Hogg S. Essential microbiology

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298 MICROBIAL GENETICS

Affected

section

removed

Nicks made in

phosphate-suga

backbone either

side of mutation

Mutation

New strand

synthesised

with correct

sequence

Figure 11.22 Excision repair. A section of the affected strand either side of the mutation

is removed and replaced by the action of DNA polymerase and DNA ligase

called DNA photolyase, which breaks the bonds formed between adjacent thymine

bases by UV light (see above) before replication takes place. Unusually, the photolyase

is dependant on visible light (>300 nm) for activation. Another form of direct repair,

involving the enzyme methylguanine transferase allows the reversal of the effects of

alkylating agents.

Loss of repair mechanisms can allow mutations to become established which would

normally be corrected. A harmful strain of E. coli which emerged in the 1980s was

shown to have developed its pathogenicity due to a deﬁciency in its repair enzymes.

Carcinogenicity testing: the Ames test

The great majority of carcinogenic substances, that is, substances that cause cancer

in humans and animals, are also mutagenic in bacteria. This fact has been used to

develop an initial screening procedure for carcinogens; instead of the expensive and

time-consuming process of exposing laboratory animals (not to mention the moral

issues involved), a substance can be tested on bacteria to see if it induces mutations.

The Ames Test assesses the ability of a substance to cause reverse mutations in aux-

otrophic strains of Salmonella that have lost the ability to synthesise the amino acid his-

tidine (his

−

). Rates of back mutation (assessed by the ability to grow in a histidine-free

medium) are compared in the presence and absence of the test substance (Figure 11.23).

A reversion to his

at a rate higher than that of the control indicates a mutagen. Many

substances are procarcinogens, only becoming mutagenic/carcinogenic after metabolic

conversion by mammals; in order to test for these, an extract of rat liver is added to the

experimental system as a source of the necessary enzymes.

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GENETIC TRANSFER IN MICROORGANISMS 299

his

strain of Salmonella

liver homogenate

Plate onto

minimal

medium (no

histidine)

Test

compound

Control

Some spontaneous

His+ colonies

Significantly greater number

of colonies than control

suggests a mutag

Incubate

Figure 11.23 The Ames test. The test assesses the ability of a mutagen to bring about reverse

mutations in a strain of S. typhimurium auxotrophic for histidine. Since it is possible that a

small number of revertants may occur due to spontaneous mutation, results are compared

with a control plate with no added mutagen

Genetic transfer in microorganisms

The various mechanisms of mutation described above result in an alteration in the

genetic make-up of an organism. This can also occur by recombination, in which genetic

material from two cells combines to produce a variant different to either parent cell.

In bacteria, this involves the transfer of DNA from one cell (donor) to another

(recipient). Because transfer occurs between cells of the same generation (unlike the ge-

netic variation brought about by sexual reproduction in eucaryotes, where it is passed

to the next generation), it is sometimes referred to as horizontal transfer. There are

three ways in which gene transfer can occur in bacteria, which we shall explore in the

following sections.

Transformation

Transformation is the simplest of these, and also the ﬁrst to have been described. We have

already referred to the classic experiment of Fred Grifﬁth in 1928, the ﬁrst demonstration

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300 MICROBIAL GENETICS

Injection

Bacteria

Live smooth

Heat-killed smooth

Live rough

Dead mouse

has live smooth

bacteria

Mouse dies

Mouse lives

Mouse dies

Figure 11.24 Transformation: the Grifﬁth experiment. See the text for details. The result

of the fourth experiment showed for the ﬁrst time that genetic material could be passed from

one bacterium to another. From Reece, RJ: Analysis of Genes and Genomes, John Wiley &

Sons, 2003. Reproduced by permission of the publishers.

that genetic transfer can occur in bacteria. Grifﬁth had previously demonstrated the

existence of two strains of the bacterium Streptococcus pneumoniae, which is one of

the causative agents of pneumonia in humans, and is also extremely virulent in mice.

The S (smooth)-form produced a polysaccharide capsule, whilst the R (rough)-form

did not. These formed recognisably different colonies when grown on a solid medium,

but more importantly, differed in their ability to bring about disease in experimental

animals. The R-form, lacking the protective capsule, was easily destroyed by the defence

system of the host. Grifﬁth observed the effects of injecting mice with bacterial cells of

both forms; these are outlined below and in Figure 11.24. The results of experiments

1–3 were predictable, those of experiment 4 startlingly unexpected:

1 Mice injected with live cells of the R-form were unaffected.

2 Mice injected with live cells of the S-form died, and large numbers of S-form bacteria

were recovered from their blood.

3 Mice injected with cells of the S-form that had been killed by heating at 60

◦

C were

unharmed and no bacteria were recovered from their blood.

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GENETIC TRANSFER IN MICROORGANISMS 301

4 Mice injected with a mixture of living R-form and heat-killed S-form cells died, and

living S-form bacteria were isolated from their blood.

The S-form bacteria recovered from the mice in the crucial fourth experiment possessed

a polysaccharide capsule like other S-forms, and, critically, were able to pass on this

characteristic to subsequent generations. This ﬁnding went against the prevailing view

that bacteria simply underwent binary ﬁssion, a completely asexual process involving

no genetic transfer. Grifﬁth deduced that some as yet unknown substance had passed

from the heat-killed S-form cells to some of the living R-forms and conferred on them

the ability to make capsules (see Box 11.7). Not long afterwards it was shown that this

process of transformation could happen in the test tube, without the involvement of a

host animal, and, as we have seen, it was eventually shown that Grifﬁth’s ‘transforming

principle’ was DNA.

How does transformation occur?

The uptake of foreign DNA from the environment is known to occur naturally in a num-

ber of bacterial types, both Gram-positive and Gram-negative, by taking up fragments

of naked DNA released from dead cells in the vicinity. Being linear and very fragile,

the DNA is easily broken into fragments, each carrying on average around 10 genes.

Transformation will only happen at a speciﬁc stage in the bacterial life cycle, when cells

are in a physiological state known as competence. This occurs at different times in dif-

ferent bacteria, but is commonly during late log phase. One of the reasons why only a

low percentage of recipient cells become transformed is that only a small proportion of

them are at any one time in a state of competence. The expression of proteins essential

to the transformation process is dependant on the secretion of a competence factor.

The exact mechanism of transformation varies somewhat according to species; the

process for Bacillus subtilis is shown in Figure 11.25. Mere uptake of exogenous DNA

is not enough to cause transformation; it must also be integrated into the host genome,

displacing a single strand, which is subsequently degraded. Upon DNA replication

and cell division, one daughter cell will inherit the parent genotype, and the other

Box 11.7 Transformation is not due to reverse mutation

At the time of Griffith’s experiment, it was already known that the wildtype S-form

could mutate to the R-form and vice versa. It might therefore be argued that the

results of his fourth experiment could easily be explained away in this way. Griffith’s

experiment was sufficiently well designed to refute this argument, however, because

the bacteria he used were of two different serotypes, meaning that they produced

different types of capsule (types II and III). Even if the IIR-form cells had mutated

back to the wildtype (IIS), they could not have produced the type III capsule Griffith

observed in the bacteria he recovered from the mice. The only explanation is that

the ability to make type III capsules had been acquired from the heat-killed type III

cells.

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302 MICROBIAL GENETICS

Donor DNA

Host

chromosome

Replacement of

host strand by

donor DNA

Degraded host strand

Degraded

second strand

Recombination

ith homologous

sequence

Figure 11.25 Transformation in Bacillus subtilis. (a) A fragment of donor DNA become

bound to the recipient cell surface by means of a DNA-binding protein. (b) After binding,

a nuclease contained at the cell surface degrades one strand of the donor DNA, leaving the

other strand to be ferried, by other transformation-speciﬁc proteins, to the interior of the cell.

chromosome. (d) The donor fragment becomes integrated by a process of non-reciprocal

recombination. At the next cell division, one daughter cell is a transformant, whilst the

other retains the parental genotype

the recombinant genotype. In the latter, there is a stable alteration of the cell’s genetic

composition, and a new phenotype is expressed in subsequent generations.

Uptake of donor DNA from an unrelated species will not result in transformation;

this is due to a failure to locate a homologous sequence and integrate into the host’s

chromosome, rather than an inability to gain entry to the cell.

Induced competence

Transformation is not thought to occur naturally in E. coli, but, if subjected to certain

treatments in the laboratory, cells of this species can be made to take up DNA, even from

a completely unrelated source. This is done by effecting a state of induced or artiﬁcial

competence, and by introducing the foreign DNA in a self-replicating vector molecule,

which does not depend on integration into the host chromosome. As we shall see in the

next chapter, this is of enormous signiﬁcance in the ﬁeld of genetic engineering.

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GENETIC TRANSFER IN MICROORGANISMS 303

Box 11.8 Auxotrophic mutants

A type of mutant that has proved to be of great use to the microbial geneticist is

the auxotrophic mutant. Here, the mutation causes the organism to lack a gene

product, usually an enzyme, involved in the synthesis of a nutrient such as an amino

acid or vitamin. If the nutrient in question is supplied in the culture medium, the

auxotroph can survive quite happily, as its ‘handicap’is not exposed. If the nutrient

is not provided however, as in a minimal medium, the cells would be unable to grow.

Thus microbiologists can detect the existence of an auxotrophic mutant by use of

selective media.

Conjugation

In 1946, Edward Tatum and Joshua Lederberg (the latter aged only 21 and still a

student!) demonstrated a second form of genetic transfer between bacteria. These ex-

periments involved the use of auxotrophic mutants (Box 11.8), which have lost the

ability to make a particular enzyme involved in the biosynthesis of an essential nutri-

ent. These can be recognised experimentally by their inability to grow on a medium

lacking the nutrient in question (Figure 11.26). The results of Tatum and Lederberg’s

experiments suggested that a process was taking place in bacteria akin to sex in eucary-

otes, in which there was a recombination of the cells’ genetic material (Figure 11.27).

Transformation, as demonstrated by Grifﬁth, could not explain their results, since the

addition of a DNA-containing extract of one strain to whole cells of the other did not

result in prototroph formation. This was conﬁrmed in an ingenious experiment in which

Bernard Davis showed that Tatum and Lederberg’s results were only obtained if direct

cell-to cell contact was allowed (Figure 11.28).

Figure 11.26 Auxotrophic mutants provide useful genetic markers. They are unable to

synthesise a particular nutrient, and can be detected by their inability to grow on a minimal

medium (one containing only inorganic salts and a carbon source such as glucose)

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304 MICROBIAL GENETICS

Figure 11.27 Tatum and Lederberg: sexual recombination in bacteria. Two strains with

complementary genotypes (auxotrophic for certain genes, prototrophic for others) are each

unable to grow on a minimal medium. When the two are mixed, however, colonies are

formed, indicating that recombinant bacteria prototrophic for all the genes have been

formed. The bacteria are washed before plating out to avoid the carry-over of nutrients

from the initial broth to the minimal medium. From Black, JG: Microbiology: Principles

and Explorations, 4th edn, John Wiley & Sons Inc., 1999. Reproduced by permission of the

publishers

Gene transfer in conjugation is one way only

The process of conjugation was initially envisaged as a fusion of the two partner cells to

give a diploid zygote, which subsequently underwent meiosis to give haploid offspring

with modiﬁed genotypes. The work of William Hayes, however, showed that the devel-

opment of colonies of recombinant cells was dependant on the survival of only one of

the participating strains, the other strain being required only as a donor of DNA.

It became apparent that in E. coli, there are two distinct mating types, which became

known as F

and F

−

, depending on whether or not they possessed a plasmid called

the F (fertility) plasmid. This contains some 30 or 40 genes responsible for its own

replication and for the synthesis of a thread-like structure expressed on the cell surface

called a sex pilus. In a mixture of F

and F

−

cells, the sex pilus contacts an F

−

cell, then

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GENETIC TRANSFER IN MICROORGANISMS 305

Application of

alternating suction

and pressure

Cells of

genotype

a b

Cells of

genotype

Glass

filter

Figure 11.28 The Davis U-tube experiment. Two bacterial strains were placed in two arms

of a U-tube, separated by a ﬁlter which did not permit the passage of cells. Although suction

was used to transfer medium between compartments, no recombinants resulted. The results

provided direct evidence that cell-to-cell contact is essential for conjugation to occur

contracts, to pull the two cells together. A single strand of plasmid DNA is then passed

across a channel made between the two cells, and enters the F

−

cell (Figure 11.29). This

then serves as a template for the production of the complementary second strand, and

similarly the single strand left behind in the donor cell is replicated, leaving us with a

double-stranded copy of the F plasmid in both cells. By acquiring the F plasmid, the

recipient F

−

cell has been converted to F

When conjugation involves a form of donor cell called Hfr (high frequency of recom-

bination), genes from the main bacterial chromosome may be transferred. In these, the

F plasmid has become integrated into the main bacterial chromosome; and thus loses its

ability to replicate independently (Figure 11.30). It behaves just like any other part of

the chromosome, although of course it still carries the genes for conjugation and pilus

formation. When conjugation occurs, a single strand of Hfr DNA is broken within the F

sequence, and is transferred in a linear fashion, carrying behind it chromosomal DNA.

Recombination in the recipient cell results in the replacement of a homologous segment

of DNA by the transferred material, which is then faithfully replicated in subsequent

generations. If transfer is uninterrupted, a process which takes around 100 minutes in

E. coli, eventually the remaining stretch of F plasmid will enter the F

−

cell, bringing up

the rear. The fragile nature of the pilus means, however, that transfer is rarely complete,

and only a limited portion of the bacterial genome is transferred. This means that those

−

cells receiving DNA from Hfr cells usually remain F

−

, unlike those in a cross with F

cells, because the remainder of the F sequence is not transferred. It soon became clear

that this phenomenon afforded a great opportunity to determine the relative positions of

genes on the bacterial chromosome. This was done by interrupted mating experiments,

in which the time allowed for conjugation is deliberately limited by mechanical break-

age of the sex pili, and correlated with the phenotypic traits transferred to the recipient

cells (Figure 11.31). By these means a genetic map of the bacterial chromosome could

be developed.

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306 MICROBIAL GENETICS

F+ F-

Transfer continues

into the F- cell.

F+ F-

One strand of the

plasmid DNA in the

F+ cell is nicked,

and passes 5’ end

first into the

conjugation tube.

F plasmid

Bacterial

chromosome

F+ F+

Upon completion of

replication, the cells

separate. By gaining

the F plasmid, the F-

cell has been converted

to F+.

= new strand

F+ F-

The single strand in

each cell acts as a

template for the

production of a

second strand by

the ‘rolling circle’

mechanism

Figure 11.29 Conjugation in bacteria. A single-stranded copy of the F plasmid passes

across a conjugation tube from an F

to an F

−

cell. Both this and the copy left behind act

as templates for their own replication, leaving both cells with a complete F plasmid. This

means that the recipient cell is converted from F

−

to F

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F+

F-

Hfr

F factor integrates into

chromosome

Single strand of

F episome

nicked and

passed to F-

cell.

Chromosomal

DNA not

incorporated

is degraded

F factor

Chromosomal

gene transfer

Last part of F

factor. Recipient

cell only becomes

Hfr if this is

transferred.

Hfr

F-

Transferred chromosomal

is replicated and may be

incorporated into F-

chromosome by

recombination

Figure 11.30 Conjugation with an Hfr cell results in transfer of chromosomal genes. Hfr

cells are formed by the integration of the F factor into the bacterial chromosome. Dur-

ing conjugation, transfer begins part of the way along the F episome, and continues with

chromosomal DNA. The amount of chromosomal material transferred depends on how long

conjugation is able to proceed. Conjugation may be followed by recombination of transferred

chromosomal material with its homologous sequence on the recipient cell’s chromosome

307