A cartoon schematic (top) and raw data (bottom) showing a lambda bacteriophage attached to an E. coli cell with the phage’s DNA labeled with a fluorescent dye. The phage injects its viral DNA into the cell, and as the ejection proceeds, the dye molecules are transferred. Once inside the cell, the dye redistributes to the bacterium’s genome, causing the whole cell to light up. Credit: Nigel Orme |
Researchers
at the California Institute of Technology (Caltech) have been able, for
the first time, to watch viruses infecting individual bacteria by
transferring their DNA, and to measure the rate at which that transfer
occurs. Shedding light on the early stages of infection by this type of
virus—a bacteriophage—the scientists have determined that it is the
cells targeted for infection, rather than the amount of genetic material
within the viruses themselves, that dictate how quickly the
bacteriophage’s DNA is transferred.
“The
beauty of our experiment is we were able to watch individual viruses
infecting individual bacteria,”says Rob Phillips, the Fred and Nancy
Morris Professor of Biophysics and Biology at Caltech and the principal
investigator on the new study. “Other studies of the rate of infection
have involved bulk measurements. With our methods, you can actually
watch as a virus shoots out its DNA.”
The
new methods and results are described in a paper titled “A
Single-Molecule Hershey–Chase Experiment,” which will appear in Current Biology and
currently appears online. The lead authors of that paper, David Van
Valen and David Wu, completed the work while graduate students in
Phillips’s group.
In
the well-known 1952 Hershey-Chase experiment, Alfred Hershey and Martha
Chase of the Carnegie Institution of Washington in Cold Spring Harbor
convincingly confirmed earlier claims that DNA—and not protein—was the
genetic material in cells. To prove this, the researchers used
bacteriophages, which are able to infect bacteria using heads of tightly
bundled DNA coated in a protein shell. Hershey and Chase radiolabeled
sulfur, contained in the protein shell but not in the DNA, and
phosphorous, found in the DNA but not in the protein shell. Then they
let the bacteriophages infect the bacterial cells. When they isolated
the cells and analyzed their contents, they found that only the
radioactive phosphorous had made its way into the bacteria, proving that
DNA is indeed the genetic material. The results also showed that,
unlike the viruses that infect humans, bacteriophages transmit only
their genetic information into their bacterial targets, leaving their
“bodies” behind.
“This
led, right from the get-go, to people wondering about the
mechanism—about how the DNA gets out of the virus and into the infected
cell,” Phillips says. Several hypotheses have focused on the fact that
the DNA in the virus is under a tremendous amount of pressure. Indeed,
previous work has shown that the genetic material is under more pressure
within its protein shell than champagne experiences in a corked bottle.
After all, as Phillips says, “There are 16 microns [16,000 nm] of DNA
in a tiny 50-nm-sized shell. It’s like taking 500 m of cable from the
Golden Gate Bridge and putting it in the back of a FedEx truck.”
Phillips’s
group wanted to find out whether that pressure plays a dominant role in
transferring the DNA. Instead, he says, “What we discovered is that the
thing that mattered most was not the pressure in the bacteriophage, but
how much DNA was in the bacterial cell.”
The
researchers used a fluorescent dye to stain the DNA of two mutants of a
bacteriophage known as lambda bacteriophage—one with a short genome and
one with a longer genome—while that DNA was still inside the phage.
Using a fluorescence microscope, they traced the glowing dye to see when
and over what time period the viral DNA transferred from each phage
into an E. coli bacterium. The mean ejection time was about five minutes, though that time varied considerably.
This
was markedly different from what the group had seen previously when
they ran a similar experiment in a test tube. In that earlier setup,
they had essentially tricked the bacteriophages into ejecting their DNA
into solution—a task that the phages completed in less than 10 sec.
In that case, once the phage with the longer genome had released enough
DNA to make what remained inside the phage equal in length to the
shorter genome, the two phages ejected DNA at the same rate. Therefore,
Phillips’s team reasoned, it was the amount of DNA in the phage that
determined how quickly the DNA was transferred.
But Phillips says, “What was true in the test tube is not true in the cell.” E. coli
cells contain roughly 3 million proteins within a box that is roughly
one micron (1,000 nm) on each side. Less than 10 nm separate each
protein from its neighbors. “There’s no room for anything else,”
Phillips says. “These cells are really crowded.”
And
so, when the bacteriophages try to inject their DNA into the cells, the
factor that limits the rate of transfer is how jam-packed those cells
are. “In this case,” Phillips says, “it had more to do with the
recipient, and less to do with the pressure that had built up inside the
phage.”
Looking
toward the future, Phillips is interested in using the methods he and
his team have developed to study different types of bacteriophages. He
also wants to investigate various molecules that could be helping to
actively pull the viral DNA into the cells. In the case of a
bacteriophage called T7, for instance, previous work has shown that the
host cell actually grabs onto the DNA and begins making copies of its
genes before the virus has even delivered all the DNA into the cell.
“We’re curious whether that kind of mechanism is in play with the lambda
bacteriophage,” Phillips says.
The
current findings have implications for the larger question of how
biomolecules like DNA and proteins cross membranes in general, and not
just into bacteria. Cells are full of membranes that divide them into
separate compartments and that separate entire cells from the rest of
the world. Much of the business of cellular life involves getting
molecules across those barriers. “This process that we’ve been studying
is one of the most elementary examples of what you could call polymer
translocation or getting macromolecules across membranes,” Phillips
says. “We are starting to figure out the physics behind that process.”
In addition to Phillips, Van Valen, and Wu, the other authors on the Current Biology
paper are graduate student Yi-Ju Chen; Hannah Tuson of the University
of Wisconsin at Madison; and Paul Wiggins of the University of
Washington. Van Valen is currently a medical student at UCLA’s David
Geffen School of Medicine, and Wu is an intern at the University of
Chicago. The work was supported by funding from the National Science
Foundation, a National Institutes of Health Medical Scientist Training
Program fellowship, a Fannie and John Hertz Yaser Abu-Mostafa Graduate
Fellowship, and an NIH Director’s Pioneer Award.