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1: Genetic Analysis of Bacteria Requires Special Approaches and Methods

1: Genetic Analysis of Bacteria Requires Special Approaches and Methods

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Bacterial and Viral Genetic Systems
(a)

(b)

Inoculating
loop

Pipet

Inoculate medium
with bacteria.

Bacteria grow
and divide.

Glass rod

Lid

Dilute soluion
of bacterial cells
Sterile
liquid
medium

A growth medium
is suspended in
gelatin-like agar.

Petri plate

Add a dilute
solution of bacteria
to petri plate.

Spread bacterial
solution evenly
with glass rod.

After incubation for
1 to 2 days, bacteria
multiply, forming
visible colonies.

6.2 Bacteria can be grown (a) in liquid medium or (b) on solid medium.
phorus, certain vitamins, and other required ions and nutrients. Wild-type (prototrophic) bacteria can use these simple
ingredients to synthesize all the compounds that they need
for growth and reproduction. A medium that contains only
the nutrients required by prototrophic bacteria is termed
minimal medium. Mutant strains called auxotrophs lack one
or more enzymes necessary for metabolizing nutrients or
synthesizing essential molecules and will grow only on
medium supplemented with one or more nutrients. For
example, auxotrophic strains that are unable to synthesize
the amino acid leucine will not grow on minimal medium
but will grow on medium to which leucine has been added.
Complete medium contains all the substances, such as the
amino acid leucine, required by bacteria for growth and
reproduction.
Cultures of bacteria are often grown in test tubes that
contain sterile liquid medium (Figure 6.2a). A few bacteria
are added to a tube, and they grow and divide until all the
nutrients are used up or—more commonly—until the concentration of their waste products becomes toxic. Bacteria
are also grown in petri plates (Figure 6.2b). Growth medium
suspended in agar is poured into the bottom half of the petri
plate, providing a solid, gel-like base for bacterial growth. In
a process called plating, a dilute solution of bacteria is spread

(a)

141

over the surface of an agar-filled petri plate. As each bacterium grows and divides, it gives rise to a visible clump of
genetically identical cells (a colony). Genetically pure strains
of the bacteria can be isolated by collecting bacteria from a
single colony and transferring them to a new test tube or
petri plate. The chief advantage of growing bacteria on a
petri plate is that it allows one to isolate and count bacteria,
which individually are too small to see without a microscope.
Because individual bacteria are too small to be seen
directly, it is often easier to study phenotypes that affect the
appearance of the colony (Figure 6.3) or can be detected by
simple chemical tests. Auxotrophs are commonly studied
phenotypes. Suppose we want to detect auxotrophs that cannot synthesize leucine (leuϪ mutants). We first spread the
bacteria on a petri plate containing medium that includes
leucine; both prototrophs that have the leuϩ allele and auxotrophs that have leuϪ alleles will grow on it (Figure 6.4).
Next, using a technique called replica plating, we transfer a
few cells from each of the colonies on the original plate to
two new replica plates: one plate contains medium to which
leucine has been added; the other plate contains selective
medium—that is, a medium in this case lacking leucine. The
leuϩ bacteria will grow on both media, but the leuϪ mutants
will grow only on the medium supplemented by leucine,

(b)

6.3 Bacteria have a variety of
phenotypes. (a) Serratia marcescens
with color variation. (b) Bacillus cereus.
[Part a: Dr. E. Bottone/Peter Arnold. Part
b: Biophoto Associates/Photo
Researchers.]

1 Plate bacteria on medium
containing leucine. Both
leu+ and leu– colonies grow.

2 Replica plate the colonies
by pressing a velvet surface
to the plate.

3 Cells adhere
to velvet.

4 Press onto new petri plates.
Cells from each colony are
transferred to new plates.

Handle

Medium
lacking leucine

5 Leucine auxotrophs (leu–) are
recovered from the colony
growing on supplemented
medium and cultured for
further study.

Only leu +
bacteria grow
Missing
colony

Velvet surface
(sterilized)

Bacterial
culture

Culture

Both leu + and leu –
bacteria grow

Medium
with leucine

6.4 Mutant bacterial strains can be isolated on the basis of
their nutritional requirements.

because they cannot synthesize their own leucine. Any
colony that grows on medium that contains leucine but not
on medium that lacks leucine consists of leuϪ bacteria. The
auxotrophs that grow on the supplemented medium can
then be cultured for further study.

The Bacterial Genome
Bacteria are unicellular organisms that lack a nuclear membrane. Most bacterial genomes consist of a circular chromosome that contains a single DNA molecule several million

Conclusion: A colony that grows only on the supplemented
medium has a mutation in a gene that encodes the
synthesis of an essential nutrient.

base pairs (bp) in length (Figure 6.5). For example, the
genome of E. coli has approximately 4.6 million base pairs of
DNA. However, some bacteria (such as Vibrio cholerae,
which causes cholera) contain multiple chromosomes, and a
few even have linear chromosomes.

Plasmids
In addition to having a chromosome, many bacteria possess
plasmids—small, circular DNA molecules (Figure 6.6).

6.5 Most bacterial cells possess a single, circular
chromosome, shown here emerging from a ruptured
bacterial cell. [David L. Nelson and Michael M. Cox, Lehninger
Principles of Biochemistry, 4th ed. (New York: Worth Publishers, 2004),
from Huntington Potter and David Dressler, Harvard Medical School,
Department of Neurobiology.]

6.6 Many bacteria contain plasmids—small, circular molecules of DNA. [Professor Stanley N. Cohen/Photo Researchers.]

Bacterial and Viral Genetic Systems

1 Replication in a plasmid begins at the
origin of replication, the ori site.

Origin of replication
(ori site)

Strand
separation

3 …eventually producing two
circular DNA molecules.

2 Strands separate and replication
takes place in both directions,…

Newly synthesized
DNA
Separation
of daughter
plasmids

Replication

Double-stranded DNA

New strand

Strands separate
at oriV

Old strand

6.7 A plasmid replicates independently of its bacterial chromosome. Replication begins at the
origin of replication (ori) and continues around the circle. In this diagram, replication is taking place in
both directions; in some plasmids, replication is in one direction only.
Some plasmids are present in many copies per cell, whereas
others are present in only one or two copies. In general, plasmids carry genes that are not essential to bacterial function
but that may play an important role in the life cycle and
growth of their bacterial hosts. Some plasmids promote mating between bacteria; others produce compounds that kill
other bacteria. Of great importance, plasmids are used extensively in genetic engineering (see Chapter 14), and some of
them play a role in the spread of antibiotic resistance among
bacteria.
Most plasmids are circular and several thousand base
pairs in length, although plasmids consisting of several hundred thousand base pairs also have been found. Possessing
its own origin of replication, a plasmid replicates independently of the bacterial chromosome. Replication proceeds
from the origin in one or two directions until the entire
plasmid is copied. In Figure 6.7, the origin of replication is
ori. A few plasmids have multiple replication origins.
Episomes are plasmids that are capable of freely replicating and able to integrate into the bacterial chromosomes.
The F (fertility) factor of E. coli (Figure 6.8) is an episome
that controls mating and gene exchange between E. coli cells,
as will be discussed shortly.

✔ Concept Check 1
Which is true of plasmids?
a. They are composed of RNA.
b. They normally exist outside of bacterial cells.
c. They possess only a single strand of DNA.
d. They replicate independently of the bacterial chromosome.

These sequences regulate
insertion into the bacterial
chromosome.

These genes regulate
plasmid transfer to
other cells.

IS3
S
G
H
F
N
U
C
B
K
E
L
A
J
O

DI

oriT (origin
of transfer)

IS2

rep inc

Concepts
Bacteria can be studied in the laboratory by growing them on
defined liquid or solid medium. A typical bacterial genome consists
of a single circular chromosome that contains several million base
pairs. Some bacterial genes may be present on plasmids, which are
small, circular DNA molecules that replicate independently of the
bacterial chromosome.

ori (origin
of replication)

These genes control
plasmid replication.
F factor

6.8 The F factor, a circular episome of E. coli, contains a
number of genes that regulate transfer into the bacterial cell,
replication, and insertion into the bacterial chromosome.
Replication is initiated at ori. Insertion sequences IS3 and IS2 control
both insertion into the bacterial chromosome and excision from it.

143

144

Chapter 6

Gene Transfer in Bacteria
Bacteria exchange genetic material by three different mechanisms, all entailing some type of DNA transfer and recombination between the transferred DNA and the bacterial
chromosome.
1. Conjugation takes place when genetic material passes
directly from one bacterium to another (Figure 6.9a).
In conjugation, two bacteria lie close together and a
connection forms between them. A plasmid or a part
(a) Conjugation
Donor
cell

Cytoplasmic bridge
forms.

of the bacterial chromosome passes from one cell (the
donor) to the other (the recipient). Subsequent to
conjugation, crossing over may take place between
homologous sequences in the transferred DNA and the
chromosome of the recipient cell. In conjugation, DNA
is transferred only from donor to recipient, with no
reciprocal exchange of genetic material.
2. Transformation takes place when a bacterium takes
up DNA from the medium in which it is growing

DNA replicates and transfers
from one cell to the other.

A crossover
in the recipient
cell leads to…

Recipient
cell

…the creation of
a recombinant
chromosome.

Degraded
DNA

Bacterial
chromosome

Transferred DNA
replicates.

(b) Transformation

Naked DNA is
taken up by the
recipient cell.

A crossover in
the bacterium
leads to…

…the creation of
a recombinant
chromosome.

DNA
fragments

(c) Transduction
A virus attaches to
a bacterial cell,…

…injects
its DNA,…

…and replicates,
taking up bacterial
DNA. The bacterial
cell lyses.

The virus infects
a new bacterium,…

…carrying
bacterial
DNA with it.

A crossover in
the recipient
cell leads to…

6.9 Conjugation, transformation, and transduction are three processes of gene transfer in
bacteria. For the transferred DNA to be stably inherited, all three processes require the transferred DNA
to undergo recombination with the bacterial chromosome.

…the creation of
a recombinant
chromosome.

Bacterial and Viral Genetic Systems

(Figure 6.9b). After transformation, recombination may
take place between the introduced genes and those of
the bacterial chromosome.
3. Transduction takes place when bacterial viruses
(bacteriophages) carry DNA from one bacterium to
another (Figure 6.9c). Inside the bacterium, the newly
introduced DNA may undergo recombination with the
bacterial chromosome.

Experiment
Question: Do bacteria exchange genetic information?
Methods

Not all bacterial species exhibit all three types of genetic
transfer. Conjugation takes place more frequently in some
species than in others. Transformation takes place to a limited extent in many species of bacteria, but laboratory techniques increase the rate of DNA uptake. Most bacteriophages
have a limited host range; so transduction is normally
between bacteria of the same or closely related species only.
These processes of genetic exchange in bacteria differ from
diploid eukaryotic sexual reproduction in two important ways.
First, DNA exchange and reproduction are not coupled in bacteria. Second, donated genetic material that is not recombined
into the host DNA is usually degraded, and so the recipient cell
remains haploid. Each type of genetic transfer can be used to
map genes, as will be discussed in the following sections.

Y10

Y24


leu – thi bio +phe +
thr –
cys +


+
leu + thi bio phe –
thr +
cys –

Bacterial
chromosome
1 Auxotrophic bacterial strain
Y10 cannot synthesize
Thr, Leu, or Thi…

2 …and strain Y24
cannot synthesize
biotin, Phe, or Cys,…

Concepts
DNA may be transferred between bacterial cells through conjugation, transformation, or transduction. Each type of genetic transfer consists of a one-way movement of genetic information to the
recipient cell, sometimes followed by recombination. These
processes are not connected to cellular reproduction in bacteria.
3 …and so neither auxotrophic strain
can grow on minimal medium.

✔ Concept Check 2
Which process of DNA transfer in bacteria requires a virus?
a. Conjugation

c. Transformation

b. Transduction

d. All of the above

4 When strains Y10 and
Y24 are mixed,…

Conjugation
In 1946, Joshua Lederberg and Edward Tatum demonstrated
that bacteria can transfer and recombine genetic information,
paving the way for the use of bacteria in genetic studies. In the
course of their research, Lederberg and Tatum studied auxotrophic strains of E. coli. The Y10 strain required the amino
acids threonine (and was genotypically thrϪ) and leucine
(leuϪ) and the vitamin thiamine (thiϪ) for growth but did not
require the vitamin biotin (bioϩ) or the amino acids phenylalanine (pheϩ) and cysteine (cysϩ); the genotype of this strain can
be written as thrϪ leuϪ thiϪ bioϩ pheϩ cysϩ. The Y24 strain
required biotin, phenylalanine, and cysteine in its medium, but
it did not require threonine, leucine, or thiamine; its genotype
was thrϩ leuϩ thiϩ bioϪ pheϪ cysϪ. In one experiment,
Lederberg and Tatum mixed Y10 and Y24 bacteria together and
plated them on minimal medium (Figure 6.10). Each strain
was also plated separately on minimal medium.

Results
+
+
leu + thi bio phe +
thr +
cys +

5 …some colonies 6 …because genetic
recombination has
grow…
taken place and
bacteria can synthesize
all necessary nutrients.
Conclusion: Yes, genetic exchange and recombination
took place between the two mutant strains.

6.10 Lederberg and Tatum’s experiment demonstrated that
bacteria undergo genetic exchange.

145

146

Chapter 6

Alone, neither Y10 nor Y24 grew on minimal medium.
Strain Y10 was unable to grow, because it required threonine,
leucine, and thiamine, which were absent in the minimal
medium; strain Y24 was unable to grow, because it required
biotin, phenylalanine, and cysteine, which also were absent
from the minimal medium. When Lederberg and Tatum
mixed the two strains, however, a few colonies did grow on
the minimal medium. These prototrophic bacteria must
have had genotype thrϩ leuϩ thiϩ bioϩ pheϩ cysϩ. Where had
they come from?
If mutations were responsible for the prototrophic
colonies, then some colonies should also have grown on the
plates containing Y10 or Y24 alone, but no bacteria grew on
these plates. Multiple simultaneous mutations (thrϪ S thrϩ,
leuϪ S leuϩ, and thiϪ S thiϩ in strain Y10 or bioϪ S bioϩ,
pheϪ S pheϩ, and cysϪ S cysϩ in strain Y24) would have
been required for either strain to become prototrophic by
mutation, which was very improbable. Lederberg and Tatum
concluded that some type of genetic transfer and recombination had taken place:
Auxotrophic strain
Y10
Y24

Experiment
Question: How did the genetic exchange seen in
Lederberg and Tatum’s experiment take place?
Methods Auxotrophic
strain A

Airflow

Strain A

thrϪ leuϪ thiϪ

bioϩ pheϩ cysϩ

thrϩ leuϩ thiϩ

bioϪ pheϪ cysϪ

Strain B

Two auxotrophic
strains were separated
by a filter that allowed
mixing of medium but
not bacteria.
No prototrophic
bacteria were produced

thrϪ leuϪ thiϪ bioϩ pheϩ cysϩ
thrϩ leuϩ thiϩ bioϪ pheϪ cysϪ

Auxotrophic
strain B

Results

Minimal
medium

Minimal
medium

Minimal
medium

Minimal
medium

No growth

No growth

No growth

No growth

Conclusion: Genetic exchange requires direct contact
between bacterial cells.

thrϪ leuϪ thiϪ bioϪ pheϪ cysϪ
Prototrophic strain

6.11 Davis’s U-tube experiment.

thrϩ leuϩ thiϩ bioϩ pheϩ cysϩ

What they did not know was how it had taken place.
To study this problem, Bernard Davis constructed a
U-shaped tube (Figure 6.11) that was divided into two compartments by a filter having fine pores. This filter allowed liquid medium to pass from one side of the tube to the other, but
the pores of the filter were too small to allow the passage of bacteria. Two auxotrophic strains of bacteria were placed on
opposite sides of the filter, and suction was applied alternately
to the ends of the U-tube, causing the medium to flow back and
forth between the two compartments. Despite hours of incubation in the U-tube, bacteria plated out on minimal medium
did not grow; there had been no genetic exchange between the
strains. The exchange of bacterial genes clearly required direct
contact, or conjugation, between the bacterial cells.

Fϩ and FϪ cells In most bacteria, conjugation depends
on a fertility (F) factor that is present in the donor cell and

absent in the recipient cell. Cells that contain F factor are
referred to as Fϩ, and cells lacking F factor are FϪ.
The F factor contains an origin of replication and a
number of genes required for conjugation (see Figure 6.8).
For example, some of these genes encode sex pili (singular,
pilus), slender extensions of the cell membrane. A cell containing F factor produces the sex pili, one of which makes
contact with a receptor on an FϪ cell (Figure 6.12) and pulls
the two cells together. DNA is then transferred from the Fϩ
cell to the FϪ cell. Conjugation can take place only between
a cell that possesses F factor and a cell that lacks F factor.
In most cases, the only genes transferred during conjugation between an Fϩ and FϪ cell are those on the F factor
(Figure 6.13a and b). Transfer is initiated when one of the
DNA strands on the F factor is nicked at an origin (oriT ).
One end of the nicked DNA separates from the circle and
passes into the recipient cell (Figure 6.13c). Replication takes

Bacterial and Viral Genetic Systems

factor is integrated into the bacterial chromosome (Figure
6.14). Hfr cells behave as Fϩ cells, forming sex pili and
undergoing conjugation with FϪ cells.
In conjugation between Hfr and FϪ cells (Figure 6.15a),
the integrated F factor is nicked, and the end of the nicked
strand moves into the FϪ cell (Figure 6.15b), just as it does
in conjugation between Fϩ and FϪ cells. Because, in an Hfr
cell, the F factor has been integrated into the bacterial chromosome, the chromosome follows it into the recipient cell.
How much of the bacterial chromosome is transferred
depends on the length of time that the two cells remain in
conjugation.
Inside the recipient cell, the donor DNA strand is replicated (Figure 6.15c), and crossing over between it and the
original chromosome of the FϪ cell (Figure 6.15d) may
take place. This gene transfer between Hfr and FϪ cells is
how the recombinant prototrophic cells observed by
Lederberg and Tatum were produced. After crossing over
has taken place in the recipient cell, the donated chromosome is degraded, and the recombinant recipient chromosome remains (Figure 6.15e), to be replicated and passed
on to later generations of bacterial cells by binary fission
(cell division).
In a mating of Hfr ϫ FϪ, the FϪ cell almost never
becomes Fϩ or Hfr, because the F factor is nicked in the middle in the initiation of strand transfer, placing part of the F
factor at the beginning and part at the end of the strand to be
transferred. To become Fϩ or Hfr, the recipient cell must
receive the entire F factor, requiring the entire bacterial chromosome to be transferred. This event happens rarely,
because most conjugating cells break apart before the entire
chromosome has been transferred.
The F plasmid in Fϩ cells integrates into the bacterial
chromosome, causing an Fϩ cell to become Hfr, at a

6.12 A sex pilus connects Fϩ and FϪ cells during bacterial
conjugation. E. coli cells in conjugation. [Dr. Dennis Kunkel/Phototake.]

place on the nicked strand, proceeding around the circular
plasmid in the Fϩ cell and replacing the transferred strand
(Figure 6.13d). Because the plasmid in the Fϩ cell is always
nicked at the oriT (origin of transfer) site, this site always
enters the recipient cell first, followed by the rest of the plasmid. Thus, the transfer of genetic material has a defined
direction. Inside the recipient cell, the single strand is replicated, producing a circular, double-stranded copy of the F
plasmid (Figure 6.13e). If the entire F factor is transferred to
the recipient FϪ cell, that cell becomes an Fϩ cell.

Hfr cells Conjugation transfers genetic material in the F

plasmid from Fϩ to FϪ cells but does not account for the
transfer of chromosomal genes observed by Lederberg and
Tatum. In Hfr (high-frequency recombination) strains, the F

(a)
F+ cell
F– cell
(donor
(recipient
bacterium) bacterium)

(b)

(c)

F+

F–

147

(d)

F–

F+

(e)

F+

F–

F+

F+

5‘

F factor

Bacterial
chromosome
During conjugation,
a cytoplasmic
connection forms.

One DNA strand of
the F factor is nicked
at an origin and
separates.

Replication takes
place on the F
factor, replacing
the nicked strand.

The 5‘ end of
the nicked DNA
passes into the
recipient cell…

6.13 The F factor is transferred during conjugation between an Fϩ and FϪ cell.

…where the
single strand
is replicated,…

…producing a
circular, doublestranded copy
of the F plasmid.

The F– cell
now
becomes F+.

148

Chapter 6

F+ cell

Bacterial
chromosome

transfer. Characteristics of different mating types of E. coli
(cells with different types of F) are summarized in Table 6.2.
During conjugation between an FЈlac cell and an FϪ cell,
the F plasmid is transferred to the FϪ cell, which means that
any genes on the F plasmid, including those from the bacterial chromosome, may be transferred to FϪ recipient cells. This
process is called sexduction. It produces partial diploids, or
merozygotes, which are cells with two copies of some genes,
one on the bacterial chromosome and one on the newly
introduced F plasmid. The outcomes of conjugation between
different mating types of E. coli are summarized in Table 6.3.

Hfr cell

F factor
Crossing over takes place between
F factor and chromosome.

The F factor is integrated
into the chromosome.

Concepts

6.14 The F factor is integrated into the bacterial chromosome in an Hfr cell.

frequency of only about 1΋10,000. This low frequency accounts
for the low rate of recombination observed by Lederberg and
Tatum in their Fϩ cells. The F factor is excised from the bacterial chromosome at a similarly low rate, causing a few Hfr
cells to become Fϩ.

Conjugation in E. coli is controlled by an episome called the F factor. Cells containing F (Fϩ cells) are donors during gene transfer; cells
without F (FϪ cells) are recipients. Hfr cells possess F integrated into
the bacterial chromosome; they donate DNA to FϪ cells at a high
frequency. FЈ cells contain a copy of F with some bacterial genes.

✔ Concept Check 3
Conjugation between an Fϩ and an FϪ cell usually results in
a. two Fϩ cells.
Ϫ

FЈ cells When an F factor does excise from the bacterial
chromosome, a small amount of the bacterial chromosome
may be removed with it, and these chromosomal genes will
then be carried with the F plasmid (Figure 6.16). Cells containing an F plasmid with some bacterial genes are called F
prime (FЈ). For example, if an F factor integrates into a chromosome adjacent to the lac genes (genes that enable a cell to
metabolize the sugar lactose), the F factor may pick up lac
genes when it excises, becoming FЈlac. FЈ cells can conjugate
with FϪ cells, given that FЈ cells possess the F plasmid with all
the genetic information necessary for conjugation and gene
(a)
Hfr cell

(b)

b. two F cells.

c. an Fϩ and an FϪ cell.
d. an Hfr cell and an Fϩ cell.

Mapping bacterial genes with the use of interrupted
conjugation The transfer of DNA that takes place dur-

ing conjugation between Hfr and FϪ cells allows bacterial
genes to be mapped. In conjugation, the chromosome of
the Hfr cell is transferred to the FϪ cell. Transfer of the
entire E. coli chromosome requires about 100 minutes; if
conjugation is interrupted before 100 minutes have
elapsed, only part of the chromosome will pass into the FϪ

(c)

(d)

F– cell

F factor
Bacterial
chromosome

(e)
Hfr cell

F– cell

Hfr chromosome
(F factor plus
bacterial genes)
In conjugation, F is
nicked and the 5’ end
moves into the F– cell.

The transferred
strand is
replicated,…

…and crossing over takes place
between the donated Hfr
chromosome and the original
chromosome of the F – cell.

6.15 Bacterial genes may be transferred from an Hfr cell to an FϪ cell in conjugation. In an
Hfr cell, the F factor has been integrated into the bacterial chromosome.

Crossing over may lead to
the recombination of
alleles (bright blue in
place of black segment).

The linear
chromosome
is degraded.

Bacterial and Viral Genetic Systems

Crossing over
takes place
within the Hfr
chromosome.

When the F factor excises from
the bacterial chromosome, it
may carry some bacterial genes
(in this case, lac) with it.

(a)
Hfr cell

During conjugation, the
F factor with the lac gene
is transferred to the F– cell,…
(b)
F cell

F cell

(c)

…producing a partial
diploid with two copies
of the lac gene.
(d)

F– cell

lac
lac

Bacterial chromosome
with integrated F factor

Bacterial
chromosome

6.16 An Hfr cell may be converted into an FЈ cell when the F factor excises from the
bacterial chromosome and carries bacterial genes with it. Conjugation produces a partial diploid.

Table 6.2

Characteristics of E. coli cells
with different types of F factor

individual genes to be transferred indicates their relative
positions on the chromosome. In most genetic maps, distances are expressed as percent recombination; but, in bacterial maps constructed with interrupted conjugation, the
basic unit of distance is a minute.

Type

F Factor
Characteristics

Role in
Conjugation



Present as separate circular DNA

Donor

Concepts

F

Absent

Recipient

Hfr

Present, integrated into bacterial
chromosome

High-frequency
donor



Present as separate circular DNA,
carrying some bacterial genes

Donor

Conjugation can be used to map bacterial genes by mixing Hfr and
FϪ cells that differ in genotype and interrupting conjugation at
regular intervals. The amount of time required for individual genes
to be transferred from the Hfr to the FϪ cells indicates the relative positions of the genes on the bacterial chromosome.

Ϫ

cell and have an opportunity to recombine with the
recipient chromosome. Chromosome transfer always
begins within the integrated F factor and proceeds in a continuous direction; so genes are transferred according to
their sequence on the chromosome. The time required for

Table 6.3

Results of conjugation between
cells with different F factors

Conjugating

Cell Types Present after Conjugation

Fϩ ϫ FϪ

Two Fϩ cells (FϪ cell becomes Fϩ)

Hfr ϫ FϪ

One Hfr cell and one FϪ (no change)*

FЈ ϫ FϪ

Two FЈ cells (FϪ cell becomes FЈ)

*Rarely, the FϪ cell becomes Fϩ in an Hfr ϫ FϪ conjugation if the entire
chromosome is transferred during conjugation.

Natural Gene Transfer and
Antibiotic Resistance
Many pathogenic bacteria have developed resistance to
antibiotics, particularly in environments where antibiotics
are routinely used, such as hospitals and fish farms. (Massive
amounts of antibiotics are often used in aquaculture to prevent infection in the fish and enhance their growth.) The
continual presence of antibiotics in these environments creates selection for resistant bacteria, which reduces the effectiveness of antibiotic treatment for medically important
infections.
Antibiotic resistance in bacteria frequently results
from the action of genes located on R plasmids, small circular plasmids that can be transferred by conjugation. R
plasmids have evolved in the past 60 years (since the beginning of widespread use of antibiotics), and some convey
resistance to several antibiotics simultaneously. Ironic but
plausible sources of some of the resistance genes found in
R plasmids are the microbes that produce antibiotics in the
first place.

149

150

Chapter 6

Transformation in Bacteria
A second way that DNA can be transferred between bacteria is through transformation (see Figure 6.9b). Transformation played an important role in the initial identification
of DNA as the genetic material, which will be discussed in
Chapter 8.
Transformation requires both the uptake of DNA from
the surrounding medium and its incorporation into a bacterial chromosome or a plasmid. It may occur naturally when
dead bacteria break up and release DNA fragments into the
environment. In soil and marine environments, this means
may be an important route of genetic exchange for some
bacteria.
Cells that take up DNA through their envelopes are said
to be competent. Some species of bacteria take up DNA
more easily than do others; competence is influenced by
growth stage, the concentration of available DNA, and environmental challenges. The DNA taken up by a competent
cell need not be bacterial: virtually any type of DNA (bacterial or otherwise) can be taken up by competent cells under
the appropriate conditions.
As a DNA fragment enters the cell in the course of transformation (Figure 6.17), one of the strands is hydrolyzed,
whereas the other strand moves across the membrane and
may pair with a homologous region and become integrated
into the bacterial chromosome. This integration requires two
crossover events, after which the remaining single-stranded
DNA is degraded by bacterial enzymes.
Bacterial geneticists have developed techniques to
increase the frequency of transformation in the laboratory to
introduce particular DNA fragments into cells. They have
developed strains of bacteria that are more competent than
wild-type cells. Treatment with calcium chloride, heat shock,
or an electrical field makes bacterial membranes more porous
and permeable to DNA, and the efficiency of transformation
can also be increased by using high concentrations of DNA.
These techniques make it possible to transform bacteria such
as E. coli, which are not naturally competent.

Transformation, like conjugation, is used to map bacterial genes, especially in those species that do not undergo
conjugation or transduction (see Figure 6.9a and c).
Transformation mapping requires two strains of bacteria
that differ in several genetic traits; for example, the recipient
strain might be aϪ bϪ cϪ (auxotrophic for three nutrients),
with the donor cell being prototrophic with alleles aϩ bϩ cϩ.
DNA from the donor strain is isolated and purified. The
recipient strain is treated to increase competency, and DNA
from the donor strain is added to the medium. Fragments of
the donor DNA enter the recipient cells and undergo recombination with homologous DNA sequences on the bacterial
chromosome. Cells that receive genetic material through
transformation are called transformants.
Genes can be mapped by observing the rate at which
two or more genes are transferred together, or cotransformed, in transformation. When the DNA is fragmented
during isolation, genes that are physically close on the chromosome are more likely to be present on the same DNA fragment and transferred together, as shown for genes aϩ and bϩ
in Figure 6.18. Genes that are far apart are unlikely to be present on the same DNA fragment and will rarely be transferred together. Inside the cell, DNA becomes incorporated
into the bacterial chromosome through recombination. If
two genes are close together on the same fragment, any two
crossovers are likely to take place on either side of the two
genes, allowing both to become part of the recipient chromosome. If the two genes are far apart, there may be one
crossover between them, allowing one gene but not the other
to recombine with the bacterial chromosome. Thus, two
genes are more likely to be transferred together when they
are close together on the chromosome, and genes located far
apart are rarely cotransformed. Therefore, the frequency of
cotransformation can be used to map bacterial genes. If
genes a and b are frequently cotransformed, and genes b and
c are frequently cotransformed, but genes a and c are rarely
cotransformed, then gene b must be between a and c—the
gene order is a b c.

Nontransformed

Recipient
DNA
Double-stranded
fragment of DNA

Transformed
One strand of the DNA
fragment enters the cell;
the other is hydrolyzed.

The single-stranded fragment pairs
with the bacterial chromosome
and recombination takes place.

The remainder of the
single-stranded DNA
fragment is degraded.

6.17 Genes can be transferred between bacteria through transformation.

When the cell replicates and divides,
one of the resulting cells is transformed
and the other is not.

Bacterial and Viral Genetic Systems

Donor cell
1 DNA from a donor cell is
fragmented.

a+

c+

b+
a+
Recipient cell

a–

b+

Uptake of:
2 Fragments are
taken up by
the recipient cell.

a+

Transformants

a+

c–

b–

c+

b+ a+

b+

a–

c–

3 After entering the
cell, the donor DNA
becomes incorporated
into the bacterial
chromosome through
crossing over.

b+

c–

b–

c+

a–

c+

b–
b+ a+

a+

c–

4 Genes that are close
to one another on
the chromosome are
more likely to be
present on the same
DNA fragment and be
recombined together.

b+

6.18 Transformation can be used to map bacterial genes.

Concepts
Genes can be mapped in bacteria by taking advantage of transformation—the ability of cells to take up DNA from the environment
and incorporate it into their chromosomes through crossing over.
The relative rate at which pairs of genes are cotransformed indicates the distance between them: the higher the rate of cotransformation, the closer the genes are on the bacterial chromosome.

✔ Concept Check 4
DNA from a bacterial strain with genotype hisϪ leuϪ thrϪ is
transformed with DNA from a strain that is hisϩ leuϩ thrϩ. A few
leuϩ thrϩ cells and a few hisϩ thrϩ cells are found, but no hisϩ leuϩ
cells are observed. Which genes are farthest apart?

Conclusion: The rate of cotransformation is inversely
proportional to the distances between genes.

To determine if the anthrax spores from the contaminated letters and the bacteria that infected the 18 victims
came from the same source, investigators turned to DNA
typing. They examined the variable number of tandem
repeats (VNTRs, also called microsatellites), which are short
DNA sequences that are repeated different numbers of times
in different bacterial strains (see Chapter 14). This analysis
showed that all of the spores found in the letters and bacteria found in the victims were related and probably originated
from a single source, although the person or persons responsible for this act of bioterrorism have never been conclusively
identified. The entire genome of Bacillus anthracis was
sequenced in 2003 and now provides a much larger set of
variable DNA sequences that can be used to effectively trace
the origins of future disease outbreaks.

Bacterial Genome Sequences
Genetic maps serve as a foundation for more-detailed information provided by DNA sequencing. Geneticists have now
determined the complete nucleotide sequence of a number
of bacterial genomes, and many additional microbial
sequencing projects are underway. The size and content of
bacterial genomes is discussed in Chapter 14.
One practical application of bacterial DNA sequencing
is the identification and tracing of the sources of bacterial
contamination and infection. This use of DNA sequences is
illustrated by the study of anthrax-causing bacteria that were
used in bioterrorism in the United States in 2001. Anthrax is
caused by long-lasting spores of the bacterium Bacillus
anthracis and was the cause of 18 deaths shortly after the terrorist attacks on the World Trade Center and the Pentagon in
the United States on September 11, 2001. The source of the
anthrax was traced to letters sent to U.S. senators and people
in the news media.

151

Model Genetic Organism
The Bacterium Escherichia coli
The most widely studied prokaryotic organism and
one of the best genetically characterized of all species
is the bacterium Escherichia coli (Figure 6.19).
Although some strains of E. coli are toxic and cause disease,
most are benign and reside naturally in the intestinal tracts of
humans and other warm-blooded animals.

Advantages of E. coli as a model genetic organism
Escherichia coli is one of the true workhorses of genetics; its
twofold advantage is rapid reproduction and small size.
Under optimal conditions, this organism can reproduce
every 20 minutes and, in a mere 7 hours, a single bacterial
cell can give rise to more than 2 million descendants. One of
the values of rapid reproduction is that enormous numbers