Phages - if you want to read more

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ALL THE WORLD'S A PHAGE

Viruses that eat bacteria abound-and surprise BY JOHN TRAVIS Science

News, 7/12/03, v164, p26

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.

" 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 thef ormer 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 box, page

27). 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 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, Curds 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

[lO to the 31] 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[10 to the 30] bacteria. If there are 10 phages for every

bacterium, a reasonable assumption according to Hatfull, then [10 to

the 31] 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 curren tDNA-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

R.

s, a Medical Institute investigator at Albert

Einstein College of Medicine in New York, and s' sister, Debbie

s-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, 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

AStreptococcus (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. 198). 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 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, F. Prescott of the

university of Guelph in Ontario and his colleagues say that fluoro-

quinolones also induce the activity of a phage genome that typically

lies dormant within Streptococcus canus, 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. canus.

Prescott wonders, " is our use of certain antibiotics helping to

spread phages, which may also encourage the spread of virulence

genes? " -J.T.