Mechanisms of Genetic Exchange in Bacteria 177

hsdR

thr
leu
pyr

a zi

A
ton
pu

pro
B
rA
me

la c
m

tsx
alK

l
pu
ar

rE
rD
gE 0

pu
rh
po a
lA 90 10 lip

Hfr P801
Hfr H
ilv l
ga
E
chl
pyrE
rC
Hf Hf
mtl rK
L2 aroA
80 5 20 pyrD
xyl
Hfr A
plsB B313 pyrC
Hfr KL19
purB
malT Hfr G11 Hfr B7
str
trp
14
70 KL tyrR
Hfr 30
R
arg
6
L1

Hfr
Hfr KL98
rK

G
arg
KL9
Hf

lC 60 40
to m
an
C
et ar
m oD
rA

50
se

thy

pa
C

bB
cys
A

uvr
tyrA
rec

his
glyA

C
purC

nalA
ptsI
aroC

◾ FIGURE 8.19 The circular linkage map of E. coli. The inner circle shows the sites of integration of
the F factor in selected Hfr strains. The arrows indicate whether transfer by the Hfr’s is clockwise or
counterclockwise. The outer circle shows the position of selected genes. The map is divided into
100 units, where each unit is the length of DNA transferred during one minute of conjugation. The
genes shown in red were used in Wollman and Jacob’s famous interrupted mating experiment (see
Figures 8.17 and 8.18).

can integrate at many different sites in the E. coli chromosome and that the site of
integration determines where gene transfer is initiated in each Hfr strain. Moreover,
the orientation of F factor integration—either d c b a reading clockwise or a b c d read-
ing clockwise (see Figure 8.15)—determines whether the transfer of genes is clockwise
relative to the E. coli linkage map or counterclockwise (◾ Figure 8.19).
The transfer of a complete chromosome from an Hfr to an F− cell takes about
100 minutes, and transfer appears to proceed at a fairly constant rate. Thus, the time
required for transfer of genes during conjugation can be used to map genes on bacte-
rial chromosomes. A map distance of 1 minute corresponds to the length of a chromo-
somal segment transferred in 1 minute of conjugation under standard conditions. The
linkage map of E. coli is therefore divided into 100 one-minute intervals (see Figure
8.19). The zero coordinate of this circular map has been arbitrarily set at the thrA
gene. When a new mutation is identified in E. coli, its location on the chromosome is
first determined by conjugation mapping. More precise mapping can subsequently be
done using transformation or transduction. To test your understanding of conjuga-
tion mapping, deduce the chromosomal locations of the genes discussed in Problem-
Solving Skills: Mapping Genes Using Conjugation Data.

PLASMIDS AND EPISOMES

As previously mentioned, the genetic material of a bacterium is carried in one main chro-
mosome plus from one to several extrachromosomal DNA molecules called plasmids.

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 178 Chapter 8 The Genetics of Bacteria and Their Viruses

PROBLEMSOLVING SKILLS

Mapping Genes Using Conjugation Data

THE PROBLEM 5. The F factor can integrate at many sites in the E. coli chromosome
You have identified a mutant E. coli strain that cannot synthesize the and in either orientation (clockwise or counterclockwise).
amino acid tryptophan (Trp−). To determine the location of the trp− 6. The genetic map of the E. coli chromosome is divided into minutes,
mutation on the E. coli chromosome, you have carried out interrupted mat- where 1 minute is the length of DNA transferred from an Hfr strain to
an F− strain during 1 minute of conjugation.
ing experiments with four different Hfr strains. In all cases, the Hfr strains
7. Transfer of the entire chromosome from an Hfr cell to an F− cell takes
carried the dominant wild-type alleles of the marker genes, and the F−
100 minutes; therefore, the linkage map of the complete circular
strain carried the recessive mutant alleles of these genes. The following chromosome totals 100 minutes.
chart shows the time of entry in minutes (in parentheses) of the wild- 8. The thr locus has been arbitrarily assigned position “0” on the map of
type alleles of the marker genes into the Trp−F− strain. The marker genes the E. coli chromosome, with linkage distance increasing from 0 to
are thr+, aro+, his+, tyr+, met+, arg+, and ilv+ (encoding enzymes required 100 minutes moving clockwise from thr.
for the synthesis of the amino acids threonine, the aromatic amino acids
phenylalanine, tyrosine, and tryptophan, histidine, tyrosine, methionine, ANALYSIS AND SOLUTION
arginine, and isoleucine plus valine, respectively), and man+, gal+, lac+,
If we examine the sequence in which genes are transferred from each Hfr
and xyl+ (required for the ability to catabolize the sugars mannose, galac-
strain to the F− strain, we observe a linear sequence in each case.
tose, lactose, and xylose, respectively, and use them as energy sources).
Moreover, note that regardless of the sequence in which genes are
Hfr A —— man+ (1) trp+ (9) aro+ (17) gal+ (20) lac+ (29) thr+ (37) transferred by different Hfr strains, the distance between adjacent genes
Hfr B —— trp+ (6) man+ (14) his+ (22) tyr+ (34) met+ (42) arg+ (48) remains the same. The distance between man and trp is 8 minutes, for exam-
Hfr C —— thr+ (3) ilv+ (20) xyl+ (25) arg+ (33) met+ (39) tyr+ (47) ple, regardless of whether Hfr strain A or B is used in the experiment. Indeed,
Hfr D —— met+ (2) arg+ (8) xyl+ (16) ilv+ (21) thr+ (38) lac+ (46) if we combine the results obtained using the four Hfr strains and place thr at
position 0, the data yield the following circular genetic map. The circular map
On the map of the circular E. coli chromosome shown here, indicate
is a satisfying result given that we know that the chromosomal DNA of E. coli
(1) the relative location of each gene, (2) the position where the
is also circular.
F factor is integrated in each of the four Hfr’s, and (3) the direction of chro-
mosome transfer for each Hfr (clockwise or counterclockwise; indicate For further discussion visit the Student Companion site.
direction with an arrow).

thr
thr

lac
0
0/100 min lac 8
ilv gal
83 17
Hfr C aro
xyl 20
78 Hfr B

28 trp
70
arg Hfr D Hfr A
64 36
FACTS AND CONCEPTS
ma
et 56 44 n
1. The chromosome of E. coli contains a circular DNA molecule. m
2. Chromosomal DNA is transferred from Hfr donor cells to F− recipient
his
tyr

cells by rolling-circle replication.

3. Rolling-circle replication, and thus transfer of chromosomal genes, is
initiated at the origin of replication on the integrated F factor.
4. The direction of transfer (clockwise or counterclockwise) depends on
the orientation of the F factor in the Hfr chromosome.

By definition, a plasmid is a genetic element that can replicate independently of the main
chromosome in an extrachromosomal state. Most plasmids are dispensable to the host;
that is, they are not required for survival of the cell in which they reside. However, under
certain environmental conditions, such as when an antibiotic is present, they may be
essential if they carry a gene for resistance to the antibiotic.

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 Mechanisms of Genetic Exchange in Bacteria 179

There are three major types of plasmids in Hfr P3

E. coli: F factors, R plasmids, and Col plasmids. tra I
tra D

es
Fertility (F) factors were discussed earlier (see

en
tra S 8
IS3 Hfr

g
Conjugation). R plasmids (resistance plasmids) tra G

fer
tra H
carry genes that make host cells resistant to

Trans
tra F 3
tra C .7 IS 312

93.2
0/94.5
antibiotics and other antibacterial drugs. Col tra B 13 Hfr AB
tra K . 0 IS 2
plasmids (previously called colicinogenic fac- 15 .3
tra E 1617.6
tors) encode proteins that kill sensitive E. coli tra L .0 F+ cell F factor
62
cells. There are a large number of distinct Col tra A
tra J
plasmids; however, they will not be discussed
ori IS3
further here. Bacterial chromosome
Some plasmids endow host cells with the (a) IS3
ability to conjugate. All F+ plasmids, many R s
rep g i g ene
plasmids, and some Col plasmids have this enes ph
property; we say that they are conjugative
plasmids. Other R and Col plasmids do not Hfr cell
endow cells with the ability to conjugate;
we say that they are nonconjugative. The IS3 IS3
conjugative nature of many R plasmids plays
an important role in the rapid spread of Integrated F factor
antibiotic and drug resistance genes through (b)
populations of pathogenic bacteria. The evo- ◾ FIGURE 8.20 IS elements mediate the integration of the F factor. (a) An abbreviated map of
lution of R plasmids that make host bacteria the structure of the F factor in E. coli strain K12, with distances given in kilobases (1000 nucleotide
resistant to multiple antibiotics has become pairs). The locations of genes required for conjugative transfer (tra genes), replication (rep genes),
a serious medical problem, and the use of and the inhibition of phage growth (phi genes) are shown, along with the positions of three IS
antibiotics for nontherapeutic purposes has elements. The arrows denote the specific IS element that mediated the integration of the F factor
contributed to the rapid evolution of mul- during the formation of the indicated Hfr strains. (b) Recombination between IS elements inserts the
tiple drug-resistant bacteria (see the Focus on F factor into the bacterial chromosome, producing an Hfr.
Antibiotic-Resistant Bacteria on the Student
Companion site).
In 1958 François Jacob and Elie Wollman recognized that the F factor and
certain other genetic elements had unique properties. They called this class of ele-
ments episomes. According to Jacob and Wollman, an episome is a genetic element
that is unessential to the host and that can replicate either autonomously or be inte-
grated (covalently inserted) into the chromosome of the host bacterium. The terms
plasmid and episome are not synonyms. Many plasmids do not exist in integrated states
and are thus not episomes. Similarly, many lysogenic phage chromosomes, such as the
phage λ genome, are episomes but are not plasmids.
The ability of episomes to insert themselves into chromosomes depends on the
presence of short DNA sequences called insertion sequences (or IS elements). The
IS elements are present in both episomes and bacterial chromosomes. These short
sequences (from about 800 to about 1400 nucleotide pairs in length) are transpos-
able; that is, they can move from one chromosome to a different chromosome (see
Chapter 21 on the Instructor Companion site). In addition, IS elements mediate
recombination between otherwise nonhomologous genetic elements. The role of
IS elements in mediating the integration of episomes is well documented in the
case of the F factor in E. coli. Crossing over between IS elements in the F factor
and the bacterial chromosome produces Hfr’s with different origins and directions
of transfer during conjugation (◾ Figure 8.20).

F′ FACTORS AND SEXDUCTION

As discussed in the preceding section, an Hfr strain is produced by the integra-
tion of an F factor into the chromosome by recombination between IS elements in
the chromosome and IS elements in the F factor (see Figure 8.20). Do you think
that this recombination process might be reversible? Indeed, rare F+ cells are pres-
ent in Hfr cultures, indicating that excision of the F factor does occur (by a process

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