1 | a g e

Conjugation
The transfer of genetic material between bacteria in direct physical contact is called
Conjugation. This method of bacterial sexual reproduction was described in 1946 by the
elegantly simple experimental work of Joshua Lederberg and Edward Tatum, who studied
two strains of Escherichia coli with different nutritional requirements. Strain A would grow
on a minimal medium only if the medium were supplemented with methionine and biotin;
strain B would grow on a minimal medium only if it were supplemented with threonine,
leucine, and thiamine. Thus, we can designate strain A as met bio thr+ leu+ thi+ and strain
B as met+ bio+ thr leu thi. Figure 1 illustrates simplified form the concept of their
experiment. Here, strains A and B are mixed together, and some of the progeny are now wild
type, having regained the ability to grow without added nutrients.












Figure 1. Demonstration by Lederberg and Tatum of genetic recombination between bacterial cells. Cells of
type A or type B cannot grow on an unsupplemented (minimal) medium (MM), because A and B each carry
mutations that cause the inability to synthesize constituents needed for cell growth. When A and B are mixed for
a few hours and then plated, however, a few colonies appear on the agar plate. These colonies derive from single
cells in which an exchange of genetic material has occurred; they are therefore capable of synthesizing all the
required constituents of metabolism. Source: An Introduction to Genetic Analysis. 7th edition. Griffiths AJF,
Miller JH, Suzuki DT, et al. New York: W. H. Freeman; 2000.
2 | a g e

Lederberg and Tatum plated bacteria into dishes containing only unsupplemented minimal
medium. Some of the dishes were plated only with strain A bacteria, some only with strain B
bacteria, and some with a mixture of strain A and strain B bacteria that had been incubated
together for several hours in a liquid medium containing all the supplements. No colonies
arose on plates containing either strain A or strain B alone, showing that back mutations
cannot restore prototrophy, the ability to grow on unsupplemented minimal medium.
However, the plates that received the mixture of the two strains produced growing colonies at
a frequency of 1 in every 10,000,000 cells plated (in scientific notation, 1 107). This
observation suggested that some form of recombination of genes had taken place between the
genomes of the two strains to produce prototrophs.
It could be suggested that the cells of the two strains do not really exchange genes but
instead leak substances that the other cells can absorb and use for growing. This possibility of
cross feeding was ruled out by Bernard Davis. He constructed a U-tube in which the two
arms were separated by a fine filter. The pores of the filter were too small to allow bacteria to
pass through but large enough to allow easy passage of the fluid medium and any dissolved
substances (Figure 2). Strain A was put in one arm; strain B in the other. After the strains had
been incubated for a while, Davis tested the content of each arm to see if cells had become
able to grow on a minimal medium, and none were found. In other words, physical contact
between the two strains was needed for wild-type cells to form. It looked as though some
kind of gene transfer had taken place, and genetic recombinants were indeed produced.

Figure 2. Experiment demonstrating that physical contact between bacterial cells is needed for genetic
recombination to take place. Source: An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH,
Suzuki DT, et al. New York: W. H. Freeman; 2000.
3 | a g e

In 1953, William Hayes determined that genetic transfer occurred in one direction in the
above types of crosses and is polar. The transfer of genetic material in E. coli is not
reciprocal. One cell acts as donor, and the other cell acts as the recipient. This kind of
unidirectional transfer of genes was originally compared to a sexual difference, with the
donor being termed male and the recipient female. However, this type of gene transfer is
not true sexual reproduction. In bacterial gene transfer, one organism receives genetic
information from a donor; the recipient is changed by that information. In sexual
reproduction, two organisms donate equally (or nearly so) to the formation of a new
organism, but only in exceptional cases is either of the donors changed.
The process of bacterial conjugation can be described as a phenomenon that requires
the presence of a special plasmid called the F plasmid. Plasmids are small, circular pieces of
DNA that are separate and replicate indepentently from the bacterial chromosome. Plasmids
contain only a few genes that are usually not needed for growth and reproduction of the cell.
However, in stressful situations, plasmids can be crucial for survial. The F plasmid, for
example, facilites conjugation. This can give a bacterium new genes that may help it survive
in a changing environment. Some plasmids can integrate reversibly into the bacterial
chromosome. An integrated plasmid is called an episome. Bacteria that have a F plasmid are
referred to as as F+ or male. Those that do not have an F plasmid are F- of female. The F
plasmid consists of 25 genes that mostly code for production of sex pilli. A conjugation event
occurs when the male cell extends his sex pili and one attaches to the female. This attached
pilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred
from the male to the female. When transfer is complete, the result is two male cells. The F
plasmid can behave as an episome. When the F+ plasmid is integrated within the bacterial
chromosome, the cell is called an Hfr cell (high frequency of recombination cell). The F
plasmid always inserts at the same spot for a bacterial species. The Hfr cell still behaves as a
F+ cell, transferring F genes to a F-cell, but now it can take some of the bacterial
chromosome with it. Replication of the Hfr chromosome begins at a fixed point within the F
episome and the chromosome is transferred to the female as it replicates. Movement of the
bacteria usually disrupts conjugation before the entire chromosome, including the tail of the F
episome can be transferred. Therefore, the recipient remains F- because the F plasmid is not
entirely transferred. A cross over event can occur between homologous genes on the Hfr
fragment and the F- DNA. Pieces of DNA not recombined will be degraded or lost in cell
division. Now the recombinant genome can be passed on to future generations.
4 | a g e



3 | a g e



Figure 3: 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
+
X 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 X 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 recipients chromosome if it is to be stably
maintained. Source: Prescott, Harley, and Kleins Microbiology, 7
th
Ed. McGraw-Hill. 2008. New York.


6 | a g e


Figure 4: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 then transferred to a recipient during conjugation. See text for explanation. Source: Prescott,
Harley, and Kleins Microbiology, 7
th
Ed. McGraw-Hill. 2008. New York.

Determining linkage from interrupted-mating experiments
The exact nature of Hfr strains became clearer in 1957, when Elie Wollman and Franois
Jacob investigated the pattern of transmission of Hfr genes to F cells during a cross. They
crossed Hfr strains a+ b+ c+ d+ with F strains a b c d. At specific time intervals after
mixing, they removed samples. Each sample was put in a kitchen blender for a few seconds
to disrupt the mating cell pairs and then was plated onto a medium containing streptomycin to
kill the Hfr donor cells. This procedure is called interrupted mating. The str
r
cells then were
tested for the presence of marker alleles from the donor. Those str
r
cells bearing donor marker
alleles must have taken part in conjugation; such cells are called exconjugants. The Hfr
chromosome, originally circular, unwinds and is transferred to the F cell in a linear fashion.
The unwinding and transfer begin from a specific point at one end of the integrated F, called
7 | a g e

the origin or O. The farther a gene is from O, the later it is transferred to the F; the transfer
process most likely will stop before the farthermost genes are transferred. Wollman and Jacob
realized that it would be easy to construct linkage maps from the interrupted-mating results,
using as a measure of distance the times at which the donor alleles first appear after mating.
The units of distance in this case are minutes. Thus, if b+ begins to enter the F cell 10
minutes after a+ begins to enter, then a+ and b+ are 10 units apart (Figure 7-8). Like the maps
based on crossover frequencies, these linkage maps are purely genetic constructions; at the
time, they had no known physical basis.


Figure 5: Interrupted-mating conjugation experiments with E. coli. F cells that are strr are crossed with Hfr
cells that are strs. The F cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and
gal) that prevent them from carrying out specific metabolic steps. However, the Hfr cells are capable of carrying
out all these steps. At different times after the cells are mixed, samples are withdrawn, disrupted in a blender to
break conjugation between cells, and plated on media containing streptomycin. The antibiotic kills the Hfr cells
but allows the F cells to grow and to be tested for their ability to carry out the four metabolic steps. (a) A plot
of the frequency of recombinants for each metabolic marker as a function of time after mating. Transfer of the
donor allele for each metabolic step depends on how long conjugation is allowed to continue. (b) A schematic
view of the transfer of markers over time. Source: E. L. Wollman, F. Jacob, and W. Hayes, Cold Spring Harbor
Symposia on Quantitative Biology 21, 1956, 141.
8 | a g e



Figure 6: A linkage map can be constructed for the E. coli chromosome from interrupted-mating studies by
using the time at which the donor alleles first appear after mating. The units of distance are given in minutes;
arrowhead at left indicates the direction of transfer of the donor alleles. Source: An Introduction to Genetic
Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al. New York: W. H. Freeman; 2000.
Gradient of transfer
Some genes dont even get into the act. To better understand this fact, let us look again at the
consequences of gene transfer. Usually, only a fragment of the donor chromosome appears in
the recipient, owing to spontaneous breakage of the mating pairs; so the entire chromosome is
rarely transferred. The spontaneous breakage can occur at any time after transfer begins,
which creates a natural gradient of transfer and makes it less and less likely that a recipient
cell will receive later and later genetic markers. (Later here refers to markers that are
increasingly farther from the origin and hence are donated later in the order of markers
transferred.) For example, in a cross of Hfr-donating markers in the order met, arg, leu, we
would expect a distribution of fragments such as the one represented here:

Note that many more fragments contain the met locus than the arg locus and that the leu locus
is present on only one fragment. It is easy to see that the closer the marker is to the origin, the
greater the chance it will be transferred in conjugation. We can use the natural gradient of
transfer to establish the order of genetic markers, provided we select for an early marker that
enters before the markers that we are ordering. Lets see how this works. Suppose that we use
an Hfr strain that donates markers in the order met, arg, aro, his. In a cross of an Hfr that is
met+ arg+ aro+ his+ strs with an F that is met arg aro his strr, recombinants are
selected that can grow on a minimal medium without methionine but with arginine, aromatic
9 | a g e

amino acids, and histidine and in the presence of streptomycin. Here we are selecting for
recombinants in the F strain that are met+ in a cross in which the met locus is transferred as
the earliest marker. We can then score for inheritance of the other markers present in the Hfr
by testing on supplemental minimal medium lacking, in turn, one of the required nutrients.
A typical result would be:

Note how the frequency of inheritance corresponds to the order of transfer. This
correspondence is due to the fact that the frequency of inheritance is indicative of the
frequency of transfer. For this method to work, it is crucial that it be applied only to genetic
markers that enter after the selected markerin this case, after met.
Transformation
The transfer of a naked fragment of DNA between bacteria is called Transformation. After
death or cell lyses, some bacteria release their DNA into the environment. Other bacteria,
generally of the same species, can come into contact with these fragments, take them up and
incorporate them into their DNA by recombination. This method of transfer is the process of
transformation. Any DNA that is not integrated into he chromosome will be degraded. The
genetically transformed cell is called a recombinant cell because it has a different genetic
makeup than the donar and the recipient. All of the descendants of the recombinant cell will
be identical to it. In this way, recombination can give rise to genetic diversity in the
population.
The transformation process was first demonstrated in 1928 by Frederick Griffith.
Griffith experimented on Streptococcus pneumoniae, a bacteria that causes pneumonia in
mammals. When he examined colonies of the bacteria on petri plates, he could tell that there
were two different strains. The colonies of one strain appeared smooth. Later analysis
revealed that this strain has a polysaccharide capsule and is virulent, that it, it causes
pneumonia. The colonies of the other strain appeared rough. This strain has no capsules and
is avirulent. When Griffith injected living encapsulated cells into a mouse, the mouse died of
pneumonia and the colonies of encapsulated cells were isolated from the blood of the mouse.
When living nonencapsulated cells were injected into a mouse, the mouse remained healthy
10 | a g e

and the colonies of nonencapsulated cells were isolated from the blood of the mouse. Griffith
then heat killed the encapsulated cells and injected them into a mouse. The mouse remained
healthy and no colonies were isolated. The encapsulated cells lost the ability to cause the
disease. However, a combination of heat-killed encapsulated cells and living nonencapsulated
cells did cause pneumonia and colonies of living encapsulated cells were isolated from the
mouse. How can a combination of these two strains cause pneumonia when either strand
alone does not cause the disease? The living nonencapsulated cells came into contact with
DNA fragments of the dead capsulated cells. The genes that code for thr capsule entered
some of the living cells and a crossing over event occurred. The recombinant cell now has the
ability to form a capsule and cause pneumonia. All of the recombinant's offspring have the
same ability. That is why the mouse developed pneumonia and died.


Figure 7: Transformation with (a) DNA fragments and (b) plasmids. Transformation with a plasmid often is
induced artificially in the laboratory. The transforming DNA is in purple and integration is at a homologous
region of the genome. Source: Prescott, Harley, and Kleins Microbiology, 7
th
Ed. McGraw-Hill. 2008. New
York.
11 | a g e


Microbial geneticists exploit transformation to move DNA (usually recombinant DNA) into
cells. However, as already noted, many species, including E. coli, are not naturally
transformation competent. Fortunately, these bacteria can be made artificially competent by
certain treatments. Two common techniques are electrical shock and exposure to calcium
chloride. Both approaches render the cell membrane more permeable to DNA and 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 vulnerable to
attack by nucleases. It is easier to transform bacteria with plasmid DNA since plasmids are
not as easily degraded as linear fragments and can replicate within the host.

Further Readings
Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th Ed. W.
H. Freeman. 2000. New York.
Prescott, Harley, and Kleins Microbiology, 7
th
Ed. McGraw-Hill. 2008. New York.
Wollman E L, Jacob F, Hayes W. Cold Spring Harbor Symposia on Quantitative Biology 21,
1956, 141.