Huge Microbe's Value Lies In More Than Just Sheer Size

"Word's biggest bacterium found in a fish," declared the front- page headline in a March 1993 edition of USA Today, while the New York Times trumpeted its coverage with: "In the world of bacteria, a behemoth." Even the relatively staid science journals couldn't resist. Science announced the identification of "Monsters from the guts," and Nature's header read "Giant among the prokaryotes." The extraordinary flurry of interest--and imagery--was over an article in Nature (E.R. Angert, et al., 362:239-41, 1993) reporting the identification by microbiologist Norman R. Pace's laboratory at Indiana University in Bloomington of the largest bacterium yet known to science: Epulopiscium fisheloni. The organism, a member of the Clostridium group of anaerobes, had been, according to the report, isolated from the intestine of the brown surgeonfish, Acanthurus nigrofuscus, and it is, indeed, big--up to 600mm long by 80mm in diameter. That translates, the Times reported, to "about the size of a hyphen in a newspaper article." While grateful for the publicity that identifying the bacterium has gained for him and his colleagues, Pace believes that the media hype surrounding the organism's sheer bulk obscured the true value of his lab's achievement. In fact, Pace's interest in this specific organism is not nearly so great as is his interest in the use of techniques his laboratory staff has developed to screen microbes found in the environment. According to Pace, their work can be broadly described as "characterizing naturally occurring microbial organisms without cultivating them." Thus, the media attention given to his lab's latest find was most valuable in, if anything, drawing attention to the techniques' usefulness in opening the door to the world of as-yet-undiscovered microbes. "We know so little about the natural microbial world," says Pace. "Imagine if our entire view of biology was based on a visit to the zoo. That's exactly the situation we're in in the context of the natural microbial world." Need For Cultivation Until now, technology to study the microbial world has been limited. Under normal circumstances, an individual bacterium will not yield any information about itself by being taken from its natural environment, stained, and viewed under a microscope. Since it can be effectively studied only in numbers, it must be grown in media to produce colonies. Carl Woese, a professor of microbiology at the University of Illinois, Urbana-Champaign, and coauthor of several papers with Pace, explains that the problem with understanding the microbial environment is that until now, available methods limited researchers' ability to find, let alone identify, microorganisms. PAYING PROPER ATTENTION TO THE MICROBIAL WORLD Indiana University microbiologist Norman Pace has been profoundly interested in RNA since his work as a graduate student in the laboratory of Sol Spiegelman at the University of Illinois. Indeed, all of his 140 scientific publications deal, in some manner, with RNA. As time and technology have progressed, Pace has concentrated more and more on RNA as a key to molecular evolution. His laboratory currently has two foci: studying RNase P, an enzyme that, in part, is composed of a catalytic RNA; and developing methods for screening and analyzing environmental microbes. It is this latter focus that leads Pace to be a vocal advocate for the study of microbial biodiversity. "The microbial world established the biosphere; the microbial world still supports the biosphere," he says. Researchers need "to find out what's out there and what are they really doing," he says. He and University of Illinois microbiologist Carl Woese-- considered by Pace to be the father of rRNA phylogeny (L. Holland, The Scientist, May 14, 1990, page 22)--point out that people making a big ruckus today about the need for preserving biodiversity are speaking about only a very few species of plants and vertebrate animals, largely ignoring the microbial world, which is much more vast and is necessary for all life. For example, without microorganisms there would not be degradation of organic matter--no biological recycling. They further point out that nowhere on the surface of the Earth is microbe-free, including the oceans and even within rocks. "They grow throughout the first few miles of the surface of the planet," says Woese. Woese adds that by studying the origins of microbes, microbiologists also are studying human origins. "The eukaryotic cell that became a plant cell did not invent photosynthesis," says Woese. He explains that the origins of both chloroplasts and mitochondria will be found in organisms similar to the microbes that were captured by the earliest cells. For their sheer longevity, microorganisms as a group deserve more scientific attention. "How old are microorganisms?" asks Woese rhetorically. "Three point five billion [years] is a conservative estimate," he answers, with 3.8 billion years as a more realistic estimate. Scientists cannot search back farther than that because that is the age of the earliest rocks. The Earth is 4.5 billion years old. --M.E.W. Newer biochemical techniques, such as using probes with known complementary DNA or RNA codes from identified organisms, along with DNA sequencing, could revolutionize microbiology, many researchers agree. "A large fraction of the bacteria [have] been studied by enrichment culturing," Woese points out. "We artificially enrich and then select those [bacteria] which grow best on the media of our design"--that is, bacteria grown on artificial nutrient media such as agar enriched with amino acids, salts, and other molecules. The media becomes the bacteria's environment, and those bacterial strains that grow best into colonies on the media are the ones with which scientists currently are best acquainted. However, enrichment culture, although the basis of much of our knowledge of bacteriology and microbial pathology, is of very limited use in exploring the entirety of the world of living microbes. Enrichment culturing is highly selective; according to Pace, only between 1 in 1,000 and 1 in 100,000 of bacteria present in a given environment will grow on enriched culture media. Thus, while a single bacterium in a particular soil sample may be identified through enrichment culturing, the sample may well contain tens of thousands of other organisms that can't be grown in the available media and will thus remain unknown. It follows, Woese points out, that "the natural distribution of microorganisms tends to be very different from the collection we have in the laboratory." Pace's technique, however, can be used on organisms that, as yet, have not been amenable to enrichment culturing: It involves the amplification of DNA that has been extracted from a bacterial cell. The resulting clones are then sequenced and compared with known sequences for ribosomal RNA (rRNA). This allows determination of what is called a "phylotype," or the position where this organism fits in the known bacterial phylogeny. Additionally, hybridization probes complementary to specific regions of the rRNA are synthesized and used both to confirm that the DNA in question came from the specific organisms being studied and to aid in screening other organisms. In the research on Epulopiscium, DNA amplification was done in Pace's lab via polymerase chain reaction (PCR), reproducing ribosomal DNA (rDNA) that codes for small subunit rRNA. In this case, the fact that Epulopiscium hybridized with a bacteria- specific hybridization probe and not one for eukaryotes established that Epulopiscium is a bacterium. The organism tentatively had been identified as a protozoan in earlier work (L. Fishelson, et al., Science, 229:49-51, 1985). SUGGESTED READING H.W. Jannasch, et al., Nature, 342:834-6, 1989. W.L. Montgomery, P.E. Pollak, Journal of Protozoology, 35:565-9, 1988. G.J. Olson, C. Woese, FASEB Journal, 7:113, 1992. D.M. Ward, et al., Advances in Microbial Ecology, Vol. 12:219- 286 (ed. K. C. Marshall, New York, Plenum, 1992). C. Woese, Microbiological Reviews, 51:221, 1987. W. Linn Montgomery, an ichthyologist at Northern Arizona University in Flagstaff, was one of the original discoverers of the organism, and his doubts that it was a protozoan led him to persuade Pace to examine it. Pace, viewing this as a good opportunity to establish the usefulness of his technique, showed the microbe to graduate student Esther R. Angert, who was just beginning her studies with Pace at Indiana. Pace asked her: "Look here, do you want to study this?" Samples of preserved surgeonfish gut contents were supplied to the laboratory by Kendall D. Clements of James Cook University, Townsville, Queensland, Australia. The organisms are so big that Angert was able to pick them out of the intestinal samples under a microscope, impossible to do with smaller bacteria. Microbiologists, including Pace, willingly acknowledge the tech- nique's limitations. For example, Bruce Hanna, an associate professor of clinical pathology at the New York University School of Medicine, who is using probes to identify the bacillus that causes tuberculosis, says, "It's not going to replace everything. First of all, it's very laborious and very expensive." Angert's research illustrates the complexities. She began work in 1990 and finally came up with "the right ribosomal RNA" in the fall of 1991. She then had difficulties, she says, "getting a clean enough Taq polymerase." Fortunately, a colleague was able to isolate and sufficiently purify the enzyme. Pace explains that even though the work is expensive, research money is available for studying high-temperature organisms, as they are thought to hold the greatest commercial opportunities as sources of thermostable enzymes. Now that the Epulopiscium study is completed, Pace's group is concentrating on microorganisms from high-temperature environments at Yellowstone National Park. They already have identified organisms that, Pace says, "are more different from known organisms than known organisms are from one another." And what will become of the giant symbiotic Epulopiscium? Angert believes that microbiologists will be able to use this bacterium, which is large enough to allow insertion of microprobes, to directly study bacterial physiology, a process that cannot be carried out with most known bacterial species. The organism itself leaves many scientific questions unanswered. First of all, large bacteria similar to Epulopiscium have been found within intestinal tracts of herbivorous animals. For example, Angert explains, "there are similar organisms in guinea pigs. How did they get there?" Angert's study also found similar bacteria in the intestine of Naso tuberosus, the humpnose unicornfish, another herbivorous surgeonfish relative. These symbionts clearly established themselves within the animals' digestive tracts many generations ago. Researchers wonder: What is their precise physiological function in the gut--and why did they grow so large? Montgomery believes that the bacteria "may enhance lipolytic activity in the main intestine. We know they greatly suppress pH locally in the mid-intestine." This, however, is only a part of a long-term coevolu-tionary relationship. Gareth Nelson, curator of ichthyology at the American Museum of Natural History in New York, describes Acanthurus nigrofuscus, the big microbe's surgeonfish host, as an Indo-Pacific cosmopolitan fish species living throughout the Pacific and Indian Oceans. Both he and Montgomery note that the true taxonomic status of the species is unknown and it, because of its broad distribution, is unlikely to be one valid species and is more likely to be at least several separate species. Montgomery, who has been studying the surgeonfish for many years, says that Epulopiscium or similar microbes have been found in the intestinal tracts of this fish from many parts of its range-- South Africa, Hawaii, Tahiti, Australia, and the Red Sea. Angert's research showed that different types of the microorganism inhabit the intestines of Australian fish and the same purported species of fish found in the Red Sea. Questions that arise include: Did the bacteria evolve along with the fish, and can you study the phylogeny of the fish by studying the molecular evolution of the bacteria? "I think, in terms of coevolutionary stories, this has some great potential," Montgomery says. And the bacterium itself, according to microbiologist James Staley of the University of Washington in Seattle, leads to some interesting speculation: "What is the upper size limit for bacteria? Is there some sort of reason why the cell can't get any larger than this? " Some advocates of Pace's approach suspect that it will be criticized by the plate-and-cultivate researchers, who are used to working with organisms they can colonize in culture, rather than with one or two organisms, as Pace is doing. But NYU's Hanna, who runs the clinical microbiology laboratory at New York's Bellevue Hospital Center, is clearly impressed. "I think what they have done is very unique and interesting," he says. "They don't know what they're looking for .... Sometimes, it's the only thing you can do." Myrna E. Watanabe is a biotechnology consultant based in Yonkers, N.Y.

Huge Microbe's Value Lies In More Than Just Sheer Size

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