First Completed Microbial Genomes Signal Birth Of New Area Of Study

Study Author: Karen Young Kreeger Researchers have been comparing parts of genomes at the individual gene level for years. Portions of the genomes of tens of thousands of species-from bacteria to humans-reside in private and public databases around the world. Pharmaceutical scientists sift through these collections, panning for potential drugs and therapies, while basic investigators in an array of fields search for similarities and differences in the species' blueprints for life. But what researchers haven't had available to examine until a short while ago is the complete genetic road map of a free-living species. IN ITS ENTIRETY: Mycoplasma genitalium, denoted by arrow in left figure, was fully sequenced by three teams, one led by UNC's Clyde Hutchison. These much-prized data became available with the recent publication of the first two full genomes derived from independent organisms. In July, a collaboration of two teams-one headed by J. Craig Venter, president of The Institute for Genomic Research (TIGR) in Rockville, Md., and another led by Hamilton O. Smith, a professor of molecular biology and genetics at the Johns Hopkins University School of Medicine-reported the sequence of a harmless strain of the bacterium Haemophilus influenzae, whose related strains cause ear infections and meningitis (R.D. Fleischmann et al., "Whole-genome random sequencing and assembly of Haemophilus influenzae Rd," Science, 269:496-512, 1995). Three months later, the TIGR and Hopkins teams, along with a group from the University of North Carolina led by microbiologist Clyde Hutchison, published a paper describing the full genomic sequence of a second microbe-Mycoplasma genitalium, a bacterium that is closely associated with human epithelial cells and is responsible for reproductive-tract infections (C.M. Fraser et al., "The minimal gene complement of Mycoplasma genitalium," Science, 270:397-403, 1995). AWAKENED: "It dawned upon me -- this is going to be a whole new field of biology", recalls DOE's David Smith. The researchers chose M. genitalium because it is the smallest known genome of any free-living organism, thus enabling them to sequence a second microbe to compare with H. influenzae more quickly. Having the full genome of these two microbes is a mighty accomplishment in itself, say genome scientists, but where the real excitement for investigators lies is in the comparison of the genomes. In fact, Venter and colleagues have coined a new term for this area of study: comparative genomics."I started reading the portion of the Mycoplasma paper on the comparison of the genomes and all of a sudden it dawned on me-Wow, this is going to be a whole new field of biology with enormous implications," recalls David Smith, director of the health effects and life sciences research division at the Department of Energy (DOE). With the genomes of several more microbes slated to be completed in the next year through the DOE Microbial Genome Initiative (K.Y. Kreeger, The Scientist, Feb. 6, 1995, page 1), re-searchers anticipate an abundance of new information to inform such fields as evolutionary biology and to utilize in commercial and biomedical settings. "It's extremely valuable to have these bacterial sequences available to researchers," says Hopkins' Smith. He remarks that to be able to look at the full lay of the genomic land will "accelerate their work enormously." Elke Jordan, deputy director of the National Institutes of Health's National Center for Human Genome Research, agrees that having whole genomes will be a great leap forward for many disciplines, including human genomics. "I think we're going to take our baby steps on the microbial genomes and then-when the bigger, more complex ge-nomes like yeast, Caenorhabditis elegans, and Drosophila become available-we'll transfer some of that experience to them." WALK, THEN RUN: The sequencing of microbila genomes will pave the way for other species, according to NIH's Elke Jordan. Hopkins' Smith, who was a co-recipient of the 1978 Nobel Prize in physiology or medicine for his work with bacterial restriction enzymes, maintains that this enthusiastic acknowledgment by the scientific community vindicates the sequencing approaches used by Venter and TIGR. "Craig built this whole thing over the doubts of virtually everybody in the country," he remarks. "It seemed like everyone was expecting him to fail and fall on his face. But he's delivered more than anyone ever thought, and quicker." Indeed, it is ironic that this field might not have taken off as soon as it did if Venter had stayed at NIH, where he was a researcher at the National Institute of Neurological Disorders and Stroke from 1984 to 1992. He left after a disagreement about the best way to approach research at the Human Genome Project (Opinion, The Scientist, July 24, 1995, page 1). Venter later approached NIH for funds to support the H. influenzae project, but was turned down because reviewers felt his methods wouldn't pan out. Equipped with the first two full genomes, the teams of genomic scientists were able to make some direct comparisons of how the two microbes go about living. They contrasted their genetic content for such critical and basic biological functions as amino acid synthesis, energy metabolism, and DNA replication. "There were two surprises-what was missing and what was there," remarks Claire Fraser, director of the molecular and cellular biology department at TIGR and first author on the M. genitalium paper. "We didn't expect Mycoplasma to be able to synthesize much, other than DNA, RNA, and some proteins." Still, M. genitalium, with just 470 genes (as compared with H. influenzae's 1,743), is able to sustain life. Fraser points out that previous sequencing work by Hutchison and his UNC colleagues Ping-Chuan Hu and Kenneth Bott "hinted that there may be a lack of biosynthetic enzymes in Mycoplasma." "There's only one enzyme that's involved in amino acid synthesis in the Mycoplasma genome, whereas Haemophilus has 68," explains Hutchison. "Mycoplasma has evolved in such a way that it gets much of its nutrition directly from its host." One striking and unexpected finding, say the papers' authors and other researchers, is that about 5 percent of M. genitalium's genome is devoted to evading its host's immune system. "We found nine partial sequences for adhesin, the protein for attaching to the surface of epithelial cells, scattered about the genome," recounts Smith. "These versions of adhesin can recombine to generate slightly different antigens to escape the immune system of Mycoplasma's host. That capability was already known, but with the full sequence we now have a more complete picture." As Fraser puts it, M. genitalium uses the suite of adhesin genes "to stay one step ahead of the host immune response." For a minimal organism, "this is a sophisticated way to survive," she remarks. "One other surprising thing is that over 30 percent of the genes in Mycoplasma are ones for which we can't currently assign any biological role," says Fraser. In H. influenzae, more than 40 percent of the genes have no known matches with genes that have been sequenced in other organisms. "What are some of these unknown proteins doing?" she asks. "That's an important commentary on the state of biological knowledge," asserts Venter. "Here we have the absolute minimal genome and we can't even identify a third of the genes in it because we haven't seen them before. It's pretty humbling." More information on the complete genomes of the two microbes is available on TIGR's home page at: http://www.tigr.org. With the first comparison of two entire genomes under their collective belts, investigators are readying to pursue studies beyond the differences and similarities in cellular machinery and proteins. With several more organisms soon to be fully sequenced, evolutionary biologists are anxious to compare genomes from different taxonomic lineages to study the origins of life. MORE QUESTIONS THAN ANSWERS: "How does the organization of an entire genome change over evolutionary time?" asks Illinois' Carl Woese. They are especially eager to get a look at the genomes of microbes from the phylogenetic group called the Archaea (M.E. Watanabe, The Scientist, May 30, 1994, page 14), three of which are due to be fully sequenced in a matter of months. The first of them, Methanococcus jannaschii, which is being sequenced in a collaboration between TIGR and University of Illinois microbiologists Carl Woese and Gary Olsen, is expected to be done by the end of the year. The Archaea are particularly interesting because they are one of the most ancient life forms; they thrive in extreme environments; and some species are autotrophic-capable of reproducing without organic substances. "This will be the first time, as soon as an autotroph is done, that we will have the minimal biochemical pathways needed to make a living system from inorganic matter," says Woese. "That's an absolutely major step forward." Woese, who calls the Archaea "living fossils" of humans' prokaryotic ancestors, also says that by comparing archaeal genomes with those of more complex species, researchers will be able to get a better handle on the origin and nature of the eukaryotic cell. Plants and animals, including humans, are composed of eukaryotic cells-those that contain membrane-bound nuclei and complex internal organelles. Prokaryotes-less complicated species such as bacteria-lack membranous nuclei and organelles. Venter relates that the eukaryotic-prokaryotic comparison is already "looking really interesting." He reports that about two-thirds of M. jannaschii's genes are new, compared with the one-third that are unknown in H. influenzae and M. genitalium. And of the remaining third that can be identified in M. jannaschii, he says, half of the genes appear to be prokaryote-like and half eukaryote-like. The first comparison at the whole-genome level between species has already started to spark more questions than answers. "The science of comparative genome structure will now come into being," declares Woese. "What will be very interesting to study will be the evolutionary flow of the genome. How does the organization of an entire genome change over evolutionary time? "Some people attempt to ask these questions now, but the material that's available now is nothing like what will be," he says. Woese also predicts that with complete genomes, evolutionary biologists will be able to study gene flow among species: "How much DNA in a given organism is foreign, coming from such agents as transposable elements or viruses?" With all of these questions swirling in the minds of scientists, Venter believes that new evolutionary theories will be needed: "For the first time we'll be able to look at the evolution of genomes and species, not individual proteins. But how do you do it? Take the evolution of each protein and average those all together? We're the first to even have these datasets. It's truly a new field." The ability to compare entire genomes is starting to conjure up as many applied investigations as basic studies. "My special interest in complete microbial genomes would be in the sequences of the major disease-producing organisms," says Hopkins' Smith, referring to the microbes that cause such diseases as tuberculosis, cholera, syphilis, and gonorrhea. "Their genomes are small-all in the range of Haemophilus, except TB; that's about twice the size." Disease-gene hunters would target in on sets of genes unique to those organisms, which determine the disease-producing properties. By comparing virulent with avirulent strains, for example, these genes "should stick out like a sore thumb," notes Smith. That information could then be used for vaccines, particularly for surface proteins that are unique to the virulent organisms. From the perspective of John Mekalanos, a professor of microbiology and molecular genetics at Harvard University, identifying homologs of disease-causing genes that have been identified in other organisms by classical methods "would give us insight into potential modes of virulence that people wouldn't have realized." Mekalanos, an expert in bacterial virulence, says these genes may control how microbes obtain nutrition from their host environment, evade the host immune system-such as in the suite of adhesin genes in M. genitalium-or produce toxins. He adds that it's too soon tell the full extent of the biomedical payoff in comparing full genomes. "Until there are a dozen or so bacterial-pathogen genomes sequenced, we won't really know how much benefit will come from it." PAYOFFS: Proteins from microbes soon to be fully sequenced could have many industrial uses, predicts DOE's Jay Grimes. Proteins of the Archaea that are about to be completed also have potential industrial, energy-producing, and environmental applications. Jay Grimes, project manager for the DOE Microbial Genome Initiative, is particularly interested to compare the genomes of the heat-loving Archaea, or thermophiles, which thrive at temperatures up to 100oC. He says this group of microbes may contain as-yet-unknown heat-stable enzymes that could be used as chemical catalysts in heavy industry. Other potential enzymatic applications from new proteins gleaned from the archaeal genomes include toxic-waste degradation and methane production for energy. Developing components from archaeal genes for PCR kits and DNA sequencing in the research market is another potential application. Using information in public databases, Recombinant BioCatalysis Inc., a private biotech firm in Sharon Hill, Pa., is doing just that. "We've discovered 12 new high-temperature DNA polymerases from Archaea," reports CEO Barry Marrs. The company uses a variety of techniques to screen for marketable candidates, but in this case, according to Marrs, they compared the sequences of several archaeal genes. He agrees that much useful information will be revealed once M. jannaschii and other Archaea are fully sequenced. RESPECT FOR THE UNKNOWN: "We have the absolute minimum genome and we can't identify a third of the genes. It's pretty humbling," says TIGR's Craig Venter, seen here viewing the Haemophilus genome. DOE's Smith contends that the myriad practical applications that may arise with this new field is "where the rubber really meets the road."

First Completed Microbial Genomes Signal Birth Of New Area Of Study

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