GREAT EXPERIMENTS
The discovery of transduction
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Norton Zinder
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I arrived in Joshua Lederberg's laboratory in Madison, Wisconsin, in early July
of 1948. The laboratory was a 20-by-30-foot room in the basement of the genetics
building, which looked like an overgrown log cabin. At the time, little was known
about the nature of the gene, although the one gene:one enzyme hypothesis of George
Beadle and Edward Tatum (1228) gave some insight into function. The process
of transformation, whereby a bacterium incorporates DNA from the media into its
genome, had been discovered in Pneumococcus in 1944 (2361) and
was still considered questionable when I began working. This experiment showed that
DNA was the genetic material. In 1946, Lederberg had discovered that E. coli
bacteria could exchange genetic material between the cells by the process of conjugation
(2341). However, in the 1940s, most scientists thought bacteria to be asexual
and that it could only occasionally undergo morphogenetic changes to filaments or
endospores. They did not believe in these new developments and in fact, they didn't
believe bacteria had genes but rather that they inherited acquired characteristics
in the manner described by Lamarck.
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My task as graduate student was to determine whether conjugation occurs only in
E. coli or was also found in other types of bacteria. I began by studying
the closely related bacterium Salmonella, which causes typhoid fever. The
different species of Salmonella are classified on the basis of serotypes
of their surface antigens and their flagellar antigens. The sets of phenotypes looked
as if they might have been constructed by genetic recombination because they seemed
to be combinations of each other.
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Background
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Figure 1
When two different mutants are plated together on minimal medium, growth only occurs
if recombination generates a strain that has wild-type alleles at both loci.
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The method Lederberg used to demonstrate genetic recombination resulting from conjugation
was to cross two strains that contained complementary nutritional requirements and
to select for bacteria that had lost the requirements. For example, one strain might
lack the ability to grow on media that doesn't have leucine, while the other can
grow on such media but can't grow on media without phenylalanine. A bacterial cross
was accomplished by spreading one tenth of a milliliter of each parent together
or separately on the surface of a minimal medium agar plate, which lacks all of
the nutrients a wild type bacterium can synthesize. Figure 1 shows that bacterial
colonies should be seen only when recombination has taken place. Generally strains
with more than one nutritional requirement were used to prevent spontaneous reversion
back to wild type. Reversion occurs at low frequency in a growing population and
is caused by a change in DNA that either reverses the original alteration or compensates
for it.
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Figure 2
Wild-type bacteria die in penicillin, but bacteria survive if they cannot grow because
they have a mutation that creates a requirement for some nutrient.
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In order to carry out my studies on conjugation in Salmonella it was necessary
for me to isolate nutritional mutants in the strains I wanted to use. At the time,
isolation of such mutants was exceedingly difficult. However, within four weeks
of my arrival Lederberg suggested a simple means for the isolation of nutritional
mutants. The isolation was based on the fact that penicillin only kills growing
cells because it inhibits the cell wall synthesis required to accommodate a growing
cell. Thus if mutagenized bacteria were placed in a minimal medium, mutants that
could not synthesize a particular nutrient would not grow and hence would not be
killed by penicillin. The bacteria could then be plated on media that contain the
necessary nutrients, and growth would resume. Figure 2 shows how penicillin selection
is used to enrich for nutritional mutants. Within a few days I was able to show
that this technique was effective and probably isolated more mutants than I could
analyze in a lifetime (2342).
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My starting material was 22 strains of the bacterial species S. typhimurium
that we received from Sweden in the diplomatic pouch. The strains had all been isolated
from cases of Salmonella gastroenteritis and were classified into 22 strain
types on the basis of their sensitivity to a series of bacterial viruses. What with
working toward a master's degree, making the media, and cleaning glassware for the
laboratory and helping Joshua with experiments, I spent the next year-and-a-half
making complementary pairs of mutants needed for the attempt at crosses. In addition
I took the phage course.
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Finally, I had the collection of mutants I needed and began to cross them to each
other. As time passed and more of the potential crosses were tried, certain things
became apparent. There was no evidence of genetic exchange with any of the intrastrain
crosses. There were a few interstrain crosses that seemed to work and in each case
the presence of a particular parental strain was critical.
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The most productive cross was between two strains labeled LT2 and LT22. LT2 had
mutations that caused it to require the amino acids methionine and histidine. LT22
had two mutations, the first of which caused it to require the amino acids phenylalanine
and tyrosine and the second added a requirement for the related aromatic amino acid
tryptophan. These were selective markers; when plated on minimal media, no bacterial
colonies would form unless the nonmutant state of these genes was present. We found
that we could get recombination and therefore growth on minimal media when these
two strains were crossed.
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There are other genetic markers that can allow growth in either mutant or nonmutant
form and thus are not necessarily selective. For example, if Salmonella
cannot ferment a particular sugar it will use amino acids as an energy source; thus
the ability to ferment the sugar is not absolutely required for growth. Fermentation
mutants can be distinguished from wild-type bacteria by adding an indicator dye
to the medium; the nonfermenters are pink and the fermenters are purple.
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Figure 3
The transfer of selective and nonselective markers can be tested for in a single
assay.
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We examined the transfer of nonselective markers in Salmonella as a more
rigorous test of genetic exchange, since there is no pressure to acquire the new
traits. We crossed two strains that had complementary nutritional requirements (inability
to synthesize a particular amino acid) as well as complementary fermentation requirements
(inability to utilize a particular sugar as a carbon source). When plated on minimal
media supplemented with a particular sugar source, only strains that have gained
the ability to make all the essential amino acids will grow. Figure 3 shows that
if a strain has also picked up the ability to ferment the sugar, then the bacterial
colony will be purple. We found that the nonselective markers were not transferred,
as indicated by the lack of purple colonies.
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Further investigation revealed an interesting difference in the results obtained
with Salmonella compared with those obtained previously with E. coli.
With the crosses in E. coli all pairs of nonselective markers would be
seen in the recombinant progeny. This meant that both parents could contribute genes
to the progeny. However, with the Salmonella crosses in which an unselected
marker from either the LT22 or the LT2 parent could be identified in the resulting
colonies (not shown here), only the unselected markers from the LT22 parent were
seen.
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The experiment
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Figure 4
Different strains of bacteria were grown on either side of a filter. Recombinant
bacteria appeared on one side of the filter, implying that material smaller than
the bacteria must have passed through the filter from the other side.
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We asked whether the parental bacteria had to come in contact with each other for
the reaction to occur as had been shown for E. coli conjugation. We used
a U-shaped glass tube that had a bacterial sterile filter in its middle (2343).
Each arm was inoculated with a different parent in growth medium and the liquid
was flushed back and forth as the cultures grew. Figure 4 shows that recombinant
bacteria were found only in the LT22 side of the tube. We concluded that something
small could pass through the filter to produce the effect. Supernatants from our
individual cultures alone did nothing, but supernatants from mixed cultures could
produce recombinants of LT22.
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We called the activity that transferred the genetic markers FA (filterable activity).
We had demonstrated that FA did not transfer nonselective markers. However, given
the right circumstances a nonselective marker can be made selective. For example,
a xylose fermentation mutant can grow on a plate supplemented with all of the essential
amino acids. But after a while, those nutrients will be used up and only bacteria
that can ferment xylose will continue to grow; thus xylose-fermentation becomes
a selectable trait.
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Figure 5
When a filtrate (FA) is prepared from a strain that had no nutritional mutations
(prototroph) and incubated with a nutritional mutant, the nutritional mutant gains
the ability to grow on medium lacking any one of several growth factors. In each
case the bacteria have only gained the single genetic marker for which one has selected.
The genetic activity of transduction correlates with the properties of the bacteriophage.
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We tested whether FA could transfer the markers under these conditions. We isolated
FA from the supernatant of a bacterial strain that had no nutritional defects and
was able to utilize galactose and xylose as carbon sources. This was accomplished
by incubating the strain with a lysogenic phage which grew on the strain and was
released by its lysis. We incubated the FA with a bacterial strain that required
phenylalanine, tyrosine, and tryptophan in the media and was also unable to ferment
galactose and xylose. Then we plated the bacteria on three different types of plates:
minimal media, complete media supplemented with xylose, and complete media supplemented
with galactose. Figure 5 shows that in each case, only the selected marker is found
in the recombinant progeny. Thus the phenomenon resembled DNA-mediated transformation
in Pneumococcus, in that only one genetic marker at a time would be changed.
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Figure 6
Transduction works by replacing all or part of the phage genome with bacterial DNA.
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With one more fact, all of the above could be explained. Even though, at the time,
there was disagreement as to whether bacteria could carry viruses, we had found
many of them in our cultures. We reasoned that LT22 released a phage that grew on
LT2 and produced the genetic material that returned through a filter. With further
experiments it was possible to show that the active particle was the phage itself.
Figure 6 shows how bacterial DNA carried in a phage particle could give the results
we saw in the U-tube experiment.
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How was the phage eliciting specific changes in the genetic constitution of the
bacteria it infected? We now know that bacteria can be infected by phage in two
different ways: lysogenically and lytically. In the lytic pathway, phage inject
their DNA into the bacterium, take over the cell's replication machinery, produce
many new phage and rapidly cause lysis of the bacterium, releasing the newly generated
phage into the media. In the lysogenic pathway, the phage DNA is incorporated into
the bacterial host's genome. The phage suppresses the lytic pathway and reproduces
along with the host until conditions become adverse for bacterial growth. The phage
senses this and excises its DNA from the bacterial genome, begins the lytic pathway,
and the cell soon lyses.
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Figure 7
The right panel shows how a transducing particle is formed by an error in packaging
during the lytic cycle of a phage. The left panel shows how the incorrectly packaged
bacterial DNA can become incorporated in the newly infected bacterium's genome.
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It turned out, in the case of the phage growing on LT2, that when the phage was
induced to excise from the genome and repackage its DNA into phage particles, the
entire phage genome was replaced by an equivalent amount of bacterial genome (2345).
The phage carried this bacterial DNA back to the LT22 strain and injected it into
the bacterium, whereby it recombined, producing the effects we had seen. The phenomenon
was called General Transduction (to lead across) in that all bacterial genes could
be carried by the phage and recombine with the newly infected bacterium's genome
(2344). However, the phage can only package a small amount of genetic material,
thus only a few genes are carried at one time. Figure 7 illustrates the process
of transducing particle formation and function.
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Shortly thereafter, specialized transduction was discovered using the bacteriophage
lambda (2346). The temperate bacteriophages would insert themselves into
the bacterial genome at a specific site. When they excised themselves, they would
occasionally make a mistake and a proximal segment of the bacterial genome will
replace some of the phage's genes. Upon infection of a new host these bacterial
genes would replace their homologues. For the phage lambda, it was the genes affecting
galactose fermentation that were involved. Later, when its position on the chromosome
was found, PLT22 would transduce the genes for proline metabolism. Thus while lambda
could only cause specific transduction, PLT22 could do both specific and general.
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The legacy
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It was the existence of transduction that led to the idea of recombinant DNA; after
all, both involve the fusing together of two different pieces of DNA, generally
one of which can replicate (2347). The goal of recombinant DNA was to be
able to do with eukaryotic cells the kinds of genetics that transduction allowed
with bacteria. In addition, it became a way to move genes that made important proteins
to organisms in which they might produce large amounts of the important protein.
And of course it was recombinant DNA which opened the way to sequencing of DNA from
any organism.
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Production of tumors by retroviruses is also an example of transduction. These retroviruses
insert themselves into a cell's genome and again by error exchange part of the genome
for mutant cell genes (1515). The genes picked up are important cellular
genes involved in regulation of the cell cycle and gene regulation. When mutant,
they are the dominant cancer-causing genes, and are known by their acronyms such
as src, abl, and ras.
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Given that transduction is a sexual mechanism, it would be surprising if it didn't
play an important role in evolution. Many different organisms have had their DNA
sequences completed, especially microbes . It is now clear that there has been a
great deal of horizontal gene transfer in evolution. The major vehicle for such
transfers was probably transduction as sequences of bacteria contain a large number
of pieces of phage genes. When genes can move, even partial genomes, it has profound
effects on living organisms.
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The author
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Norton D. Zinder, John D. Rockefeller, Jr. Professor of The Rockefeller University
and cohead of a laboratory of genetics was the first chairman (1989-1993) of the
Program Advisory Committee on the Human Genome convened by the National Institutes
of Health. Dr. Zinder's studies of the genetics of bacteria and the properties of
bacteriophages, viruses that infect bacteria, have provided important information
on the mechanisms of heredity. In early experiments conducted in collaboration with
Dr. Joshua Lederberg, former president of The Rockefeller University, Dr. Zinder
discovered that bacteriophages can carry genetic material from donor to recipient
bacteria. This process, called transduction, is one of nature's models for recombinant
DNA technology and viral oncology and has also been widely applied in the development
of research methods for the study of bacterial genetics. With colleagues at Rockefeller,
Dr. Zinder later discovered the first bacteriophages known to contain RNA. He also
demonstrated that RNA phage replication is not dependent on DNA, a finding which
provided further evidence that RNA carries the blueprint for the manufacture of
protein. The first protein synthesized outside the cell was made in his laboratory,
providing clues as to the start and stop signals of the genetic code. Work on the
filamentous bacteriophage (F1) has led to the ability to convert any piece of DNA
to a single strand and thereby being able to sequence it. Born in New York, Dr.
Zinder received an A.B. degree from Columbia University in 1947 and a Ph.D. from
the University of Wisconsin in 1952, the same year he joined The Rockefeller University.
He was appointed professor in 1964 and John D. Rockefeller, Jr. Professor in 1977.
A member of the National Academy of Sciences, Dr. Zinder has served on many scientific
committees and received numerous awards, including the Scientific Freedom and Responsibility
Award given by the American Association for the Advancement of Science.
Norton D. Zinder
Rockefeller University
Laboratory of Genetics
1230 York Avenue
New York, New York 10021
Phone: 212 327 8644
Fax: 212 327 7850
E-mail: zinder@mail.rockefeller.edu
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Last Revised on September 10, 2004
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- 1228 Beadle, G. W., and Tatum, E. L. (1941). Genetic
control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 27,
499-506.
- 1515 Stehelin, D., Varmus, H. E., Bishop, J. M.,
and Vogt, P. K. (1976). DNA related to the transforming gene(s) of avian sarcoma
viruses is present in normal avian DNA. Nature 260, 170-173. PubMed
- 2341 Lederberg, J. (1947). Gene recombination
and linked segregation in E. coli. Genetics 32, 505-525.
- 2342 Lederberg, J., and Zinder, N. D. (1948).
Concentration of biochemical mutants of bacteria with penicillin. J.
Amer. Chem. Soc. 70, 4267-4268.
- 2343 Davis, B. D. (1950). Nonfilterability
of the agents of recombination in E. coli. J. Bacteriol. 60,
507-.
- 2344 Zinder, N. D., and Lederberg, J. (1952).
Genetic exchange in Salmonella. J. Bacteriol. 64, 679-699.
PubMed
- 2345 Ikeda, H., and Tomizawa, J. I. (1965). Transducing
fragments in generalized transduction by phage P1. I. Molecular origin of the fragments.
J. Mol. Biol. 14, 85-109. PubMed
- 2346 Morse, L., Lederberg, E., and Lederberg,
J. (1956). Transduction in E. coli K12. Genetics 41, 142-156.
Journal
- 2347 Jackson, D. A., Symons, R. H., and Berg,
P. (1972). Biochemical method for inserting new genetic information into DNA
of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and
the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 69,
2904-2909. PubMed
- 2361 Avery, O. T., Macleod, C. M., and McCarty,
M. (1944). Studies on the chemical nature of the substance inducing transformation
of pneumococcal types. J. Exp. Med. 79, 137-null. PubMed
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