Robert J. Brooker - Genetica
Esperimento di genetica 6.1
Conjugation Experiments Can Map
Genes Along the E. coli Chromosome
The first genetic mapping experiments in bacteria were carried
out by Elie Wollman and François Jacob in the 1950s. At the time
of their studies, not much information was known about the organization of bacterial genes along the chromosome. A few key advances made Wollman and Jacob realize that the process of genetic
transfer could be used to map the order of genes in E. coli. First, the
discovery of conjugation by Lederberg and Tatum and the identification of Hfr strains by Cavalli-Sforza and Hayes made it clear that
bacteria can transfer genes from donor to recipient cells in a linear
fashion. In addition, Wollman and Jacob were aware of previous
microbiological studies concerning bacteriophages—viruses that
bind to E. coli cells and subsequently infect them. These studies
showed that bacteriophages can be sheared from the surface of E.
coli cells if they are spun in a blender. In this treatment, the bacteriophages are detached from the surface of the bacterial cells, but
the bacteria themselves remain healthy and viable. Wollman and
Jacob reasoned that a blender treatment could also be used to separate bacterial cells that were in the act of conjugation without killing
them. This technique is known as an interrupted mating.
The rationale behind Wollman and Jacob’s mapping strategy is
that the time it takes for genes to enter a donor cell is directly related to their order along the bacterial chromosome. They hypothesized that the chromosome of the donor strain in an Hfr mating is
transferred in a linear manner to the recipient strain. If so, the order
of genes along the chromosome can be deduced by determining the
time it takes various genes to enter the recipient strain. Assuming
the Hfr chromosome is transferred linearly, they realized that interruptions of mating at different times would lead to various lengths
of the Hfr chromosome being transferred to the F– recipient cell. If
two bacterial cells had mated for a short period of time, only a
small segment of the Hfr chromosome would be transferred to the
recipient bacterium. However, if the bacterial cells were allowed to
mate for a longer period of time before being interrupted, a longer
segment of the Hfr chromosome could be transferred (see Figure
6.5b). By determining which genes were transferred during short
matings and which required longer mating times, Wollman and
Jacob were able to deduce the order of particular genes along the E.
coli chromosome.
As shown in the experiment of Figure EG6.1.1, Wollman and
Jacob began with two E. coli strains. The donor (Hfr) strain had the
following genetic composition:
thr+: able to synthesize threonine, an essential amino acid for
growth
leu+: able to synthesize leucine, an essential amino acid for
growth
azis: sensitive to killing by azide (a toxic chemical)
tons : sensitive to infection by bacteriophage T1. (As discussed
later, when bacteriophages infect bacteria, they may
cause lysis, which results in plaque formation.)
lac+: able to metabolize lactose and use it for growth
gal+: able to metabolize galactose and use it for growth
strs: sensitive to killing by streptomycin (an antibiotic)
The recipient (F–) strain had the opposite genotype: thr– leu– azir
tonr lac– gal– strr (r = resistant). Before the experiment, Wollman
and Jacob already knew the thr+ gene was transferred first, followed by the leu+ gene, and both were transferred relatively soon (5
to 10 minutes) after mating. Their main goal in this experiment was
to determine the times at which the other genes (azis tons lac+ gal+)
were transferred to the recipient strain. The transfer of the strs gene
was not examined because streptomycin was used to kill the donor
strain following conjugation.
Before discussing the conclusions of this experiment, let’s consider how Wollman and Jacob monitored gene transfer. To determine if particular genes had been transferred after mating, they took
the mated cells and first plated them on growth media that lacked
threonine (thr) and leucine (leu) but contained streptomycin (str).
On these plates, the original donor and recipient strains could not
grow because the donor strain was streptomycin sensitive and the
recipient strain required threonine and leucine. However, mated
cells in which the donor had transferred chromosomal DNA carrying the thr+ and leu+ genes to the recipient cell would be able to
grow.
To determine the order of gene transfer of the azis, tons, lac+,
and gal+ genes, Wollman and Jacob picked colonies from the first
plates and restreaked them on media that contained azide or
bacteriophage T1 or on media that contained lactose or galactose as
the sole source of energy for growth. The plates were incubated
overnight to observe the formation of visible bacterial growth.
Whether or not the bacteria could grow depended on their genotypes. For example, a cell that is azis cannot grow on media containing azide, and a cell that is lac– cannot grow on media containing
lactose as the carbon source for growth. By comparison, a cell that
is azir and lac+ can grow on both types of media.
THE GOAL (DISCOVERY-BASED SCIENCE)
The chromosome of the donor strain in an Hfr mating is transferred
in a linear manner to the recipient strain. The order of genes along
the chromosome can be deduced by determining the time various
genes take to enter the recipient strain.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
Starting materials: The two E. coli strains already described, one Hfr strain (thr+ leu+ azis tons lac+ gal+ strs) and one F– (thr– leu– azir
tonr lac– gal– strr).
FIGURE EG6.1.1 The use of conjugation to map the order of genes along the E. coli chromosome.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
THE DATA
Minutes That
Bacterial Cells
Were Allowed
to Mate Before
Blender Treatment
5
10
15
20
25
30
40
50
60
Percent of Surviving Bacterial Colonies with
the Following Genotypes:
azis
tons
lac+
gal+
thr+ leu+
—*
—
—
—
—
100
12
3
0
0
100
70
31
0
0
100
88
71
12
0
100
92
80
28
0.6
100
90
75
36
5
100
90
75
38
20
100
91
78
42
27
100
91
78
42
27
obtained. To determine the order of the remaining genes (azis, tons,
lac+, and gal+), each surviving colony was tested to see if it was
sensitive to killing by azide, sensitive to infection by T1 bacteriophage, able to use lactose for growth, or able to use galac- tose for
growth. The likelihood of surviving colonies depended on whether
the azis, tons, lac+, and gal+ genes were close to the origin of transfer or farther away. For example, when cells were allowed to mate
for 25 minutes, 80% carried the tons gene, whereas only 0.6%
carried the gal+ gene. These results indicate that the tons gene is
closer to the origin of transfer compared to the gal+ gene. When
comparing the data in Figure EG6.1.1, a consistent pattern emerged.
The gene that conferred sensitivity to azide (azis) was transferred
first, followed by tons, lac+, and finally, gal+. From these data, as
well as those from other experiments, Wollman and Jacob constructed a genetic map that described the order of these genes
along the E. coli chromosome.
*There were no surviving colonies within the first 5 minutes of mating.
INTERPRETING THE DATA
Now let’s discuss the data shown in Figure EG6.1.1. After the
first plating, all survivors would be cells in which the thr+ and leu+
alleles had been transferred to the F– recipient strain, which was
already streptomycin resistant. As seen in the data, 5 minutes was
not sufficient time to transfer the thr+ and leu+ alleles because no
surviving colonies were observed. After 10 minutes or longer, however, surviving bacterial colonies with the thr+ leu+ genotype were
This work provided the first method for bacterial geneticists to
map the order of genes along the bacterial chromosome. Throughout the course of their studies, Wollman and Jacob identified several different Hfr strains in which the origin of transfer had been
integrated at different places along the bacterial chromosome. When
they compared the order of genes among different Hfr strains, their
results were consistent with the idea that the
E. coli chromosome is circular (see solved problem R2).
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia