mark the damaged DNA, which is then excised and repaired by DNA polymerase I and ligase
D. Direct repair
1. thymine dimers and alkylated bases repair occurs through photoreactivation or the action of alkyl- or
methyltransferases, respectively
2. catalyzed by enzyme photolyase
E. Recombinational repair
1. Recombination with an undamaged molecule, if available, is used to restore DNA that has damage in
both strands through the action of RecA protein; an undamaged molecule can be available in rapidly
dividing cells where there is a copy of the chromosome that has not yet segregated into daughter
cells
F. The SOS response
1. SOS repair is a type of recombination repair that depends on the RecA protein; it is used to repair
excessive damage that halts replication; it is an error-prone process that results in many mutations
2. RecA derepresses the synthesis of a variety of DNA repair genes; very serious damage is treated by
translesion DNA synthesis that is highly error prone
IV. Creating Additional Genetic Variability
A. Sexual reproduction and genetic variability
1. vertical gene transfer
2. observed in all organisms capable of sexual reproduction
B. Horizontal gene transfer: creating variability the asexual way
1. Horizontal (or lateral) gene transfer moves genes from one mature, independent organism to another
(compare this to vertical gene transfer—transmission of genes from parents to offspring)
2. Exogenote—donor DNA that enters the bacterium by one of several mechanisms
a. Conjugation is direct transfer from donor bacterium to recipient while the two are temporarily
in physical contact
b. Transformation is transfer of a naked DNA molecule
c. Transduction is transfer mediated by a bacteriophage
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Prescott’s Microbiology, 9th Edition
3.
Endogenote—the genome of the recipient
a. Merozygote—a recipient cell that is temporarily diploid for a portion of the genome during the
gene transfer process
4. Intracellular fates of exogenote
a. Integration into the host chromosome
b. Independent functioning and replication of the exogenote without integration (a partial diploid
clone develops)
c. Survival without replication (only the one cell is a partial diploid)
d. Degradation by host nucleases (host restriction)
C. Molecular recombination: joining DNA molecules together
1. General recombination usually involves a reciprocal exchange in which a pair of homologous
sequences breaks and rejoins (double-stranded break model) in a crossover; nonreciprocal general
recombination involves the incorporation of a single strand into the chromosome to form a stretch of
heteroduplex DNA
2. Site-specific recombination is the nonhomologous insertion of DNA into a chromosome; often
occurs during viral genome integration into the host, a process catalyzed by enzymes specific for the
virus and its host
3. Transposition is a kind of recombination that occurs throughout the genome and does not depend on
sequence homology
V. Transposable Elements
A. Transposition is the movement of pieces of DNA around in the genome; transposons are segments of
DNA that can move about chromosomes, "jumping genes"
B. Insertion sequences (IS elements) contain genes only for those enzymes required for transposition (e.g.,
transposase); they are bound on both ends by inverted terminal repeat sequences
C. Some transposons carry other genes in addition to those needed for transposition (e.g., for antibiotic
resistance, toxin production, etc.)
D. Transposition can occur by two mechanisms:
1. Simple transposition is a cut-and-paste process involving transposase-catalyzed excision of a
transposon and insertion into a new target site
2. Replicative transposition is a mechanism during which a replicated copy of the transposon inserts at
the target site on the DNA, while the original copy remains at the parental site
E. Effects of transposable elements
1. Insertional mutagenesis can cause deletion of genetic material at or near the target site, arrest of
translation or transcription due to stop codons or termination sequences located on the inserted
material, and activation of genes near the point of insertion due to promoters located on the inserted
material
2. Fusion of plasmids and insertion of F plasmids into chromosomes
3. Generation of plasmids with resistance genes
F. Conjugative transposons can move between bacteria through the process of conjugation
VI. Bacterial Conjugation
A. The transfer of genetic information via direct cell-cell contact; this process is mediated by fertility factors
(F plasmids)
B. F+  F– mating
1. In E. coli and other gram-negative bacteria, an F plasmid moves from the donor (F+) to a recipient
(F–) while being replicated
a. Replication is by the rolling circle mechanism where the 3' end is extended from a nick in one
DNA strand, following around the circular genome, and displacing the 5' end
b. The displaced strand is transferred via a sex pilus and then copied to produce double-stranded
DNA; the donor retains the other parental DNA strand and its complement; thus the recipient
becomes F+ and the donor remains F+
c. Chromosomal genes are not transferred
C. Hfr conjugation
1. F plasmid integration into the host chromosome results in an Hfr (high frequency of recombination)
strain of bacteria
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Prescott’s Microbiology, 9th Edition
The mechanics of conjugation of Hfr strains are similar to those of F+ strains
The initial break for rolling-circle replication is at the integrated plasmid’s origin of transfer site
a. Part of the plasmid is transferred first
b. Chromosomal genes are transferred next
c. The rest of the plasmid is transferred last
4. Complete transfer of the chromosome takes approximately 100 minutes, but the conjugation bridge
does not usually last that long; therefore, the entire F factor is not usually transferred, and the
recipient remains F–
D. F conjugation
1. When an integrated F plasmid leaves the chromosome incorrectly, it may take with it some
chromosomal genes from one side of the integration site; this results in the formation of an abnormal
plasmid called an F plasmid
2. The F cell (cell harboring an F plasmid) retains its genes, although some of them are in the
chromosome and some are on the plasmid; in conjugation, an F cell behaves as an F+ cell, mating
only with F– cells
3. The chromosomal genes included in the plasmid are transferred with the rest of the plasmid, but
other chromosomal genes usually are not
4. The recipient becomes an F cell, and a partially diploid merozygote
E. Other examples of bacterial conjugation
1. Less is known about conjugative transfer in gram-positive bacteria
2. No sex pilus is formed; however, cells may directly adhere to each other using special plasmidencoded proteins
VII. Bacterial Transformation
A. Transformation—a naked DNA molecule from the environment is taken up by the cell and incorporated
into its chromosome in some heritable form
B. A competent cell is one that is capable of taking up DNA and therefore acting as a recipient; only a
limited number of species are naturally competent; the mechanics of the natural transformation process
differ from species to species
C. Species that are not normally competent (such as E. coli) can be made competent by calcium chloride
treatment and other methods that make the cells more permeable to DNA
VIII. Transduction
A. Transduction is the transfer of bacterial genes by viruses (bacteriophages); it occurs as the result of the
reproductive cycle of the virus
1. Lytic cycle—a viral reproductive cycle that ends in lysis of the host cell; viruses that use this cycle
are called virulent bacteriophages
2. Lysogeny—a reproductive cycle that involves maintenance of the viral genome (prophage) within
the host cell (usually integrated into the host cell’s chromosome), without immediate lysis of the
host; with each round of cell division, the prophage is replicated and inherited by daughter cells;
bacteriophages reproducing by this mechanism are called temperate phages; certain stimuli (e.g., UV
radiation) can trigger the switch from lysogeny to the lytic cycle
B. Generalized transduction
1. Transfer of any portion of the bacterial genome; occurs during the lytic cycle of virulent and
temperate bacteriophages
2. The phage degrades the host chromosome into randomly sized fragments
3. During assembly, fragments of host DNA of the appropriate size can be mistakenly packaged into a
phage head (generalized transducing particle)
4. When the next host is infected, the bacterial genes are injected and a merozygote is formed
a. Preservation of the transferred genes requires their integration into the host chromosome
b. Much of the transferred DNA does not integrate into the host chromosome, but is often able to
survive and be expressed; the host is called an abortive transductant
C. Specialized transduction
1. Transfer of only specific portions of the bacterial genome; carried out only by temperate phages that
have integrated their DNA into the host chromosome at a specific site in the chromosome
2.
3.
5
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Prescott’s Microbiology, 9th Edition
a.
The integrated prophage is sometimes excised incorrectly and contains portions of the bacterial
DNA that was adjacent to the phage’s integration site on the chromosome
b. The excised phage genome is defective because some of its own genes have been replaced by
bacterial genes; therefore, the bacteriophage cannot reproduce
c. When the next host is infected, the donor bacterial genes are injected, leading to the formation
of a merozygote
2. Low-frequency transduction lysates—lysates containing mostly normal phages and just a few
specialized transducing phages
3. High-frequency transduction lysates—lysates containing a relatively large number of specialized
transducing phages; created by coinfecting a host cell with a helper phage (normal phage) and a
transducing phage; the helper phage allows the transducing phage to replicate, thus increasing the
number of transducing phages in the lysate
IX. Evolution in Action: The Development of
A. Antibiotic Resistance in BacteriaSpread of drug resistant pathogens a serious threat to public health
1. MRSA- methicillin resistant S. aureus
2. VRE-vancomycin resistant enterococci
B.
Mechanisms of drug resistance
1. modify the target of the antibiotic by mutating a gene that functions in the synthesis of the target
2. drug inactivation by Beta-lactam ring of penicillins by the enzyme penicillinase
3. efflux pumps expel drugs
C. The origin and transmission of drug resistance
1. antibiotic resistance “captured” by horizontal gene transfer (HG)
2. R plasmids are resistance plasmids, code for enzymes that destroy or modify drugs
3. transposons can also carry antibiotic resistance genes
4. integron consists of a gene encoding an integrase and one or more genes that form a “gene cassette,”
which can be moved by a plasmid to another cell via conjugation
CRITICAL THINKING
1.
A strain of bacteria is protrophic. How would you isolate from this strain one that requires the amino acid
leucine (i.e., it is a leucine auxotroph)?
2.
You have a bacterial strain that is a tryptophan auxotroph and sensitive to the antibiotic streptomycin. You
expose this strain to a mutagen. How would you isolate mutants that no longer require tryptophan (i.e., strains
that have reverted to prototrophy)? How would you isolate mutants that are resistant to streptomycin? How
would you isolate mutants that no longer require leucine and that are resistant to streptomycin?
3.
Explain how cotransductional frequencies of gene markers during bacteriophage transduction or
transfer rates during conjugation can be used to produce genetic maps.
CONCEPT MAPPING CHALLENGE
Construct a concept map that describes the types of DNA repair mechanisms employed by organisms to protect their
DNA. Use the concepts that follow, any other concpts or terms you need, and your own linking words between each
pair of concepts in your map.
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Prescott’s Microbiology, 9th Edition
Proofreading Excision repair Mismatch repair SOS response Direct Repair
Homologous recombination DNA synthesis Error prone repair Thymine dimer
Rec A protein
7
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manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.