Bacterial Conjugation 337

F factor

oriT

Region of F factor with genes

needed for conjugation
tra G

C
trbD

trbQ
trbB

G
A
C

D
B

tra E
traA

S
M

I
traY

X
H
trbJ
F

T
traL
traiT

traJ

tra
tra I
trb

trb

trb

trb

trb
tra

tra
tra
tra

tra

tra

tra

tra

tra

tra

tra
tra

tra

tra
trb
tra
or

Encodes Encode proteins that Encodes Encodes

pilin are components coupling relaxase
protein of the type IV protein
secretion system

Figure 13.23 The F plasmid. Genes that play a role in conjugation are shown, and some of their functions are indicated. The plasmid
also contains three insertion sequences and a transposon. The site for initiation of rolling-circle replication and gene transfer during conju-
gation is oriT.

several hours in nutrient medium, and then plated it on minimal

medium. To reduce the chance that their results were due to sim-
ple reversion, they used double and triple auxotrophs on the as-
sumption that two or three simultaneous reversions would be
extremely rare. For example, one strain required biotin (Bio),
phenylalanine (Phe), and cysteine (Cys) for growth, and an-
other needed threonine (Thr), leucine (Leu), and thiamine
(Thi). Recombinant prototrophic colonies appeared on the min-
imal medium after incubation (figure 13.26). Thus the chromo-
somes of the two auxotrophs were able to associate and undergo
recombination.
Lederberg and Tatum did not directly prove that physical con-
tact of the cells was necessary for gene transfer. This evidence
was provided by Bernard Davis (1950), who constructed a U tube
consisting of two pieces of curved glass tubing fused at the base
Figure 13.24 Bacterial Conjugation. An electron micro- to form a U shape with a fritted glass filter between the halves.
graph of two E. coli cells in an early stage of conjugation. The F+ The filter allowed the passage of media but not bacteria. The U
cell to the right is covered with small pili or fimbriae, and a sex tube was filled with nutrient medium and each side inoculated
pilus connects the two cells. with a different auxotrophic strain of E. coli (figure 13.27). Dur-
ing incubation, the medium was pumped back and forth through
the filter to ensure medium exchange between the halves. After a
4 hour incubation, the bacteria were plated on minimal medium.
Davis discovered that when the two auxotrophic strains were sep-
13.7 BACTERIAL CONJUGATION arated from each other by the fine filter, gene transfer could not
The initial evidence for bacterial conjugation, the transfer of take place. Therefore direct contact was required for the recombi-
DNA by direct cell to cell contact, came from an elegant experi- nation that Lederberg and Tatum had observed. F factor-mediated
ment performed by Joshua Lederberg and Edward Tatum in 1946. conjugation is one of the best-studied conjugation systems. It is
They mixed two auxotrophic strains, incubated the culture for the focus of this section.
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338 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

▼
F factor
1 2

IS
A B
Bacterial chromosome
IS

1 2

A B

A IS 2 O 1 IS B
▼

Integrated F factor

Figure 13.25 F Plasmid Integration. The reversible integration of an F plasmid or factor into a host bacterial chromosome. The
process begins with association between plasmid and bacterial insertion sequences. The O arrowhead (white) indicates the site at which
oriented transfer of chromosome to the recipient cell begins. A, B, 1, and 2 represent genetic markers.

F  F Mating nicks one strand of the F factor at a site called oriT (for origin of
In 1952 William Hayes demonstrated that the gene transfer ob- transfer). Relaxase, an enzyme associated with the relaxosome,
served by Lederberg and Tatum was polar. That is, there were def- remains attached to the 5' end of the nicked strand. As F factor is
inite donor (F, or fertile) and recipient (F, or nonfertile) strains, replicated, the displaced strand and the attached relaxase enzyme
and gene transfer was nonreciprocal. He also found that in F  move through the type IV secretion system to the recipient cell.
F mating the progeny were only rarely changed with regard to Because the pilus is embedded in the secretion apparatus, it has
auxotrophy (that is, chromosomal genes were not often trans- been suggested that the DNA moves through a lumen in the pilus.
ferred), but F strains frequently became F. However, studies of a related conjugation system, that of the plant
These results are readily explained in terms of the F factor pathogen Agrobacterium tumefaciens, provide strong evidence
previously described (figure 13.23). The F strain contains an ex- that the DNA does not move through the sex pilus. However, it
trachromosomal F factor carrying the genes for sex pilus forma- should be noted that although the F factor system and the
tion and plasmid transfer. The sex pilus is used to establish Agrobacterium system are related, there is one important differ-
contact between the F and F cells (figure 13.28a). Once con- ence between the two. The F factor system is used to transfer
tact is made, the pilus retracts, bringing the cells into close phys- DNA from one bacterium to another, whereas the Agrobacterium
ical contact. The F cell then prepares for DNA transfer by system moves DNA from the bacterium into its plant host. What-
assembling a type IV secretion apparatus, using many of the same ever the route of transfer, as the plasmid is transferred, the enter-
genes used for sex pilus biogenesis; the sex pilus is embedded in ing strand is copied to produce double-stranded DNA. The
the secretion structure (figure 13.29). The F factor then replicates recombination frequency is low because chromosomal genes are
by a rolling-circle mechanism (see figure 11.12). Replication is rarely transferred with the independent F factor. Microorganism as-
initiated by a complex of proteins called the relaxosome, which sociations with vascular plants: Agrobacterium (section 29.5)
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Bacterial Conjugation 339

Bio- Phe- Cys- Thr+ Leu+ Thi+ Bio+ Phe+ Cys+ Thr- Leu- Thi-
conjugation was not known, eventually it was determined that Hfr
strains contain the F factor integrated into their chromosome,
rather than free in the cytoplasm (figure 13.28b). When inte-
grated, the F plasmid’s tra operon is still functional; the plasmid
can direct the synthesis of pili, carry out rolling-circle replication,
Mixture
and transfer genetic material to an F recipient cell. However,
rather than transferring itself, the F factor directs the transfer of
host chromosome. DNA transfer begins when the integrated F fac-
tor is nicked at its site of transfer origin. As it is replicated, the
chromosome moves to the recipient (figure 13.28c). Because only
part of the F factor is transferred, the F recipient does not become
F unless the whole chromosome is transferred. Transfer of the
entire chromosome with the integrated F factor requires about 100
minutes in E. coli, and the connection between the cells usually
breaks before this process is finished. Thus a complete F factor
usually is not transferred, and the recipient remains F.
As mentioned earlier, when an Hfr strain participates in con-
jugation, bacterial genes are frequently transferred to the recipient.
Gene transfer can be in either a clockwise or counterclockwise di-
rection around the circular chromosome, depending on the orien-
Minimal medium without supplements tation of the integrated F factor. After the replicated donor
Bio+ Phe+ Cys+ Thr+ Leu+ Thi+ chromosome enters the recipient cell, it may be degraded or in-
Prototrophic colonies corporated into the F genome by recombination.

Figure 13.26 Evidence for Bacterial Conjugation. F′ Conjugation

Lederberg and Tatum’s demonstration of genetic recombination
Because the F plasmid is an episome, it can leave the bacterial
using triple auxotrophs. See text for details.
chromosome and resume status as an autonomous F factor. Some-
times during this process the plasmid makes an error in excision
and picks up a portion of the chromosome. Because it is now geno-
Pressure or suction typically distinct from the original F factor, it is called an F′ plas-
mid (figure 13.30a). It is not unusual to observe the inclusion of
one or more chromosomal genes in excised F plasmids. A cell con-
taining an F′ plasmid retains all of its genes, although some of
them are on the plasmid. It mates only with an F recipient. F′ 
F conjugation is similar to F  F mating. Once again, the plas-
mid is transferred as it is copied by rolling-circle replication. How-
Met– Thr+ Leu+Thi+ Met + Thr– Leu–Thi– ever, bacterial genes on the chromosome usually are not
transferred (figure 13.30b). Bacterial genes acquired during exci-
sion of the F′ plasmid are transferred with it and need not be in-
Fritted glass filter corporated into the recipient chromosome to be expressed. The
recipient becomes F′ and is a partially diploid merozygote because
Figure 13.27 The U-tube Experiment. The U-tube experi- the same bacterial genes present on the F′ plasmid are also found
ment used to show that genetic recombination by conjugation on the recipient’s chromosome. In this way specific bacterial genes
requires direct physical contact between bacteria. See text for details. may spread rapidly throughout a bacterial population.
F′ conjugation is very important to the microbial geneticist. A
partial diploid’s behavior shows whether the allele carried by an
Hfr Conjugation F′ plasmid is dominant or recessive to the chromosomal gene.
The formation of F′ plasmids also is useful in mapping the chro-
Not long after the discovery of F  F mating, a second type
mosome because if two genes are picked up by an F factor they
of F factor-mediated conjugation was discovered. In this type of
must be neighbors.
conjugation, the donor transfers chromosomal genes with great
efficiency, but does not change the recipient bacteria into F
cells. Because of the high frequency of recombinants produced Other Examples of Bacterial Conjugation
by this mating, it is referred to as Hfr conjugation and the donor Although most research on plasmids and conjugation has been done
is called an Hfr strain. Although initially the mechanism of Hfr using E. coli and other gram-negative bacteria, self-transmissible
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340 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

F factor Bacterial chromosome

F+ Coupling
Origin of
cell factor
transfer
Type IV
secretion
system

Sex pilus makes contact Recipient

with F- recipient cell. cell (F–)
Relaxosome makes a
cut at the origin of transfer
Relaxosome and begins to separate one
DNA strand. The intact
F- strand is replicated by the
recipient rolling circle mechanism.

Sex pilus contracts

bringing cells together.
Accessory proteins of the
relaxosome are released.
The DNA/relaxase complex
is recognized by the coupling
factor and transferred to the
Relaxase secretion system.

Coupling
factor

Secretion
system
The secretion system
Type IV secretion system pumps the DNA/relaxase
is constructed and joins complex into the recipient
two cells. cell.

(a) F  F conjugation
(a) F+ x F- conjugation

As the DNA enters, the

F + cell Hfr cell F-factor DNA is replicated
to become double stranded.

Bacterial F+ cell
chromosome
pro+
lac+ Integration of pro+
F factor into
chromosome lac+
F+ cell

Origin of Origin of
transfer transfer
F factor

(b) Insertion of F factor into chromosome

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Bacterial Conjugation 341

New strand synthesized

–
by rolling circle replication
Hfr cell F cell
Sex pilus makes contact
with F– cell and contracts
to pull Hfr and F– cell
together. Type IV secretion
system connects cells. pro– pro–
pro+ pro+
Transfer of Hfr
lac – chromosome lac–
lac+ lac+
lac+

pro+

Origin of
transfer Conjugation for
(toward lac+) longer times

Hfr cell F – recipient Conjugation for Hfr cell F – recipient

short time

pro– pro–
pro+ pro+
lac – lac –
lac+ lac+

pro+
lac + lac +

Recombination Recombination
between exogenote between exogenote
and endogenote and endogenote

pro+ pro– pro+ pro+

lac+ lac+ lac+ lac+

(c) Hfr  F conjugation

Figure 13.28 F Factor-Mediated Conjugation. The F factor encodes proteins for building the sex pilus and proteins needed to
construct the type IV secretion system that will transfer DNA from the donor to the F recipient. One protein, the coupling factor, is
thought to guide the DNA to the secretion system. (a) During F  F conjugation, only the F factor is transferred because the plasmid is
extrachromosomal. The recipient cell becomes F. (b) Integration of the F factor into the chromosome creates an Hfr cell. (c) During Hfr  F
conjugation, some plasmid genes and some chromosomal genes are transferred to the recipient. Note that only a portion of the F factor
moves into the recipient. Because the entire plasmid is not transferred, the recipient remains F. In addition, the incoming DNA must
recombine into the recipient’s chromosome if it is to be stably maintained.
342 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

Tip Hfr F
TraA
(pilin) A
Sex pilus

A De-integration
including part of
LPS bacterial
OM chromosome

PG P
L D
coupling (a)
TraQ, X
C PM Cells connected by type IV
secretion system
ATP ADP+P F F
ATP ADP+P
A
Figure 13.29 The Type IV Secretion System Encoded by
F Factor. The F factor-encoded type IV secretion system is
composed of numerous Tra proteins, including TraA proteins, a
which form the sex pilus, and TraD, which is the coupling factor.
Some Tra proteins are located in the plasma membrane (PM),
others extend into the periplasm (P) and pass through the pepti-
doglycan layer (PG) into the outer membrane (OM) and its F plasmid begins
lipopolysaccharide (LPS) layer. replication and transfer

A
plasmids are present in gram-positive bacterial genera such as
Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Strep-
tomyces. Much less is known about these systems. It appears that
fewer transfer genes are involved, possibly because a sex pilus may a
not be required for plasmid transfer. For example, Enterococcus
faecalis recipient cells release short peptide chemical signals that
activate transfer genes in donor cells containing the proper plasmid.
Donor and recipient cells directly adhere to one another through F plasmid replicated
special plasmid-encoded proteins released by the activated donor and transferred
cell. Plasmid transfer then occurs. F F

A A
1. What is bacterial conjugation and how was it discovered?
2. Distinguish between F,Hfr,and F strains of E.coli with respect to their
physical nature and role in conjugation.
3. Describe in some detail how F  F and Hfr conjugation processes pro- a
ceed,and distinguish between the two in terms of mechanism and the final
results.
4. What is F′ conjugation and why is it so useful to the microbial geneticist?
How does the F′ plasmid differ from a regular F plasmid?
(b)

Figure 13.30 F' Conjugation. (a) Due to an error in excision,

the A gene of an Hfr cell is picked up by the F factor. (b) The A gene is
13.8 DNA TRANSFORMATION
then transferred to a recipient during conjugation. See text for expla-
The second way DNA can move between bacteria is through nation.
transformation, discovered by Fred Griffith in 1928. Transfor-
mation is the uptake by a cell of a naked DNA molecule or frag- transformation the DNA comes from a donor bacterium. The
ment from the medium and the incorporation of this molecule process is random, and any portion of a genome may be trans-
into the recipient chromosome in a heritable form. In natural ferred between bacteria. DNA as genetic material (section 11.1)
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DNA Transformation 343

When bacteria lyse, they release considerable amounts of

DNA into the surrounding environment. These fragments may be
relatively large and contain several genes. If a fragment contacts a DNA fragments
competent cell, a cell that is able to take up DNA and be trans-
formed, the DNA can be bound to the cell and taken inside (figure
13.31a). The transformation frequency of very competent cells is
around 103 for most genera when an excess of DNA is used. That
Bacterial
is, about one cell in every thousand will take up and integrate the chromosome
gene. Competency is a complex phenomenon and is dependent on
several conditions. Bacteria need to be in a certain stage of growth;
for example, Streptococcus pneumoniae becomes competent dur-
ing the exponential phase when the population reaches about 107 Uptake
to 108 cells per ml. When a population becomes competent, bac- of DNA
teria such as S. pneumoniae secrete a small protein called the com-
petence factor that stimulates the production of 8 to 10 new
proteins required for transformation. Natural transformation has
been discovered so far only in certain genera including Strepto-
coccus, Bacillus, Thermoactinomyces, Haemophilus, Neisseria,
Moraxella, Acinetobacter, Azotobacter, Helicobacter, and OR
Integration by
Pseudomonas. Gene transfer by this process occurs in soil and nonreciprocal
aquatic ecosystems and may be an important route of genetic ex- recombination Degradation
change in biofilm and other microbial communities.
The mechanism of transformation has been intensively stud-
ied in S. pneumoniae (figure 13.32). A competent cell binds a
double-stranded DNA fragment if the fragment is moderately
large; the process is random, and donor fragments compete with
Stable transformation Unsuccessful transformation
each other. The DNA then is cleaved by endonucleases to double-
stranded fragments about 5 to 15 kilobases in size. DNA uptake (a) Transformation with DNA fragments
requires energy expenditure. One strand is hydrolyzed by an
envelope-associated exonuclease during uptake; the other strand
associates with small proteins and moves through the plasma
DNA plasmid
membrane. The single-stranded fragment can then align with a
homologous region of the genome and be integrated, probably by
a mechanism similar to that depicted in figure 13.18. Bacterial
chromosome
Transformation in Haemophilus influenzae, a gram-negative
bacterium, differs from that in S. pneumoniae in several respects. H.
influenzae does not produce a competence factor to stimulate the de-
velopment of competence, and it takes up DNA from only closely
related species (S. pneumoniae is less particular about the source of Uptake
its DNA). Double-stranded DNA, complexed with proteins, is taken of plasmid
in by membrane vesicles. The specificity of H. influenzae transfor-
mation is due to a special 11 base pair sequence (5′AAGTGCG-
GTCA3′) that is repeated over 1,400 times in H. influenzae DNA.
DNA must have this sequence to be bound by a competent cell.
The protein complexes that take up free DNA must be able to
move it through gram-negative and gram-positive walls, which Stable transformation
may be both thick and complex. As expected, the machinery is
quite large and complicated and appears related to protein secre- (b) Transformation with a plasmid
tion systems. Figure 13.33a shows a schematic diagram of the
complex used by the gram-negative bacterium Neisseria gonor- Figure 13.31 Bacterial Transformation. Transformation
rhoeae. PilQ aids in the movement across the outer membrane, with (a) DNA fragments and (b) plasmids. Transformation with a
and the pilin complex PilE moves the DNA through the plasmid often is induced artificially in the laboratory. The trans-
periplasm and peptidoglycan. ComE is a DNA binding protein; N forming DNA is in purple and integration is at a homologous
is the nuclease that degrades one strand before the DNA enters region of the genome.
the cytoplasm through the transmembrane channel formed by
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344 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

Receptor lac+

_
lac
DNA fragment
binds to a cell
surface receptor. Outer
PilQ PilQ
membrane

An extracellular PilE
endonuclease ComE
cuts the DNA into
lac+ Peptidoglycan
smaller fragments.
N
_ Plasma
lac ComA ComA membrane

(a)
One strand is
degraded and a
single strand is
ComGC
transported into
Uptake the cell.
system
_ lac+ ComEA Peptidoglycan
lac
N
The DNA strand
aligns itself with a Plasma
homologous ComEC ComEC membrane
region on the
bacterial ComFA
chromosome.
lac+
_
lac (b)

The DNA strand is

incorporated into Figure 13.33 DNA Uptake Systems. (a) DNA uptake
the bacterial machinery in N.gonorrhoeae. (b) Uptake machinery in B.subtilis. See
chromosome via
homologous text for details.
recombination.
plasm. A gram-negative equivalent of ComFA has not been identi-
Heteroduplex
fied yet in N. gonorrhoeae.
Microbial geneticists exploit transformation to move DNA
The heteroduplex (usually recombinant DNA) into cells. However, as already noted,
DNA is repaired in many species, including E. coli, are not naturally transformation
a way that _ competent. Fortunately, these bacteria can be made artificially
changes lac
strand to create competent by certain treatments. Two common techniques are
a lac+ gene. electrical shock and exposure to calcium chloride. Both ap-
lac+ proaches render the cell membrane more permeable to DNA and
Transformed
cell both have been used to make artificially competent E. coli cells.
To increase the transformation frequency with E. coli, strains that
lack one or more nucleases are used. These strains are especially
important when transforming the cells with linear DNA, which is
Figure 13.32 Bacterial Transformation as Seen in vulnerable to attack by nucleases. It is easier to transform bacteria
S. pneumoniae. with plasmid DNA since plasmids are not as easily degraded as
linear fragments and can replicate within the host (figure 13.31b).
ComA. The machinery in the gram-positive bacterium Bacillus sub-
1. Define transformation and competence.
tilis is depicted in figure 13.33b. It is localized to the poles of the
2. Describe how transformation occurs in S.pneumoniae. How does the process
cell, and as can be seen, many of the components are similar to those
differ in H.influenzae?
of N. gonorrhoeae: the pilin complex (ComGC), DNA binding pro-
3. Discuss two ways in which artificial transformation can be used to place
tein (ComEA), nuclease (N), and channel protein (ComEC).
functional genes within bacterial cells.
ComFA is a DNA translocase that moves the DNA into the cyto-
 Transduction 345

13.9 TRANSDUCTION diation. When this occurs, the prophage is excised from the bac-
terial genome and the lytic cycle proceeds.
The third mode of bacterial gene transfer is transduction. It is a Transduction is the transfer of bacterial genes by viruses.
frequent mode of horizontal gene transfer in nature and is medi- Bacterial genes are incorporated into a phage capsid because of
ated by viruses. The morphology and life cycle of bacterial viruses errors made during the virus life cycle. The virus containing these
or bacteriophages is not discussed in detail until chapter 17. Nev- genes then injects them into another bacterium, completing the
ertheless, it is necessary to briefly describe the life cycle here as transfer. There are two different kinds of transduction: general-
background for a consideration of their role in gene transfer. ized and specialized.
Viruses are structurally simple, often composed of just a nu-
cleic acid genome protected by a protein coat called the capsid.
They are unable to replicate autonomously. Instead, they infect Generalized Transduction
and take control of a host cell, forcing the host to make many Generalized transduction occurs during the lytic cycle of viru-
copies of the virus. Viruses that infect bacteria are called bacte- lent and some temperate phages and can transfer any part of the
riophages, or phages for short. Some phages are replicated by bacterial genome (figure 13.35). During the assembly stage,
their bacterial host immediately after entry. After the number of when the viral chromosomes are packaged into protein capsids,
replicated phages reaches a certain number, they cause the host to random fragments of the partially degraded bacterial chromo-
lyse, so they can be released and infect new host cells (figure 13.34). some also may be packaged by mistake. Because the capsid can
These phages are called virulent bacteriophages and the process contain only a limited quantity of DNA, the viral DNA is left be-
is called the lytic cycle. Other bacteriophages do not immediately hind. The quantity of bacterial DNA carried depends primarily on
kill their host. Many of these viruses enter the host bacterium and, the size of the capsid. The P22 phage of Salmonella enterica
instead of replicating, insert their genomes into the bacterial chro- serovar Typhimurium usually carries about 1% of the bacterial
mosome. Once inserted, the viral genome is called a prophage. genome; the P1 phage of E. coli and a variety of gram-negative
The host bacterium is unharmed by this, and the phage genome is bacteria carries about 2.0 to 2.5% of the genome. The resulting
passively replicated as the host cell’s genome is replicated. These virus particle often injects the DNA into another bacterial cell but
bacteriophages are called temperate bacteriophages and the re- cannot initiate a lytic cycle. This phage is known as a generalized
lationship between these viruses and their host is called lysogeny transducing particle or phage and is simply a carrier of genetic
(figure 13.34). Bacteria that have been lysogenized are called information from the original bacterium to another cell. As in
lysogens. Temperate phages can remain inactive in their hosts for transformation, once the DNA has been injected, it must be in-
many generations. However, they can be induced to switch to a corporated into the recipient cell’s chromosome to preserve the
lytic cycle of growth under certain conditions, including UV irra- transferred genes. The DNA remains double stranded during

Phage DNA Bacterial

chromosome

Phage injects Exposure to

New phages its DNA into stress such as
can bind to cytoplasm. UV light triggers
bacterial excision from host
cells. chromosome.
Lytic cycle Lysogenic cycle

Phage DNA
Cell lyses integrates
and releases into host
the new phages. chromosome.
Phage DNA Prophage DNA
directs the is copied when
synthesis of cell divides.
many new
phages. Prophage

Figure 13.34 Lytic and Lysogenic Cycles of Temperate Phages. Virulent phages undergo only the lytic cycle. Temperate phages
have two phases to their life cycles. The lysogenic cycle allows the genome of the virus to be replicated passively as the host cell’s genome is
replicated. Certain environmental factors such as UV light can cause a switch from the lysogenic cycle to the lytic cycle. In the lytic cycle,
new virus particles are made and released when the host cell lyses. Virulent phages are limited to just the lytic cycle.
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346 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

Phage DNA and indeed it was, but their initial conclusion that the transfer re-
sulted from conjugation was not borne out. When these investi-
his+ lys+ Phage infects gators performed the U-tube experiment (figure 13.27) with
bacterial cell. Salmonella, they still recovered prototrophs. The filter in the U
tube had pores that were small enough to block the movement of
Host DNA is hydrolyzed
bacteria between the two sides but allowed phage P22 to pass.
into pieces, and phage Lederberg and Zinder had intended to confirm that conjugation
DNA and proteins are was present in another bacterial species but instead discovered a
made.
completely new mechanism of bacterial gene transfer. This seem-
his+ lys+ ingly routine piece of research led to surprising and important re-
sults. A scientist must always keep an open mind about results
and be prepared for the unexpected.

Phages assemble;
occasionally a phage Specialized Transduction
carries a piece of the
host cell chromosome. In specialized transduction, the transducing particle carries only
specific portions of the bacterial genome. Specialized transduction
lys+ is made possible by an error in the lysogenic life cycle of phages
that insert their genomes into a specific site in the host chromo-
Transducing some. When a prophage is induced to leave the host chromosome,
his+
phage with excision is sometimes carried out improperly. The resulting phage
host DNA Transducing phage genome contains portions of the bacterial chromosome (about 5 to
injects its DNA into
a new recipient cell.
10% of the bacterial DNA) next to the integration site, much like
the situation with F′ plasmids (figure 13.36). A transducing phage
his+ genome usually is defective and lacks some part of its attachment
Crossing over site. The transducing particle will inject bacterial genes into an-
_ _
Recipient cell
_ _ his lys other bacterium, even though the defective phage cannot repro-
(his lys ) duce without assistance. The bacterial genes may become stably
The transduced DNA incorporated under the proper circumstances.
is recombined into the The best-studied example of specialized transduction is car-
chromosome of the ried out by the E. coli phage lambda. The lambda genome inserts
recipient cell.
into the host chromosome at specific locations known as attach-
_
his+ lys ment or att sites (figure 13.37, see also figures 17.19 and 17.22).
Recombinant
bacterium The phage att sites and bacterial att sites are similar and can com-
plex with each other. The att site for lambda is next to the gal and
bio genes on the E. coli chromosome; consequently, specialized
_ transducing lambda phages most often carry these bacterial
The recombinant bacterium has a genotype (his+lys )
_ _
that is different from recipient bacterial cell (his lys ).
genes. The lysate, or product of cell lysis, resulting from the in-
duction of lysogenized E. coli contains normal phage and a few
Figure 13.35 Generalized Transduction in Bacteria. defective transducing particles. These particles are called either
lambda dgal because they carry the galactose utilization genes or
lambda dbio because they carry the bio from the other side of the
transfer, and both strands are integrated into the endogenote’s att site (figure 13.37). Because these lysates contain only a few
genome. About 70 to 90% of the transferred DNA is not integrated transducing particles, they often are called low-frequency trans-
but often is able to survive temporarily and be expressed. duction lysates (LFT lysates). Whereas the normal phage has a
Abortive transductants are bacteria that contain this noninte- complete att site, defective transducing particles have a nonfunc-
grated, transduced DNA and are partial diploids. tional hybrid integration site that is part bacterial and part phage
Generalized transduction was discovered in 1951 by Joshua in origin. Integration of the defective phage chromosome does not
Lederberg and Norton Zinder during an attempt to show that con- readily take place. Transducing phages also may have lost some
jugation, discovered several years earlier in E. coli, could occur genes essential for reproduction. Stable transductants can arise
in other bacterial species. Lederberg and Zinder were repeating only if there is a double cross-over event on each side of the gal
the earlier experiments with S. enterica serovar Typhimurium. site (figure 13.37). Temperate bacteriophages and lysogeny (section 17.5)
They found that incubation of a mixture of two multiply aux- Defective lambda phages carrying the gal or bio genes can in-
otrophic strains yielded prototrophs at the level of about one in tegrate if there is a normal lambda phage in the same cell. We will
105. This seemed like good evidence for bacterial recombination, continue our discussion of this with a phage carrying the gal gene.
wil92913_ch13.qxd 8/16/06 9:37 AM Page 347

Transduction 347

Prophage

Lysogenized cell
with prophage

Induction

Rare-deintegration that
includes some
bacterial genes

Replication of defective
virus DNA with incorporated
host genes

Assembly and release of

transducing phage particles

Infection of recipient cell

Crossover to integrate
bacterial genes

Integration as prophage

Bacterial chromosome
containing both virus Bacterial
and donor DNA chromosome
containing only
donor DNA

Figure 13.36 Specialized Transduction by a Temperate Bacteriophage. Recombination can produce two types of transductants.
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348 Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation

2
3 1
λ
att sites

gal +
Integration to
form prophage

1 2 3
gal + bio+

Normal excision Error in excision

2 1
3 1
gal +
2
3
gal +

2 1
3 1 λ
λdgal
2 gal +

gal + 3

2
1
gal + 1
2
gal +

1 2
gal – 3
gal –
λdgal
λ

1 2 gal + 1 2
– 3
gal
gal +
Unstable transductant Stable transductant

Figure 13.37 The Mechanism of Transduction for Phage Lambda and E. coli. Integrated lambda phage lies between the gal and
bio genes. When it excises normally (top left), the new phage is complete and contains no bacterial genes. Rarely excision occurs asymmetrically
(top right), and either the gal or bio genes are picked up and some phage genes are lost (only aberrant excision involving the gal genes is
shown).The result is a defective lambda phage that carries bacterial genes and can transfer them to a new recipient.

The normal phage integrates, yielding two bacterial/phage hybrid These transductants are unstable because the prophages can be in-
att sites where the defective lambda dgal phage can insert (figure duced to excise by agents such as UV radiation. Excision, however,
13.37). It also supplies the genes missing in the defective phage. produces a lysate containing a fairly equal mixture of defective
The normal phage in this instance is termed the helper phage be- lambda dgal phage and normal helper phage. Because it is very ef-
cause it aids integration and reproduction of the defective phage. fective in transduction, the lysate is called a high-frequency trans-
 Mapping the Genome 349

duction lysate (HFT lysate). Reinfection of bacteria with this Gene linkage, or the proximity of two genes on a chromo-
mixture will result in the generation of considerably more trans- some, can be determined from transformation by measuring the
ductants. LFT lysates and those produced by generalized transduc- frequency with which two or more genes simultaneously trans-
tion have one transducing particle in 105 or 106 phages; HFT lysates form a recipient cell. Consider the case for cotransformation by
contain transducing particles with a frequency of about 0.1 to 0.5. two genes. In theory, a bacterium could simultaneously receive
two genes, each carried on a separate DNA fragment. However,
1. Briefly describe the lytic and lysogenic viral reproductive cycles. Define it is much more likely that genes residing on the same fragment
lysogeny, lysogen, temperate phage, prophage, and transduction. will be simultaneously transferred. If two genes are closely linked
2. Describe generalized transduction,how it occurs,and the way in which it on the chromosome, then they should be able to cotransform. The
was discovered.What is an abortive transductant? closer the genes are together, the more often they will be carried
3. What is specialized transduction and how does it come about? Distinguish on the same fragment and the higher will be the frequency of co-
between LFT and HFT lysates and describe how they are formed. transformation. If genes are spaced a great distance apart, they
4. How might one tell whether horizontal gene transfer was mediated by gen- will be carried on separate DNA fragments and the frequency of
eralized or specialized transduction? double transformants will equal the product of the individual
5. Why doesn’t a cell lyse after successful transduction with a temperate phage? transformation frequencies.
6. Describe how conjugation, transformation, and transduction are similar. Generalized transduction can be used to obtain linkage infor-
How are they different? mation in much the same way as transformation. Linkages usu-
ally are expressed as cotransduction frequencies, using the
argument that the closer two genes are to each other, the more
likely they both will reside on the DNA fragment incorporated
13.10 MAPPING THE GENOME into a single phage capsid. The E. coli phage P1 is often used in
Before the advent of genome sequencing, microbial geneticists such mapping because it can randomly transduce up to 1 to 2% of
only had one general approach for elucidating the organization of the genome (figure 13.39).
genes in a bacterial chromosome—to carry out linkage analysis. Specialized transduction is used to find which phage attach-
Such analyses yield a genetic map showing the position of genes ment site is close to a specific gene. The relative locations of spe-
relative to each other. Genetic mapping using linkage analysis is cific phage att sites are known from conjugational mapping, and
a very complex task. This section surveys approaches to mapping the genes linked to each att site can be determined by means of
the bacterial genome, using E. coli as an example. All three modes specialized transduction. These data allow precise placement of
of gene transfer and recombination have been used in mapping. genes on the chromosome.
Hfr conjugation is frequently used to map the relative location A simplified genetic map of E. coli K12 is given in figure 13.40.
of bacterial genes. This technique rests on the observation that dur- Because conjugation data are not high resolution and cannot be used
ing conjugation, the chromosome moves from donor to recipient at to position genes that are very close together, the map was devel-
a constant rate. In an interrupted mating experiment the conju- oped using several mapping techniques. Interrupted mating data
gation bridge is broken and Hfr  F mating is stopped at various were combined with those from cotransduction and cotransforma-
intervals after the start of conjugation by mixing the culture vigor- tion studies. Data from recombination studies also were used. New
ously in a blender (figure 13.38a). The order and timing of gene genetic markers in the E. coli genome were located within a rela-
transfer can be determined because they are a direct reflection of tively small region of the genome (10 to 15 minutes long) using a
the order of genes on the bacterial chromosome (figure 13.38b). series of Hfr strains with F factor integration sites scattered through-
For example, extrapolation of the curves in figure 13.38b back to out the genome. Once the genetic marker was located with respect
the x-axis gives the time at which each gene just began to enter the to several genes in the same region, its position relative to nearby
recipient. The result is a circular chromosome map with distances neighbors was more accurately determined using transformation
expressed in terms of the minutes elapsed until a gene is trans- and transduction studies. Such analyses are no longer performed in
ferred. This technique can fairly precisely locate genes 3 minutes E. coli and other microbes for which a genome sequence has been
or more apart. The heights of the plateaus in figure 13.38b are published.
lower for genes that are more distant from the F factor (the origin Using these techniques, researchers mapped about 2,200
of transfer) because there is an ever-greater chance that the conju- genes of E. coli K12 and compared this with the actual nucleotide
gation bridge will spontaneously break before these genes are sequence of the genome (i.e., a physical map of the genome).
transfered. Because of the relatively large size of the E. coli Genome sequencing has revealed about 4,300 possible genes.
genome, it is not possible to generate a map from one Hfr strain. Thus genetic analysis defined over half of the potential genes.
Therefore several Hfr strains with the F plasmid integrated at dif- The genetic map approximates the physical map, but they do not
ferent locations must be used and their maps superimposed on one correspond perfectly. This is because the genetic map is derived
another. The overall map is adjusted to 100 minutes, although com- from genetic linkage frequencies that do not correlate exactly
plete transfer may require somewhat more than 100 minutes. In a with the number of nucleotides that separate two genes. Roughly
sense, minutes are an indication of map distance and not strictly a speaking, one minute of the E. coli genetic map corresponds to 40
measure of time. Zero time is set at the threonine (thr) locus. kilobases of DNA sequence.