Welcome back! On today’s aGENEda is an accidental, yet profound, scientific discovery.
It all began in 1948 when Norton Zinder arrived at the University of Wisconsin-Madison,
to join Joshua Lederberg’s laboratory as a fresh graduate. Two years prior to his arrival,
Lederberg and Tatum had discovered that conjugation was the mechanism of genetic
transfer in E. coli. Zinder’s task was now to help build upon this discovery and investigate
whether conjugation was E. coli-specific, or if it also occurred in other bacteria.
They started off by looking for conjugation in Salmonella Typhimurium. What they found instead
was “entertainment” – well we can’t call it that now because they ended up naming the phenomena
“transduction” – but yes, “entertainment” was up there on the suggestions list.
Okay, let’s cut to the chase. So they began by using UV radiation to create doubly auxotrophic
mutant strains, which lacked the ability to synthesise two different amino acids.
Then they isolated these strains on minimal media using penicillin. Penicillin interferes with the
synthesis of new peptidoglycan cell walls by blocking the activity of the transpeptidase
enzyme in growing bacteria. This causes osmotic lysis of the bacterial cells. However,
since mutant strains cannot grow on minimal media and therefore aren’t synthesising any
peptidoglycan cell walls, penicillin is unable target them. Hence, they survive.
Next, they cultured pairs of these isolated mutants with complementary
nutritional requirements, and found that the only cross showing above background level
prototrophs was that between the parental strain labelled LT-2, which was auxotrophic
for methionine and histidine, and LT-22, which was auxotrophic for tryptophan, phenylalanine,
and also tyrosine (as phenylalanine is a precursor for tyrosine synthesis).
To confirm if these prototrophs were arising through cell-to-cell
contact - which is necessary for conjugative transfer - they set up a U-tube experiment.
The two strains were cultured in minimal growth media inside the separate arms of the tube. At
the bottom of the tube was a glass filter with a very small pore size, which prevented bacteria
from passing through. During the experiment, the growth medium was flushed from side to side,
and after the cells had grown to saturation, they were plated out on minimal agar.
If it was conjugation that elicited the prototrophs earlier, then the formation
of colonies was not expected now as the two strains had been separated in the
U-tube by the glass filter. However, a small number of colonies did arise from the LT-22
side of the tube. From this, it was clear that cell-to-cell contact was not being required.
So what was going on then? And why were they only seeing LT-22 prototrophs? Zinder and
Lederberg hypothesised that this asymmetric transfer of genetic material was occurring
via some filterable agent (or FA) that was being produced by LT-2 under stimulation from the LT-22,
which then gave rise to LT-22 prototrophs. It did cross their minds that perhaps it
was transformation that was taking place and the FA was simply free DNA fragments. However, this
hypothesis was disproved when they added DNase to the U-tube and saw that LT-22 protorophs were
still being produced. Subsequent experiments then revealed that the chemical, physical and genetic
characteristics of the FA were similar to that of bacteriophage (i.e. viruses that infect bacteria).
It later became clear that LT-22 was lysogenic for a bacteriophage now called P22. That is,
P22 could infect LT-22 and incorporate its genome into the LT-22 DNA to silently
replicate along with it, until the bacteria experienced some sort of stress. Therefore,
some of the LT-22 cells being used in this experiment must have harboured a P22 infection,
and when the bacterial cells were subjected to UV radiation (and perhaps due to the lack of
nutrition available to the mutant strains), the phage genome excised from the LT-22 DNA
and entered the lytic cycle. Whereby, it overtook the cell’s replication machinery and assembled new
phage particles (or virions) which eventually caused cell lysis. Some of these virions then
crossed the filter and subsequently infected LT-2 cells. P22 is mostly lytic towards LT-2,
thus, after causing LT-2 cell lysis, new virions returned through the filter to infect
intact LT-22 cells. Since LT-22 is lysogenic for the phage, it underwent recombination.
One thing I didn’t mention is that when the phage overtakes the host cell machinery during the lytic
cycle, it firstly creates nucleases which begin hydrolysing the bacterial DNA into fragments.
What ends up happening is that around 1 in 10,000 phage accidentally package some of these fragments
along with their own genome. So, among the virions that accidentally packaged LT-2 genes,
there were some that incorporated the wild-type genes required for the synthesis of tryptophan,
phenylalanine, and tyrosine. Because remember, LT-2 could synthesise all amino acids – just
not methionine and histidine. When such virions then infected LT-22 and underwent recombination,
auxotrophy was reversed, and protrophy restored.
The discovery of transduction created new avenues for genetic engineering and
modern biotechnology. Around two decades after Zinder and Lederberg published their findings,
scientists had discovered how to use viruses to insert DNA fragments with known sequences and
functions, into a bacterial genome. During the 1990s viral vectors, isolated from human tissue,
were used to deliver healthy genes into cells lacking them. This formed the basis
of gene therapy which, although is a field still currently under research,
holds the potential for treating various diseases and cancers.