Hogg S. Essential microbiology

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288 MICROBIAL GENETICS

Figure 11.16 The trp operon. Five structural genes A-E encode enzymes necessary for

the synthesis of tryptophan. (a) In the absence of tryptophan, transcription of the operon

proceeds unhindered. Although a repressor protein is produced, it is inactive and unable to

bind to the operator sequence. (b) Tryptophan activates the repressor by binding to it. This

prevents RNA polymerase from binding to the promoter, and transcription is blocked

own, so the operon is switched off. This is achieved by tryptophan binding to and

activating a repressor protein, which in turn binds to the operator of the trp operon and

prevents transcription of the synthetic enzymes. The tryptophan here is said to act as a

corepressor. As tryptophan is used up and its level in the cell falls, the repressor reverts

to its inactive form, allowing transcription of the tryptophan-synthesising enzymes to

go ahead unhindered.

Global gene regulation

The systems of gene regulation discussed above apply to individual operons; sometimes,

however, a change in environmental conditions necessitates the regulation of many genes

at once. These global regulatory systems respond to stimuli such as oxygen depletion

and temperature change, and utilise a number of different mechanisms.

The molecular basis of mutations

Any alteration made to the DNA sequence of an organism is called a mutation. This may

or may not have an effect on the phenotype (physically manifested properties) of the

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THE MOLECULAR BASIS OF MUTATIONS 289

organism. It may for example enable bacteria to grow without the need for a particular

growth supplement, or confer resistance to an antibiotic. Just how this happened was

for many years a source of debate. Since the mutant forms only became apparent after

the change in conditions (e.g. withdrawal of a nutrient, addition of antibiotic), have

some of the bacteria been induced to adapt to the new conditions, or are mutant forms

arising all the time at a very low frequency, and merely selected by the environmental

change? In 1943, Salvador Luria and Max Delbr

uck devised the ﬂuctuation test to settle

the matter (see Box 11.6). Results of the ﬂuctuation test together with other evidence

led to the understanding that mutations occur spontaneously in nature at a very low

frequency. As we shall see later on in this section, however, they can also be induced

by a variety of chemical and physical agents. Any change to the DNA sequence is

heritable, thus mutations represent a major source of evolutionary variation. Bacteria

make marvellous tools for the study of mutations because of their huge numbers and

very short generation times.

Since the DNA sequence of a gene represents highly ordered coded information,

most mutations have a neutral or detrimental effect on the organism’s phenotype, but

occasionally a mutation occurs which confers an advantage to an organism, making

it better able to survive and reproduce in a particular environment. Mutants that are

favoured in this way may eventually become the dominant type in a population, and,

by steps like this, evolution gradually takes place.

Mutations occur spontaneously in any part of an organism’s genome. Spontaneous

mutations causing an inactivation of gene function occur in bacteria at the rate of about

one in a million for a given gene at each round of cell division. Most genes within a

given organism show similar rates of mutation, relative to their gene size; clearly a larger

‘target’ will be ‘hit’ more often than a small one.

How do mutations occur?

Figure 11.17(a) reminds us how the code in DNA is transcribed into messenger RNA

and then translated into a sequence of amino acids. Each time the DNA undergoes repli-

cation, this same sequence will be passed on, coding for the same sequence of amino

acids. Occasionally, mistakes occur during replication. Cells have repair mechanisms

to minimise these errors, but what happens if a mistake still slips through? In Figure

11.17(b), we can see the effect of one nucleotide being inserted into the strand instead

of another. When the next round of replication occurs, the modiﬁed DNA will act as

a template for a newly synthesised strand, which at this position will be made comple-

mentary to the new, ‘wrong’ base, instead of the original one, and thus the mistake will

be perpetuated.

A missense mutation al-

ters the sense of the mes-

sage encoded in the

DNA, and results in an in-

correct amino acid be-

ing produced at the

point where it occurs.

This is an example of the simplest type of mutation,

a point mutation, where one nucleotide has been substi-

tuted by another. The example shown is a missense muta-

tion, which has resulted in the affected triplet coding for

a different amino acid; this may or may not have an ef-

fect on the phenotype of the organism. RNA polymerase,

which transcribes the DNA sequence into mRNA, is un-

able to tell that an error has occurred, and faithfully

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290 MICROBIAL GENETICS

Box 11.6 Settling an argument: the fluctuation test

Luria and Delbr

uck designed the fluctuation test to show whether resistance in E.

coli to the bacteriophage T1 was induced or occurred spontaneously.

Suppose we divide a broth culture of bacteria into two, then inoculate half into

a flask of fresh broth, and divide the other half between a large number of smaller

cultures in tubes

1 2 3 50 1 2 3 50

Half of culture grown

up in a single large

flask.

Half of culture divided

between 50 small tubes.

1 2 3 50

E. coli

culture

50 samples

plated out

onto agar

pates

Each sample

plated out onto

an agar plate.

Colony counts quite similar Colony counts highly variable

After allowing the bacteria to grow, samples are taken from tubes and flask, and

spread onto a selective agar medium (one covered with T1 phage). All the plates

deriving from the single bulk culture have roughly the same number of resistant

colonies, but the plates resulting from the smaller, tube cultures show very variable

colony counts. Although the average number of colonies across the 50 tubes is

similar to that obtained from the bulk culture, the individual plate counts varies

greatly, from none on several plates to over a hundred on another.

If the phage-resistance was induced by the presence of the phage, we would

expect all plates in the experiment to give rise to approximately the same number

of resistant colonies, as all cultures experienced the same exposure. If however, the

resistant forms were spontaneously arising all the time at a low level in the population,

the numbers of resistant colonies would be dependent on when, if at all, a mutation

had taken place in a particular tube culture. A mutant arising early in the incubation

period would give rise to more resistant offspring and therefore more colonies than

one that arose later.

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THE MOLECULAR BASIS OF MUTATIONS 291

Figure 11.17 Mutations can alter the sense of the DNA message. (a) A short sequence of

DNA is transcribed into mRNA and then transcribed into the corresponding amino acids. (b)

A single base change from T to A results in a missense mutation, as a histidine is substituted

for a leucine. (c) A silent mutation has altered the DNA (and therefore mRNA) sequence, but

has not changed its sense. Both AGG and AGA are mRNA triplets that code for arginine. (d)

A nonsense mutation has replaced a tryptophan codon with a STOP codon, hence bringing

the peptide chain to a premature end

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292 MICROBIAL GENETICS

transcribes the misinformation. The machinery of translation is similarly ‘unaware’ of

the mistake, and as a consequence, a different amino acid will be inserted into the

polypeptide chain. The consequence of expressing a ‘wrong’ amino acid in the protein

product could range from no effect at all to a total loss of its biological properties. This

can be understood in terms of protein structure (Chapter 2), and depends on whether the

amino acid affected has a critical role (such as part of the active site of an enzyme), and

whether the replacement amino acid has similar or different polar/non-polar properties.

You may recall from the beginning of this chapter that the genetic code is degenerate,

and that most amino acids are coded for by more than one triplet; this means that some

mutations do not affect the amino acid produced; such mutations are said to be silent,

as in Figure 11.17(c). These most commonly occur at the third nucleotide of a triplet.

A nonsense mutation re-

sults in a ‘stop’ codon

being inserted into the

mRNA at the point

where it occurs, and

the premature termina-

tion of translation.

Another type of point mutation is a nonsense muta-

tion. Remember that of the 64 possible triplet permuta-

tions of the four DNA bases, three are ‘stop’ codons,

which terminate a polypeptide chain. If a triplet is

changed from a coding to a ‘stop’ codon as shown in

Figure 11.17(d), then instead of the whole coding se-

quence being read, translation will end at this point, and

a truncated (and probably non-functional) protein will

result.

Mutations can add or remove nucleotides

Other mutations involve the insertion or deletion of nucleotides. This may involve

anything from a single nucleotide up to millions. Deletions occur as a result of the

A frameshift mutation re-

sults in a change to

the reading frame, and

an altered sequence of

amino acids results

downstream of the point

where it occurs.

replication machinery somehow ‘skipping’ one or more

nucleotides. If the deletion is a single nucleotide, or any-

thing other than a multiple of three, the ribosome will be

thrown out of its correct reading frame, and a completely

new set of triplet codons will be read (Figure 11.18). This

is known as a frameshift mutation, and will in most cases

result in catastrophic changes to the ﬁnal protein prod-

uct. If the deletion is a multiple of three nucleotides, the

reading frame will be preserved and the effect on the

protein less drastic.

Mutations can be reversed

Just as it is possible for a mutation to occur spontaneously, so it is possible for the

nucleotide change causing it to be spontaneously reversed – in other words, a mutant

can mutate back to being a wildtype. This is known for obvious reasons as a reverse or

back mutation. When this happens, the original genotype and phenotype are restored.

Whereas a forward mutation results from any change that inactivates a gene, a back

mutation is more speciﬁc; it must restore function to a protein damaged by a speciﬁc

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THE MOLECULAR BASIS OF MUTATIONS 293

Figure 11.18 Frameshift mutations. (a) A short sequence of DNA is transcribed into

mRNA and then transcribed into the corresponding amino acids. (b) The fourth nucleotide

in the sequence is deleted, upsetting the groups of triplets or reading frame. This alters the

sense of the remainder of the message, leading to a completely different sequence of amino

acids

mutation. Not surprisingly, given this speciﬁcity, the rate of back mutations is much less

frequent.

It is possible for the wildtype phenotype to be restored, not through a reversal of the

original base change, but due to a second mutation at a different location. The effect of

this second mutation is to suppress the effects of the ﬁrst one. These are called suppressor

or second site mutations. They are double mutants that produce a pseudowildtype; the

phenotype appears to be wildtype, but the genotype differs.

Mutations have a variety of mechanisms

Why are mistakes made every so often during DNA replication? One source of erro-

neous base incorporation is a phenomenon called tautomerism. Some of the nucleotide

bases occur in rare alternative forms, which have different base-pairing properties. For

example, as you will recall from Chapter 2, cytosine normally has an amino group that

provides a hydrogen atom for bonding with the complementary keto group of gua-

nine. Occasionally, however (once in about every 10

–10

molecules), the cytosine may

undergo a rearrangement called a tautomeric shift, which results in the amino group

changing to an imino group (

NH), and this now behaves in pairing terms as if it were

thymine, and therefore pairs with adenine (Figure 11.19a). Similarly, thymine might

undergo a tautomeric shift, changing its usual keto form (C

O) to the rare enol form

(COH). This then takes on the pairing properties of cytosine and pairs with guanine (Fig-

ure 11.19b). The result of such mispairing is that at subsequent rounds of replication,

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294 MICROBIAL GENETICS

Figure 11.19 Rare forms of nucleotides may cause mispairing. If DNA replication takes

place whilst a base is in its rare alternative form, an incorrect base will be incorporated into

the newly synthesised second strand. (a) In its normal keto form, thymine pairs with adenine

in keeping with the rules of base-pairing. In its rare enol form, however, it preferentially

pairs with guanine. (b) Cytosine can likewise assume the rare imino form and mispair with

adenine

one half of the DNA molecules will contain a wrong base pair at that point. The fact

that spontaneous mutations occur much less frequently than the rate just quoted is due

to the DNA repair mechanisms discussed earlier in this chapter.

Mutations also occur in viruses

As we saw in Chapter 10, the genome of viruses can be composed of either DNA or

RNA, and these are subject to mutation just like cellular genomes. The rate of mutation

in RNA viruses is much higher than in those containing DNA, about a thousand times

higher, in fact. This is signiﬁcant, because by frequently changing in this way, pathogenic

viruses are able to stay one step ahead of host defence systems.

Mutagenic agents increase the rate of mutations

In the early part of the 20th century, the existence of mutations was appreciated, but

attempts to gain a fuller understanding of them were hampered by the fact that they

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THE MOLECULAR BASIS OF MUTATIONS 295

occurred so rarely. In the 1920s, however, it was shown that the mutation rate in both

barley plants and the fruit ﬂy Drosophila was greatly increased as a result of exposure to

X-rays. In the following decades, a number of chemical and physical agents were shown

Chemical or physical

agents capable of in-

ducing mutations are

termed mutagens.

also to cause mutations. Mutagens such as these raise

the general level of mutations in a population, rather

than the incidence of mutation in at particular location

in the genome. They are extremely useful tools for the

microbial geneticist, but need to be treated with great

care because they are also mutagenic (and in most cases

carcinogenic) towards humans.

Chemical mutagens can be divided into ﬁve classes:

Base analogues

A base analogue is able to ‘mimic’ one of the four normal DNA bases by having a

chemical structure sufﬁciently similar for it to be incorporated into a DNA molecule

instead of that base during replication. One such base is 5-bromouracil (5-BU); in its

usual keto form, it acts like thymine (which it closely resembles – see Figure 11.20), and

therefore pairs with adenine, and is not mutagenic. However 5-BU is capable of tau-

tomerising to the enol form, which, remember, pairs with G, not A. Whereas for thymine

the enol form is a rarity, for 5-BU it is more common, hence mispairing with guanine

occurs more frequently. So, we get the same outcome as with spontaneous mutation –

the TA has been replaced by a CG – but its frequency is much increased. Several other

base analogues, such as 2-aminopurine, which mimics adenine, have similar effects to

5-BU. Mutations brought about by base analogues all result in a purine being replaced

by another purine, or a pyrimidine being replaced by another pyrimidine; this type of

mutation is called a transition.

Alkylating agents

As the name suggests, this group of mutagens act by adding an alkyl group (e.g. methyl,

ethyl) at various positions on DNA bases. Ethylethanesulphonate (EES) and ethyl-

methanesulphonate (EMS) are alkylating agents, used as laboratory mutagens. They

act by alkylating guanine or thymine at the oxygen atom involved in hydrogen bond-

ing. This leads to an impairment of the normal base-pairing properties, and causes

guanine for example to mispair with thymine.

Substitutions brought about by alkylating agents can be either transitions or trans-

versions.

Deaminating agents

Nitrous acid is a potent mutagen, which alters the base-pairing afﬁnities of cytosine and

adenine by replacing an amino group with an oxygen atom. Transitions occur in both

directions, AT → GC and GC → AT. Note that although guanine is deaminated, its

base pairing properties are not affected.

Intercalating agents

These mutagens exert their effect by inserting themselves between adjacent nucleotides

in a single strand of DNA. They distort the strand at this site and cause either the addition

or, less commonly, deletion of a nucleotide. This leads to frameshift mutations, and often

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296 MICROBIAL GENETICS

Figure 11.20 Base analogues mimic DNA base structure. In its keto form, the base analogue

5-bromouracil (5-BU) forms base pairs with adenine, but in the enol form it mispairs with

guanine. 5-BU differs from thymine only in the substitution of a bromine atom in place of a

methyl group. This makes the occurrence of a tautomeric shift more likely

during replication the bound mutagen interferes with new strand synthesis, leading to a

gross deletion of nucleotides. Ethidium bromide, commonly used for the visualisation of

small amounts of DNA in the molecular biology laboratory, is an intercalating agent; it

has a planar structure with roughly the same dimensions as a purine–pyrimidine pairing.

Hydroxylating agents

Hydroxylamine has a speciﬁc mutagenic effect, hydroxylating the amino group of cy-

tosine to cause the transition of GC → AT.

Base analogues and certain intercalating agents can only exert their mutagenic effects

if they are incorporated into DNA while it is replicating. Others, which depend on

altering base pairing by modifying the structure of DNA bases, are effective on both

replicating and non-replicating DNA.

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THE MOLECULAR BASIS OF MUTATIONS 297

Figure 11.21 Thymine dimer formation. Absorption of UV light results in the formation

of dimers between adjacent thymine residues in the same strand. This can lead to errors

being introduced into the DNA sequence. From Gardner, EJ, Simmons MJ and Snustad, DP:

Principles of Genetics, 8th edn, John Wiley & Sons Inc., 1991. Reproduced by permission

of the publishers.

Physical mutagens

As mentioned at the start of this section, the ﬁrst mutagenic agent to be demonstrated

were X-rays; along with ultraviolet light, they are the most commonly used physical

mutagen. X-rays, like other forms of ionising radiation, cause the formation of highly

reactive free radicals. These can bring about changes in base structure as well as gross

chromosomal alterations. DNA strands can be broken and reannealed incorrectly, to

produce errors in the sequence.

In recent years, we have all become much more aware of the possible perils of ex-

cessive exposure to sunlight. Ultraviolet light (UV) from the sun damages DNA in skin

cells, which can lead to them becoming cancerous. The speciﬁc action is on adjacent

pyrimidine bases (usually thymines) on the same strand, which are cross-linked to form a

dimer (Figure 11.21). This results in a distortion of the helix and interferes with replica-

tion. UV light is most effective at wavelengths around 260 nm, as this is the wavelength

most strongly absorbed by the DNA bases.

DNA damage can be repaired

All organisms have developed the means to repair damage to their DNA, in addition to

the proofreading mechanism described earlier

The most common way of dealing with mutations is by means of a method called

excision repair, in which enzymes recognise and cut out the altered region of DNA

and then ﬁll in the missing bases, using the other strand as a template (Figure 11.22).

Mismatch repair is used to repair the incorporation of a base that has escaped the

proofreading system. In a situation such as this, it is not immediately obvious which

is the correct base and which is the mistake, so how does the cell know which strand

to replace? In E. coli, the old strand is distinguished from the new by the fact that

some of its bases are methylated. This only occurs some time after replication, so newly

synthesised strands will not have the methyl groups and can thus be recognised.

Less frequently, the alteration in DNA structure is simply reversed by direct repair

mechanisms, the best known of which is photoreactivation. This involves an enzyme