Harnessing the Microbial World: Big Info in Small Packages

Using genomcs to develop new antibiotics and vaccines could help prepare for potential bioterrorist actions Although the Human Genome Project now draws more attention, the genomes of smaller organisms collectively contain more information. The American Association for the Advancement of Science's second annual Genome Seminar, held at this year's annual meeting in January in Anaheim, Calif., focused on analyzing that information for a myriad of purposes ranging from developing malaria vaccine strategies to discerning the origins of life. Genomic data will continue rapidly accumulating as sequencing techniques and computing power keep advancing, noted J. Craig Venter, the two-day seminar's cochairman and chief scientific officer of Celera Genomics Corp., of Rockville, Md. Venter noted that since he and a team of scientists at The Institute for Genomic Research (TIGR), in Rockville, Md., finished the first genome, Haemophilus influenzae, in 1995,1 about 20 genomes have been completed, 80 more microbial genomes are under way, and about 50 of those will be completed in 1999. "We're in an exponential, explosive period of knowledge," Venter said in a news conference preceding his talk. But how scientists interpret, manage, and use that knowledge may be more important than the base pairs by themselves, speakers noted throughout the two-day seminar. The following are highlights from those presentations. Tuberculosis (TB) may be coming back, but new sequencing information may help keep it from reemerging completely, noted Thomas Shinnick, director of the U.S. Centers for Disease Control and Prevention. "Genomics has opened up a new array of approaches to conquering TB." Although the incidence of TB decreased from 1953 to 1985 in the United States, there was a 20 percent resurgence between 1988 and 1992. Tuberculosis still accounts for 3 million deaths per year and 26 percent of all preventable adult deaths in the world. The disease has been notoriously hard to genetically analyze and has been characterized by a dearth of selectable genetic markers. But that may change, since the complete genome sequence of M. tuberculosis strain H37Rv has been published.2 And a second sequence--TIGR's version of strain CDC155--is nearing completion. Initial comparisons of the two have already yielded about 100 differences, including the unexpected finding that two large families of glycine-rich proteins with a repetitive structure may provide the source of antigenic variation. Genomic research has the potential to identify new drugs and to understand vaccine efficacy and virulence mechanisms, Shinnick concluded. CUTTING AWAY: Blackened areas indicate the portions of the HIV genome that have been removed in the most recently developed HIV lentivirus vectors. Knowledge about the HIV genome has allowed researchers to take the virus apart and potentially turn it upon itself. Inder Verma, American Cancer Society professor of molecular biology at the Salk Institute for Biological Studies, and his colleagues first reported the retrovirus as a vector system in 1996.3 HIV, a lentivirus, can target both dividing and nondividing cells, such as neurons and macrophages.4 Before the development of the so-called "lentivirus" vector, scientists could only infect nondividing cells ex vivo, meaning diseases infecting the liver and brain couldn't be reached by gene therapy. Verma's method changes that. He and colleagues deleted all the genes that allow the HIV virus to replicate, leaving only the genetic sequences that control integration into the target cell's genome. So far, genes delivered with the lentiviral system have integrated into nondividing cells without adverse immunological reactions in mouse and rat models.5 "Viruses are the most efficient way to get into genes, because we can make them in large amounts," Verma said. "They can be used in a novel form of gene therapy by expressing the gene into the cell to ameliorate, prevent, or slow down the progression of various kinds of disease." To better probe distant planets for signs of life, scientists must develop "biosignatures," general features of life that are non-Earth-specific, reported Kenneth H. Nealson, faculty associate at the California Institute of Technology in Pasadena, and a researcher with the Jet Propulsion Lab. Those criteria may be very broad, due to more understanding of life forms that can survive in both extreme heat and cold. "Our definition of tenacity--the ability of organisms to hold on--has changed drastically." He pointed to TIGR's nearly complete sequence of Shewanella putrefaciens as an example, noting that the microorganism thrives on manganese rather than oxygen. "It's a remarkable organism," Nealson noted. "It will grow on almost anything you give it." That knowledge broadens, rather than narrows the search, though, he admitted. However, that might not be such a bad thing, since some seemingly obvious targets, like Europa, the ice-covered Jovian moon, may not be as viable as it appears: "The assumption is, there's ice, there's water--there must be life," Nealson commented, but added that, without proper CO2 exchange, the ice and water could be a red herring. But other potential biosignatures, hinted at by extreme organisms, like S. putrefaciens, could provide more telling clues. Genomics could provide a new way to develop DNA vaccines for malaria, which, if successful, could be applied toward other diseases. "DNA vaccines would offer this flexibility because of the ease of production, stability, and versatility of use," reported Stephen L. Hoffman of the Naval Medical Research Institute. DNA vaccines are fundamentally different from traditional ones, Hoffman notes. "The difference in this approach is that we would be receiving DNA that encodes a substance and asking our bodies to make a protein in response to it. However, this approach, while potentially flexible, is also more complex. It requires assessing potential antigen proteins encoded by the malaria genome, using that "vaccinome" to induce antibodies, judging the accuracy of expression, immunizing mice with each plasmid, and ultimately, developing a multigene DNA vaccine. Also, the malaria genome can hardly be described as simple. It contains 14 chromosomes, 30 megabases, and 6,000 genes (of which only 1,000 have been identified), and contains widely varying alleles. Still, the international $28 million Malaria Genome Sequencing Project has so far yielded 209 new genes and an attempt to create two kinds of vaccines against malaria--one for travelers and one for children in sub-Saharan Africa. Inder Verma "While the risk of bioterrorism is relatively low, the consequences are so severe that no prudent nation can afford not to prepare for such an event," warned Frank E. Young, former commissioner of the Food and Drug Administration and executive director of the Reformed Theological Seminary. Preparation involves three interrelated strategies: using genomic information to develop new antibiotics and vaccines, enhancing civil defense until new measures of counteracting biological agents become available, and negotiating an international treaty to reduce the use of pathogens. Genomic science can play a key role in the first strategy. "As we look at the outbreak of infection, we first have to ask ourselves, 'Are they man-made or are they natural?'"Having sequence information of pathogens such as botulism and hantavirus could help answer that question quickly. That information could quickly help health workers decide what, if any, existing vaccines could be deployed, and provide clues about how to make new ones. Young emphasized the need to build up a stockpile of vaccines and antibacterials. Artist's drawing of models of the subsurface structure of Europa: Is there a "biosignature?" As part of an ad hoc committee, Young presented his recommendations to President Bill Clinton in 1998. The president directed that $300 million be spent to support programs to meet the threat of weapons of mass destruction. Congress approved 75 percent of the funds recommended by the ad hoc panel, and the National Institutes of Health received $24 million for genome sequence information on organisms--including anthrax, tularemia, and plague--that are potentially useful in bioterrorism. Such steps will help researchers to understand the pathogenicity of infectious disease.6 "It behooves scientists to understand the various world views that motivate mankind, develop sound ethical principles for the conduct of research, and ensure the safety of both lab workers and the public from the threat of accidental release and inappropriate shipment of organisms. As scientists, we can no longer focus narrowly on our own research divorced from the conflicts within society," Young concluded. Applying genetics to taxonomy can provide a better view of biodiversity, noted Norman R. Pace, University of California at Berkeley professor of microbial biology. Perhaps the most dramatic example was when Carl R. Woese, professor of microbiology at the University of Illinois at Urbana-Champaign, cited genetic evidence in 1978 that archaea represented the third major branch of life, next to bacteria and eukaryotes. "Our textbooks haven't yet recovered," Pace said, adding he'd like to see more taxonomy conducted on a genetic basis. He suggested using ribosomal RNA (rRNA) gene cloning and sequencing; single-cell hybridization probes; and polymerase chain reaction (PCR) techniques to do comparative sequence interpretation. Such analysis--which is not yet widely used, Pace noted in a subsequent interview--would provide a clearer idea about both the nature and the breadth of biodiversity: "For all the talk of biodiversity that's been around, I've never heard anyone define it, except in nebulous terms." Pace said that, if the technique would be more widely used, people might be surprised at the amount of variation in life. For example, analyzing bacterial samples recently collected from an obsidian pool in Yellowstone National Park "expanded dramatically our view of microbial diversity." Examining the gene function of the genetically simplest known organism could answer a question with profound biological and philosophical implications. "Can we come up with the molecular definition of life?" Venter asked rhetorically. Venter and TIGR colleagues began their search by first comparing Mycoplasma genitalium's 476 genes with the next-simplest genome, Mycoplasma pneumoniae, which has about 600. They found about 300 genes in common. The similarities in both simple organisms "imply that these genes are the essential genes for life." However, questions remained. "One hundred of those 300 genes, we as scientists have no idea what they do," Venter said. The TIGR scientists have begun systematically inserting transposons into the M. genitalium genome. That technique shuts off single genes temporarily. Disrupting genes whose functions are not key allows the organism to continue growth and division. But even that technique has limits, noted Venter, since even in the simplest organisms, several genes work together to create products. "The problem is, we don't know if all those genes can be knocked out simultaneously." Scientists could also use existing information to build a "minimal" organism from the ground up. But besides presenting technical difficulty, that approach provokes another question. "Is it ethical to synthesize life in the laboratory?" Venter asked. R.D. Fleischmann et al., "Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.," Science, 269:496-512, 1995. S.T. Cole et al., "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence," Nature, 393:537-44, June 1998. L. Naldini et al., "In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector," Science, 272:263-7, 1996. T. Kafri et al., "A packaging cell line for lentivirus vectors," Journal of Virology, 73:576-84, Jan. 1999. P. Smaglik, "Gene therapy--the next generation," The Scientist, 12[10]:1, May 11, 1998. R. Lewis, "Bioweapons research proliferates," The Scientist, 12[9]:1, April 27, 1998.

Harnessing the Microbial World: Big Info in Small Packages

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