The History of the Study of Bacterial Conjugation:
A Prominent Mechanism of Horizontal Gene Transfer
By: Andy Moeller
Horizontal gene transfer (HGT) is a relatively well studied mechanism by which
genetic material is transferred from organism to organism through transduction,
transformation, and conjugation. Transduction is the process by which genetic material is
transferred from one organism to another by way of a viral agent, transformation is the
process by which an organism obtains foreign genetic material from the environment, and
conjugation is the process by which one organism transfers genetic material directly to
another organism. In this review, the history of HGT will be told through the examination
of three landmark papers, with a specific focus on the history of bacterial conjugation.
Horizontal gene transfer as a method for bacterial sexual recombination was first
supported experimentally by E.L. Tatum and Joshua Lederberg in 1947 in their paper
Gene Recombination in the Bacterium Escherichia Coli. The experiment described in this
paper showed that recombinant phenotypes could be created through the exchange of
genetic material between E. coli strains.
During the first step of the experiment, X-rays, ultraviolet light, and nitrogenmustard were used to create genetically and phenotypically unique mutants of
Escherichia coli K-12. Each mutant had specific nutritional requirements for its
individual survival in growth media. Some strains required biotin to survive, while others
needed arginine, arginine and methionine, or threonine and leucine, amongst various
other amino acid combinations. (Tatum and Lederberg, 1947)
Once created, the mutants were paired and grown together in minimal media
lacking any and all of the growth factors required by the mutants. The pairings always
included strains with completely different growth requirements. For example, a strain that
required biotin to survive was mixed with a strain that required arginine, but never with a
strain that required both arginine and biotin. Without the option of genetic recombination,
all of the bacteria should have died in minimal media because they would not have had
access to their specific growth factors. What was observed, however, was the generation
of new phenotypes that were able to survive in the minimal media regardless of the
absence of specific growth factors. (Tatum and Lederberg, 1947)
For example, when a strain that required biotin was grown in a minimal growth
media with a strain that required arginine, new bacterial strains that didn’t require either
biotin or arginine were produced. Since one strain was originally able to produce biotin
but not arginine, and the other was able to produce arginine but not biotin, Tatum and
Lederberg hypothesized that the strains capable of surviving in the minimal growth media
were recombinants capable of producing both arginine and biotin. This experiment was
performed many times with many different E. coli mutants, and recombination always
seemed to occur.
The alternative explanation for the observations was that the E. coli strains were
randomly mutating to become capable of surviving in minimal growth media. However,
by growing individual strains in media devoid of their specific nutritional requirements
and consistently observing cell culture death, Tatum and Lederberg showed that
spontaneous mutation rates were too low to account for the creation of strains capable of
surviving in minimal growth media.
This study was a paradigm shift in bacterial genetics. It was the first to show that
bacteria are capable of genetic recombination, and eventually led to the coining of the
term “horizontal gene transfer.” Later experiments used similar experimental methods to
further investigate the nature of HGT. One such experiment focused on HGT in
Rhodopseudomonas capsulata.
In 1973, Barry Marrs published a paper entitled Genetic Recombination in
Rhodopseudomonas capsulata. This study provided further evidence for methods of
genetic exchange between bacteria. The experiment isolated two strains of the
Rhodopseudomonas capsulata, each resistant to a particular type of antibiotic. One strain
was resistant to rifampicin, the other resistant to streptomycin. The two strains where
then mixed and grown together in media. Also, control groups containing bacterial strains
resistant to only one antibiotic were grown in media separate from the mixed
experimental group. After several generations of growth, the frequencies of strains
resistant to both streptomycin and rifampicin were determined in the experimental and
control groups. This procedure revealed two strains (H9 and B10) that, when mixed,
consistently gave significantly more rifampicin and streptomycin-resistant bacterial
colonies than the control cultures. Results from the experiment can be seen in Table 1.
(Marrs, 1974)
Table 1: A mixture of whole cultures of H9 and B10 yielded 847 rifampicin and
streptomycin resistant bacterial colony forming units, while cultures containing just one
strain of either H9 or B10 yielded only 18 and 4 rifampicin and streptomycin resistant
cultures. (Marrs, 1974)
The results from this experiment showed that a mechanism for genetic
recombination exists in Rhodopseudomonas capsulata. Somehow, genes for resistance to
rifampicin and streptomycin were exchanged in the cultures containing both H9 and B10
strains. This led to a very high number of colonies resistant to both types of antibiotics in
the experimental cultures. The control cultures, which contained only one strain of
bacteria, yielded low numbers of colonies resistant to both types of antibiotics because
there was no opportunity for genetic recombination to occur. (Marrs, 1974)
Marrs was also interested in further describing the mechanism by which genetic
recombination occurs in Rhodopseudomonas capsulata. To do this, he gathered media
from an H9 bacterial culture, ran it through a filter fit to remove only cells, and added the
product to a B10 bacterial culture. He then let the newly infused B10 culture grow for
several generations. When he treated this culture with rifampicin and streptomycin, Marrs
found that a large number of colony forming units had developed resistances,
demonstrating that cell to cell contact is not needed for genetic recombination to occur in
Rhodopseudomonas capsulata. Marrs then repeated the experiment, this time adding
DNase to the cell-free filtrate. Suprisingly, rifampicin and streptomycin resistant strands
were still generated. This led Marrs to hypothesize the existence of a releasable, DNAcontaining sex pilus capable of transmitting DNA from cell to cell. He was partially
correct in this hypothesis, as it is now known that a sex pilus functions in bacterial
conjugation. However, since a bacterium’s sex pilus is not releasable, its existence does
not explain how DNA was transferred by the cell-free filtrate. Based on what has been
learned since this experiment, it is likely that an undetected viral vector transported the
H9 DNA through the filter and into the B10 bacterial colony.
Besides clarifying Marrs’ pilus hypothesis, science has come a long way in
describing HGT since the two studies mentioned above. With the advent of extensive
online genome libraries, it has become possible to phylogenetically pinpoint evolutionary
examples of HGT. By comparing the genetic makeup of different organisms, scientists
can determine which genes have been horizontally transferred, and, in some cases, rates
of HGT can be calculated. These types of bioinformatic/phylogenetic studies currently
dominate the study of HGT. Yet, wet lab HGT science is not dead, as new techniques
have recently been used to more accurately describe the mechanism of bacterial
conjugation.
The paper Direct Visualization of Horizontal Gene Transfer by Ana Babic et al.
was published in March of 2008. The study provided solid visual evidence for bacterial
conjugation in Escherichia coli. The experiment also answered questions about the fate
of horizontally transferred DNA and the mechanism of bacterial conjugation. Before this
experiment, it was known that a sex pilus mediates the transfer of single stranded DNA
from donor cell to recipient cell. It was also known that the single stranded DNA
(ssDNA) is converted into double stranded DNA (dsDNA) by complementary base
pairing after it enters recipient cell. In order to answer their questions about the fate of
DNA transferred through conjugation, the experimenters needed to be able to see the
transferred DNA inside living cells. To do this, they developed a novel technique that
utilizes natural protein fluorescence as a means of DNA visualization. (Babic et al.,
2008.)
First, E. coli cells that contained the DAM methylase enzyme were mated with
cells that did not contain the DAM methylase enzyme. DAM methylase is a protein that
adds methyl groups to the adenine of the DNA sequence 5'-GATC-3'. So, cells containing
the protein have methylated 5'-GATC-3' DNA while the cells lacking the protein have
unmethylated 5'-GATC-3' DNA. Since the sex pilus merely allows for the passage of
ssDNA, only a single strand of methylated DNA was transferred from the DAM
methylase proficient bacteria into the DAM methylase deficient bacteria. The ssDNA was
then converted into dsDNA by complementary base pairing. However, since the recipient
cell lacked the DAM methylase enzyme, the resulting dsDNA molecule consisted of one
strand of methylated DNA and one strand of non-methylated DNA. Furthermore, the
bacterial strains used in this experiment all containted the protein SeqA-YFP, which
fluoresces yellow and has a high binding affinity for DNA that has been hemimethylated.
Hemimethylated DNA consists of one methylated strand and one non-methylated strand.
Since the recently transferred DNA became hemimethylated after conjugate base pairing
in the recipient cell, SeqA-YFP bound to this DNA sequence very frequently, allowing it
to be viewed under a fluorescent microscope. (Babic et al., 2008.)
Figure 2:
Figure 2: “(A) Dam-proficient cells. (B) Dam-deficient cells. (C) Recipient with
SeqA-YFP focus after conjugation with donor. Left, phase contrast image; center,
fluorescence image; right, overlay between the phase contrast image and the fluorescence
image represented in green. (D and E) Real-time conjugation [(D), phase contrast; (E),
fluorescence overlay] of donors (red cells) with recipient (green cells) as followed by
time-lapse microscopy at 0, 10, and 30 min after plating on nutrient-agarose cavity slide.”
(Babic et al., 2008.)
The results of this experiment showed that pilus-mediated conjugation can occur
at distances up to 12 micrometers, and that transferred DNA is incorporated into the
recipient cell 96.7% of the time, ± 0.83%. The other 3.3% of the time the DNA was
degraded by the RecBCD exonuclease before it could form a stable circular plasmid or be
incorporated into the main bacterial chromosome. These results quantified aspects of
bacterial conjugation that were previously unquantifiable, and provide support for a
method that could be used to study conjugation in a variety of organisms. (Babic et al.,
2008.)
Horizontal Gene Transfer is an area of genetics that remains poorly understood. In
the last 60 years, however, scientific understanding of bacterial conjugation has gone
from nonexistent to relatively complex. From comparative phenotypic analysis to
advanced fluorescent microscopy, innovative techniques and technologies have allowed
for the ever expanding understanding of HGT. Surely, as science moves into the 21st
century, these trends will continue to permit the clarification of HGT’s role in life on
earth.
References:
Babic, Ana et al., Science 319, 5869 (2008).
E. L. Tatum and Joshua Lederberg, Journal of Bacteriology 53, pp. 673-684 (1947).
Marrs, Barry, Proc. Nat. Acad. Sci. 71, 3 (1974).