All the World’s a Phage

Viruses that eat bacteria abound — and surprise

Smaller than bacteria, some of them look like microscopic spacecraft. You can find them almost anywhere: under a rosebush or miles out to sea. These strange entities are bacteriophages, viruses that prey upon bacteria, and there’s a staggering number of them. A pinch of soil or drop of seawater, for example, contains many millions of bacteriophages.

PHAGE POWER. Bacteriophages, such as these, infect bacteria all over the planet. The abundance and diversity of these bacteria-killing viruses has stunned scientists. Jacobs

“They’re nature’s most successful experiment,” says Marisa Pedulla of the University of Pittsburgh. “They outnumber all the bacteria, all the humans, whales, trees, et cetera, put together.”

Bacteriophages, also known simply as phages, came to light around 90 years ago, when two European scientists independently discovered that there are viruses that kill bacteria. Like an Apollo spacecraft landing on the moon, these viruses settle onto the surface of a bacterium. Next, they inject their genes. They reproduce inside the microbe, and eventually their multitudinous descendants explode out of the host.

One of the discoverers of these odd viruses was Felix d’Herelle of the Pasteur Institute in Paris. He coined the word bacteriophage, which translates to “eater of bacteria,” and began to promote the viruses as treatments for infectious diseases, such as cholera and bubonic plague, caused by bacteria.

Because of inconsistent results, phage therapy never took root in the United States, especially after powerful antibiotics such as penicillin emerged. Yet many physicians in the former Soviet Union continue to use bacteriophages. And with the rise of antibiotic-resistant bacteria, some investigators and biotech firms in the United States are trying to resurrect d’Herelle’s dream (SN: 6/1/96, p. 350).

Bacteriophage researchers, however, say that these viruses are of interest beyond medicine. At this year’s American Society for Microbiology meeting in Washington, D.C., in May, bacteriophages dominated the agenda of several symposia, only one of which was focused on medical therapy. Several talks concentrated on the total number of bacteriophages in nature and their impact on bacteria and the environment in general. Another series of lectures revolved around phages that can make bacteria more virulent (see “Phages Behaving Badly,” below). And a number of talks made the point that bacteriophages have an amazing amount of genetic diversity and possess an untold number of novel genes.

Bacteriophages represent a “vast, untapped wealth of genetic information,” says Pedulla. They’re “the pinnacle of creation,” adds Pedulla’s colleague Graham Hatfull, a Howard Hughes Medical Institute investigator at the University of Pittsburgh. “Phages represent the major form of life in the biosphere.”

They’re everywhere

Bacteriophages are drawing renewed interest in part because scientists are only now coming to appreciate how many of these viruses exist. It was just over a decade ago that scientists realized the amazing number of phages in oceans, Curtis Suttle of the University of British Columbia in Vancouver recalled at the recent microbiology meeting. The realization occurred after several investigators training powerful transmission electron microscopes on drops of seawater and that viral particles, most of them bacteriophages, flooded the images.

“Believe it or not, nobody had looked before,” says Suttle. “On average, there are 50 million viruses per milliliter in seawater. The question is, What the heck they’re doing there?”

Microbiologists then documented similar, and even higher, concentrations of phages in soil samples. This led to estimates of 1031 bacteriophages worldwide, a staggeringly large number that many scientists initially dismissed. “We can’t wrap our brains around it,” says Pedulla. “If phages were the size of a beetle, they would cover the Earth and be many miles deep.”

An independent line of reasoning, however, lends support to such a phage tally. Other microbiologists have recently estimated the planet harbors 1030 bacteria. If there are 10 phages for every bacterium, a reasonable assumption according to Hatfull, then 1031 is a fair estimate for the number of bacteriophages in the world.

These plentiful viruses could have a profound impact on their environment, especially in water. According to estimates put forth by Suttle, phages destroy up to 40 percent of the bacteria in Earth’s oceans each day. In doing so, bacteriophages may influence the oceans’, and perhaps the entire world’s food supply by limiting the volume of bacteria available for other organisms to eat.

Bacteria destroyed by phages fill the water with organic matter that’s either consumed by other bacteria or settles to the ocean floor.

“If we’ve got 40 percent of bacterial cells dying each day, that’s certainly going to be important to carbon cycling,” says Suttle. Viruses are “major players” in the global exchange of carbon between organisms and the environment, he says.

“Phages, being so numerous and such excellent predators of bacteria, are going to be very involved in bacterial turnover. That’s a large amount of carbon being recycled,” agrees Pedulla.

What’s inside

Almost as staggering as the number of bacteriophages is their genetic diversity, according to scientists at the microbiology meeting. “Phages are probably the most diverse things on the planet,” says Forest Rohwer of San Diego State University.

Scientists usually study a phage’s DNA after it has reproduced within a bacterial host. This provides many copies of a phage’s genome, enabling researchers to read the virus’ full genetic sequence. The strategy, however, may limit the types of phages examined, since up to 99 percent of bacteria have yet to be grown in a laboratory environment.

Rohwer’s group has instead directly isolated individual bacteriophages free-floating in the ocean waters around San Diego and La Jolla, Calif. Given current DNA-analysis techniques, having a single copy of a phage doesn’t permit a complete reading of its genome, but the investigators can determine the sequence of a few fragments of its DNA. With this approach, Rohwer and his colleagues compiled nearly 2,000 of these partial sequences from seawater phage genomes and ran them through a computer database of all known genetic sequences of plants, animals, fungi, bacteria, viruses, and other microbes. Only 28 percent of the phage sequences bore similarities to previously documented genes. “Most phages’ sequences are unknown,” concludes Rohwer.

With the help of high school students in Pennsylvania and New York, Hatfull and Pedulla have reached a similar conclusion. Working with William R. Jacobs, a Howard Hughes Medical Institute investigator at Albert Einstein College of Medicine in New York, and Jacobs’ sister, Debbie Jacobs-Sera, who is a high school biology teacher in Pennsylvania, Hatfull and Pedulla asked the teenagers to take soil samples.

The students collected soil from barnyards, gardens, and even the monkey pit at the Bronx Zoo. The scientists then taught the students how to isolate a bacteriophage from the soil by growing the viruses in Mycobacterium smegmatis, a harmless bacterial relative of the microbe that causes tuberculosis.

“We guarantee them that the bacteriophage they find will never have been discovered before. We know that because the diversity is so high, and we’ve never isolated the same bacteriophage twice,” says Hatfull.

In the April 18 Cell, Hatfull and his professional and teenage collaborators describe the genomes of 10 soil-dwelling bacteriophages that they had isolated. Of the more than 1,600 genes that the team identified, about half are novel, that is, they don’t match any previously described genes in any other organism. “For a very large number of genes, we just don’t have a clue what they do. They don’t look like anything else we’ve seen before,” says Hatfull.

The University of Pittsburgh team has also recently deciphered the DNA sequence of a bacteriophage with a relatively massive genome. Known as bacteriophage G, this virus has nearly 700 genes, many more than some bacteria. And the proteins encoded by almost 500 of those genes don’t match any known proteins, the scientists discovered.

Equally bewildering, however, are some of the newfound bacteriophage genes that do have matches. One phage contains a gene for a molecule that resembles a human protein called Ro. People with the autoimmune disease known as lupus often have antibodies to Ro, Hatfull points out.

The phage finding raises the possibility that the virus has a role in lupus. There’s been conflicting evidence about whether bacterial infections can trigger the disorder, notes Hatfull. Perhaps, he says, it takes the combination of a bacterium and a certain phage.

Another genetic puzzle comes from a phage called Rosebush, which a high school student isolated from the soil around such a plant. It has two genes resembling those used by many animal immune systems to defend against mycobacterial infections such as tuberculosis and leprosy. Other phages contain genes for proteins that the mycobacteria produce to manipulate immune responses in their hosts. All these phage genes may influence how the microbes cause illness, the scientists suggest.

“We see these [genes], and we don’t know what they do or why they’re there. Speculation becomes rife,” says Hatfull. “All we can say is that there are genes that we didn’t really expect” in bacteriophages.

If that sounds as if Hatfull is saying that he and his colleagues remain largely ignorant when it comes to phages, so be it. “I can’t think of a better word to describe our state of knowledge of the bacteriophage population,” he admits. “We are thoroughly ignorant.”

Phages Behaving Badly

Viruses can control how dangerous some bacteria are

For almost a century, some physicians have championed the medical uses of bacteriophages, but others have been sobered by these viruses’ darker side. Acting as gene-delivery vans, phages can shuttle genetic sequences among different bacterial species and strains — and that can be bad news for people.

Not all phages destroy the bacteria they invade — at least not immediately. Some infect bacteria and then lie dormant. The phages sometimes insert their own genes, and any they’ve acquired by reproducing inside other bacteria, into the chromosomes of their new host. These phage-delivered genes make some bacteria dangerous, scientists have found.

In the 1950s, for example, investigators realized that the bacterium Corynebacterium diphtheriae causes the upper respiratory illness known as diphtheria only if a certain bacteriophage infects the microbe. The phage, in fact, contains the gene for the toxin that triggers diphtheria. A similar story emerged in the 1990s for cholera. This deadly disease is attributed to infections by Vibrio cholera, but it’s actually bacteriophages genes inside the bacterium that carry the instructions for cholera toxin (SN: 6/29/96, p. 404).

More recently, James M. Musser of the National Institute of Allergy and Infectious Diseases in Hamilton, Mont., and his colleagues have fingered phages as co-conspirators with bacteria known as group A Streptococcus (GAS) in illnesses ranging from simple sore throats to heart-damaging rheumatic fever and deadly toxic shock syndrome. When the researchers probed the full genetic sequences, or genomes, of several GAS strains, they were surprised to find that a significant part of each one’s genome consists of phage genes. Indeed, bacteriophages are the major source of genetic differences among GAS strains and seem to account in large part for strain differences in virulence, Musser reported in May at the American Society for Microbiology meeting in Washington, D.C.

Last year, for example, his team determined that the GAS strain M18, which causes acute rheumatic fever, contains phage genes that encode toxins, but that another strain, which causes strep throat, doesn’t have those genes (SN: 3/30/02,

p. 197: Available to subscribers at Deciphering Virulence: Heart-harming bacteria flaunt unique viral genes). The investigators also reported in the July 23, 2002 Proceedings of the National Academy of Sciences that M3, an unusually deadly strain of GAS that produces toxic shock syndrome, has yet a different set of phage genes. And in the Feb. 18, 2003, issue of that journal, Musser and his colleagues revealed that when certain immune cells begin to engulf GAS bacteria, the microbes activate several phage-derived genes. The function of these genes remains unknown, but they appear to be part of the bacterium’s coordinated response to avoid destruction.

Scientists have also recently discovered that bacteriophages may do more than just hand over toxin genes to a bacterium — sometimes, they control the release of those toxins. Take the case of Escherichia coli, a normally harmless gut bacterium. Strains of E. coli that produce a molecule known as Shiga toxin can cause a deadly form of food poisoning. The Shiga-toxin gene turns out to be a part of a phage genome that has integrated itself into the DNA of some E. coli strains. The toxin gene becomes active only when phage begin to reproduce inside an E. coli, says Matthew K. Waldor of Tufts University School of Medicine in Boston.

Moreover, intact bacteria carrying the gene don’t secrete Shiga toxin into people. It’s the rupture of the bacterial cells by phages that releases the toxin, Waldor and his colleagues report in the May 2002 Molecular Microbiology. If there’s a mutation in the gene that the bacteriophages use to disrupt bacterial membranes, the toxin merely builds up inside the E. coli.

“Phages not only disseminate virulence genes but also regulate the production of the virulence factors,” says Waldor.

This discovery has brought a disconcerting fact about certain antibiotics to light. Fluoroquinolones, the class of antibiotics that includes the anthrax-fighting drug Cipro, actually trigger the activity of phage genes — and thus can increase production of Shiga toxin, notes Waldor.

In the June Infection and Immunity, John F. Prescott of the University of Guelph in Ontario and his colleagues say that fluoroquinolones also induce the activity of a phage genome that typically lies dormant within Streptococcus canis, a bacterium that normally harmlessly infects dogs and some other animals.

The recent use of these antibiotics in dogs may therefore explain why veterinarians have recently reported some severe cases of toxic shock syndrome and flesh-eating infections in dogs infected with S. canis.

Prescott wonders, “Is our use of certain antibiotics helping to spread phages, which may also encourage the spread of virulence genes?”

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