9.

4 Mutations: Changes in the Genetic Code 255 256 Chapter 9 Microbial Genetics

blocking the initiation step of transcription, and are selec- type as the trait present in the highest numbers in a popula- Causes of Mutations cation. Exposure to large doses of radiation can be fatal, which
tively more active against bacterial RNA polymerase than tion. If a microorganism bears a mutation, it is called a mutant is why radiation is so effective in microbial control; it can also
Mutations can be spontaneous or induced, depending upon
the corresponding eukaryotic enzyme. Actinomycin D binds strain. Mutant strains can show variance in morphology, be carcinogenic in animals. (The intentional use of UV to control
their origin. A spontaneous mutation is a random change in
to bacterial DNA and halts mRNA chain elongation, but it nutritional characteristics, genetic control mechanisms, resist- microorganisms is described further in chapter 11.)
the DNA arising from errors in replication that occur ran-
also binds to human DNA. For this reason, it is very toxic ance to chemicals, temperature preference, and nearly any
and never used to treat bacterial infections, though it can type of enzymatic function. Mutant strains are very useful
domly. The frequency of spontaneous mutations has been Categories of Mutations
measured for a number of organisms. Mutation rates vary
be applied in tumor treatment. for tracking genetic events, unraveling genetic organization, Mutations range from large mutations, in which large genetic
tremendously, from one mutation in 105 replications (a high
The ribosome is a frequent target of antibiotics that inhibit and pinpointing genetic markers. A classic method of detect- sequences are gained or lost, to small ones that affect only a
rate) to one mutation in 1010 replications (a low rate). The rapid
ribosomal function and ultimately protein synthesis. The value ing mutant strains involves addition of various nutrients to a single base on a gene. These latter mutations, which involve
rate of bacterial reproduction allows these mutations to be
and safety of these antibiotics again depend upon the differen- culture to screen for its use of that nutrient. For example, in a addition, deletion, or substitution of single bases, are called
observed more readily in bacteria than in most eukaryotes.
tial susceptibility of prokaryotic and eukaryotic ribosomes. One culture of a wild-type bacterium that is lactose-positive (mean- point mutations.
Induced mutations result from exposure to known
problem with drugs that selectively disrupt prokaryotic ribos- ing it has the necessary enzymes for fermenting this sugar), a To understand how a change in DNA in uences the cell,
mutagens, which are primarily physical or chemical agents
omes is that the mitochondria of humans contain a prokaryotic small number of mutant cells have become lactose-negative, remember that the DNA code appears in a particular order of
that interact with DNA in a disruptive manner (table 9.3).
type of ribosome, and these drugs may inhibit the function of having lost the capacity to ferment this sugar. If the culture is triplets (three bases) that is transcribed into mRNA codons,
The carefully controlled use of mutagens has proved a useful
the host’s mitochondria. One group of antibiotics (including plated on a medium containing indicators for fermentation, each of which speci es an amino acid. A permanent alteration
way to induce mutant strains of microorganisms for study.
erythromycin and spectinomycin) prevents translation by inter- each colony can be observed for its fermentation reaction in the DNA that is copied faithfully into mRNA and trans-
Chemical mutagenic agents act in a variety of ways to
fering with the attachment of mRNA to ribosomes. Chloram- and the negative strain isolated. Another standard method of lated can change the structure of the protein. A change in a
change the DNA. Agents such as acridine dyes insert com-
phenicol, lincomycin, and tetracycline bind to the ribosome in a detecting and isolating microbial mutants is by replica plating protein can likewise change the morphology and physiology
pletely across the DNA helices between adjacent bases to
way that blocks the elongation of the polypeptide, and ( gure 9.20). of a cell. Some mutations have a harmful effect on the cell,
produce mutations that distort the helix. Analogs4 of the
aminoglycosides (such as streptomycin) inhibit peptide initia- leading to cell dysfunction or death; these are called lethal
Culture of bacteria nitrogen bases (5-bromodeoxyuridine and 2-aminopurine,
tion and elongation. It is interesting to note that these drugs with discrete colonies mutations. Neutral mutations produce neither adverse nor
for example) are chemical mimics of natural bases that are
have served as important tools to explore genetic events helpful changes. A small number of mutations are bene cial
incorporated into DNA during replication. Addition of these
because they can arrest speci c stages in these processes. in that they provide the cell with a useful change in structure
abnormal bases leads to mistakes in base-pairing. Many
Plate exposed to a
or physiology.
9.3 Learning Outcomes—Can You . . . chemical mutagens also act as carcinogens, or cancer-causing
mutagenic agent Any change in the code that leads to placement of a dif-
agents, when vertebrates are exposed to them (see the discus-
ferent amino acid is called a missense mutation. A missense
13. . . . list one or two important advantages to arranging genes in sion of the Ames test in a later section of this chapter).
mutation can do one of the following:
an operon? Physical agents that alter DNA are primarily types of
14. . . . differentiate between repressible and inducible operons? radiation. High-energy gamma rays and X rays introduce major 1. create a faulty, nonfunctional (or less functional) protein,
15. . . . name some antibiotic targets of the transcription and physical changes into DNA, and it accumulates breaks that may 2. produce a protein that functions in a different manner, or
translation machinery? not be repairable. Ultraviolet (UV) radiation induces abnormal 3. cause no signi cant alteration in protein function (see
Special velveteen replica bonds between adjacent pyrimidines that prevent normal repli- table 9.4 to see how missense mutations look).
plating carrier (sterile)
picks up tiny bits of colony
when pressed upon agar 4. An analog is a chemical structured very similarly to another chemical
9.4 Mutations: Changes in the and removed. except for minor differences in functional groups.
Table 9.4 Categories of Point Mutations
Genetic Code and Their Effects
As precise and predictable as the rules of genetic expression Table 9.3 Selected Mutagenic Agents
seem, permanent changes do occur in the genetic code. and Their Effects DNA TAC TGG CTG CTC TAC TTT...
Pressed down Normal gene
RNA AUG ACC GAC GAG AUG AAA...
Indeed, genetic change is the driving force of evolution. Agent Effect
Protein Met Thr Asp Glu Met Lys...
In microorganisms, such changes may become evident in (a)
Chemical
altered gene expression, such as the appearance or disap- Missense mutation:
Medium that Nitrous acid, bisulfite Removes an amino group from DNA TAC TGG CTT CTC TAC TTT...
pearance of anatomical or physiological traits. For example, selects for Nonselective leading to amino acid
RNA AUG ACC GAA GAG AUG AAA...
a pigmented bacterium can lose its ability to form pigment, mutants medium some bases switch (may or may
(b) Protein Met Thr Glu Glu Met Lys... not function well)
or a strain of the malarial parasite can develop resistance to a Ethidium bromide Inserts between the paired
drug. Any change to the nucleotide sequence in the genome bases DNA TAC TGG CTA CTC TAC TTT...
Base substitution:
is called a mutation. Mutations are most noticeable when the Acridine dyes Cause frameshifts due to
RNA AUG ACC GAU GAG AUG AAA... silent (no change in
(c) function)
genotypic change leads to a change in phenotype. Mutations insertion between base pairs
Protein Met Thr Asp Glu Met Lys...

can involve the loss of base pairs, the addition of base pairs, G
FRAMESHIFT MUTATION
Nitrogen base analogs Compete with natural bases for
or a rearrangement in the order of base pairs. Do not confuse DNA TAC TGC TGC TCT ACT TT
Deletion mutation (d)
sites on replicating DNA
this with genetic recombination, in which microbes transfer RNA AUG ACG ACG AGA UGA AA...
Both lead to frameshifts
(d)
whole segments of genetic information among themselves. Radiation Protein Met Thr Thr
Frameshift and premature stop
Arg STOP
and can lead to
premature stop codons
A microorganism that exhibits a natural, nonmutated Colonies of Mixture of wild-type Ionizing (gamma rays, X rays) Form free radicals that cause and/or poorly
characteristic is known as a wild type, or wild strain with mutant strains and mutant strains DNA TAC TGG GCT GCT CTA CTT...
functioning protein
single or double breaks in DNA
respect to that trait. You might ask: “In a constantly chang- Figure 9.20 The general basis of replica plating. This
RNA AUG ACC CGA CGA GAU GAA...
Insertion mutation (e)
Ultraviolet Causes cross-links between (e) Protein Met Thr Arg Arg Asp Glu...
ing population of microbes, what is the natural, nonmutated method was developed by Joshua Lederberg for detecting and adjacent pyrimidines Frameshift
state?” For that reason most scientists prefer to de ne wild isolating mutant strains of microorganisms.

A nonsense mutation, on the other hand, changes a nor-
mal codon into a stop codon that does not code for an amino
acid and stops the production of the protein wherever it
occurs. A nonsense mutation almost always results in a non-
functional protein. (Table 9.4, row d, shows a nonsense muta-
tion resulting from a frameshift [described below].) A silent
mutation (table 9.4, row c) alters a base but does not change
the amino acid and thus has no effect. For example, because
of the redundancy of the code, ACU, ACC, ACG, and ACA
all code for threonine, so a mutation that changes only the
last base will not alter the sense of the message in any way. A
back-mutation occurs when a gene that has undergone muta-
tion reverses (mutates back) to its original base composition.
Mutations also occur when one or more bases are
inserted into or deleted from a newly synthesized DNA
strand. This type of mutation, known as a frameshift
(table 9.4, rows d and e), is so named because the read-
ing frame of the mRNA has been changed. Frameshift
mutations nearly always result in a nonfunctional protein
because every amino acid after the mutation is different
from what was coded for in the original DNA. Also note
that insertion or deletion of bases in multiples of three (3, 6,
9, etc.) results in the addition or deletion of amino acids but
does not disturb the reading frame. The effects of all of these
types of mutations can be seen in table 9.4.
Repair of Mutations
Earlier we indicated that DNA has a proofreading mecha-
nism to repair mistakes in replication that might otherwise
become permanent (see page 240). Because mutations are
potentially life-threatening, the cell has additional systems
for nding and repairing DNA that has been damaged by
various mutagenic agents and processes. Most ordinary
DNA damage is resolved by enzymatic systems specialized
for nding and xing such defects.
DNA that has been damaged by ultraviolet radiation
can be restored by photoactivation or light repair. This
repair mechanism requires visible light and a light-sensitive
enzyme, DNA photolyase, which can detect and attach to the
damaged areas (sites of abnormal pyrimidine binding). Ultra-
violet repair mechanisms are successful only for a relatively
small number of UV mutations. Cells cannot repair severe,
widespread damage and will die. In humans, the genetic dis-
ease xeroderma pigmentosa is due to nonfunctioning genes for
enzymes responsible for excising pyrimidine dimers caused
by UV light. Persons suffering from this rare disorder develop
severe skin cancers; this relation provided early strong evi-
dence for a link between cancer and mutations.
Mutations can be excised by a series of enzymes that
remove the incorrect bases and add the correct ones. This pro-
cess is known as excision repair. First, enzymes break the bonds
between the bases and the sugar-phosphate strand at the site of
the error. A different enzyme subsequently removes the defec-
tive bases one at a time, leaving a gap that will be lled in by
DNA polymerase I and ligase ( gure 9.21). A repair system can
also locate mismatched bases that were missed during proof-
9.4 Mutations: Changes in the Genetic Code 257
Enzyme complex I
Removed
(a)
Enzyme complex II
Added
(b)
(c)
Figure 9.21 Excision repair of mutation by enzymes.
(a) The first enzyme complex recognizes one or several incorrect bases
and removes them. (b) The second complex (DNA polymerase I and
ligase) places correct bases and seals the gaps. (c) Repaired DNA.
reading: for example, C mistakenly paired with A, or G with T.
The base must be replaced soon after the mismatch is made, or
it will not be recognized by the repair enzymes.
The Ames Test
New agricultural, industrial, and medicinal chemicals are
constantly being added to the environment, and exposure
to them is widespread. The discovery that many such com-
pounds are mutagenic and that many of these mutagens
are linked to cancer is signi cant. Although animal testing
has been a standard method of detecting chemicals with
carcinogenic potential, a more rapid screening system called
the Ames test 5 is also commonly used. In this ingenious test,
the experimental subjects are bacteria whose gene expression
and mutation rate can be readily observed and monitored.
5. Named for its creator, Bruce Ames.
258 Chapter 9 Microbial Genetics
The premise is that any chemical capable of mutating has proved invaluable for screening an assortment of envi-
bacterial DNA could similarly mutate mammalian (and thus ronmental and dietary chemicals for mutagenicity and carci-
human) DNA and is therefore potentially hazardous. nogenicity without resorting to animal studies.
One indicator organism in the Ames test is a mutant
strain of Salmonella typhimurium6 that has lost the ability to
synthesize the amino acid histidine, a defect highly suscep- Positive and Negative Effects of Mutations
tible to back-mutation because the strain also lacks DNA Many mutations are not repaired. How the cell copes
repair mechanisms. Mutations that cause reversion to the with them depends on the nature of the mutation and the
wild strain, which is capable of synthesizing histidine, occur strategies available to that organism. Mutations are perma-
spontaneously at a very low rate. A test agent is considered nent and heritable and will be passed on to the offspring of
a mutagen if it enhances the rate of back-mutation beyond organisms and new viruses and become a long-term part of
levels that would occur spontaneously. One version of this the gene pool. Most mutations are harmful to organisms; oth-
testing procedure is outlined in gure 9.22. The Ames test ers provide adaptive advantages.
If a mutation leading to a nonfunctional protein occurs
6. S. typhimurium inhabits the intestine of poultry and causes food poisoning in a gene for which there is only a single copy, as in haploid
in humans. It is used extensively in genetic studies of bacteria.
or simple organisms, the cell will probably die. This happens
when certain mutant strains of E. coli acquire mutations in
Culture of Salmonella the genes needed to repair damage by UV radiation. Muta-
bacteria, histidine (–) tions of the human genome affecting the action of a single
protein (mostly enzymes) are responsible for more than 3,500
diseases.
Although most spontaneous mutations are not bene cial,
In the control setup, bacteria The experimental plate is a small number contribute to the success of the individual
are plated on a histidine-free prepared the same way
medium containing liver except that it contains the test and the population by creating variant strains with alternate
enzymes but lacking the agent. ways of expressing a trait. Microbes are not “aware” of this
test agent. advantage and do not direct these changes; they simply
respond to the environment they encounter. Those organisms
with bene cial mutations can more readily adapt, survive,
and reproduce. In the long-range view, mutations and the
variations they produce are the raw materials for change in
the population and, thus, for evolution.
(a) Control Plate (b) Test Plate
Mutations that create variants occur frequently enough
Minimal medium Minimal medium
lacking histidine with test chemical that any population contains mutant strains for a number of
and test chemical and no histidine characteristics, but as long as the environment is stable, these
mutants will never comprise more than a tiny percentage of
Incubation (12 h)
the population. When the environment changes, however,
Any colonies that form have
back-mutated to his(+) it can become hostile for the survival of certain individuals,
and only those microbes bearing protective mutations will
be equipped to survive in the new environment. In this way,
the environment naturally selects certain mutant strains that
will reproduce, give rise to subsequent generations, and in
time, be the dominant strain in the population. Through
his(+) colonies arising from his(+) colonies in presence these means, any change that confers an advantage during
spontaneous back-mutation of the chemical
selection pressure will be retained by the population. One of
(c) The degree of mutagenicity of the chemical agent can be the clearest models for this sort of selection and adaptation
calculated by comparing the number of colonies growing on the is acquired drug resistance in bacteria (see chapter 12 ). Bac-
control plate with the number on the test plate. Chemicals that teria have also developed a mechanism for increasing their
induce an increased incidence of back-mutation (right side) are
adaptive capacity through genetic exchange, called genetic
considered carcinogens.
recombination, discussed in the next section.
Figure 9.22 The Ames test. This test is based on a strain of
Salmonella typhimurium that cannot synthesize histidine [his( )]. It lacks 9.4 Learning Outcomes—Can You . . .
the enzymes to repair DNA so that mutations show up readily, and it
has leaky cell walls that permit the ready entrance of chemicals. Many 16. . . . de ne the term “mutation” and discuss its importance?
potential carcinogens (benzanthracene and aflatoxin, for example) 17. . . . differentiate among frameshift, nonsense, silent, and
are mutagenic agents only after being acted on by mammalian liver missense mutations?
enzymes, so an extract of these enzymes is added to the test medium.