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by Wallace Ravven, Senior Science Writer, San Francisco State University The ability to splice genes and deduce their functions has revolutionized microbial biology. Researchers now have fundamental access to the life processes, ecology and evolution of organisms in ways they never did before. These same tools of molecular genetics also spawned the biotechnology industry, and potent biotech products are already at work in medicine, industry, agriculture and environmental cleanup. Research in the College of Natural Resources' Microbial Biology Division lies at the heart of many of these efforts, both through basic discoveries and their practical uses. Faculty interests range from studies of microbial diversity, ecology, physiology and genetics to plant and insect virology, bioremediation, fungal ecology and evolution. Eight faculty hold primary appointments in the division, three of them members of the National Academy of Sciences. A ninth colleague joins the division next academic year, and five more will be recruited over the next five years. These are productive days for the division, as for the field, of microbial biology. This article profiles research under way in the division-its intellectual roots as well as its applications. They can survive inside rocks, at toxic deep-sea vents and even under crushing pressure. They are known as extremophiles-mic-robes adapted to Earth's extreme environments-and many of them are just now coming under scrutiny. Extremophiles demonstrate both the phenomenal tenacity of life and our scant understanding of microbial diversity. By most estimates there are millions, if not billions, of species of single-celled bacteria and related microbes. A gram of soil is thought to contain 10,000 to 100,000 different types, yet few have been cultured in a laboratory and most never will be. So sketchy is our view of microbial life that scientists only recently realized the so-called bacteria actually comprise two fundamentally different types of organisms: the Bacteria and the Archaea (are KEE ya). These distinct divisions represent two of the three major evolutionary branches of all life on Earth. (In this schema, we belong to the third branch, Eukarya, along with the beloved slime molds, sea cucumbers, salamanders and sequoias.) Discovery of the Archaea emerged from a powerful insight: that the most refined record of any organism's evolutionary history lies not in its anatomy or protein structure, but in the precise makeup of its genes. Today, "bioprospectors" can search for new species of microbes by reading this evolutionary history. They retrieve DNA from organisms living in any environment and look for novel se-quences in a certain region of the DNA. If they find an unfamiliar sequence, they may have a new species. By comparing this new genetic fingerprint to those of known microbes, they can trace a specimen to its rightful branch on the evolutionary tree. Identifying New Species Norman Pace, a professor in the division, and his colleagues have used this strategy to identify hundreds of new species of Archaea and Bacteria in a single scalding mineral pool in Yellowstone National Park. "We're trying to understand who we're sharing the Earth with," he says. The research promises to reveal organisms important to agriculture, medicine and environmental restoration. Biotechnology already employs bacteria to churn out valuable products, from industrial solvents to insulin. Since many chemical production steps require high temperatures or pressure, microbes adapted to extreme environments are often particularly useful. The extraordinarily rapid gene-amplifying procedure known as PCR that has revolutionized cloning efforts relies on an enzyme that can function at 70 degrees Celsius (158 F). It, too, was isolated from a microbe discovered in thermal pools at Yellowstone. Microbial Biology In microbial biology, basic discovery and application often have been closely linked. Bob Buchanan, professor and chair of the Division of Microbial Biology, is fond of this statement by Louis Pasteur: "There does not exist a category of science to which we can give the name applied science. There are science and the application of science, bound together as the fruit to a tree which bears it." Battling Pollution with Bacteria Buchanan's lab team, along with colleagues in the Department of Molecular and Cell Biology and the Lawrence Berkeley National Laboratory, have used their understanding of bacterial metabolism and biochemistry to address serious pollution problems. They have determined what nutrient conditions allow naturally occurring water and soil bacteria to convert toxic selenium in agricultural wastewater into non-toxic forms and to remove toxic nitrates from wastewater. The process is already being employed on a pilot scale for cleaning up agricultural runoff in the San Joaquin Valley, and Exxon Corporation is interested in possible commercial-scale use. The microbes practice a kind of altruism, Buchanan says. Only about a fourth of the bacteria in any population detoxify selenium, and they die in the process. The rest of the population benefits from the detoxification. "How the bacteria segregate themselves into altruists and survivors is a total mystery," he says. His lab is now trying to tease apart, at the biochemical level, how the bacteria convert toxic compounds into non-toxics. Buchanan oversees the division at a time when the university is re-instituting a microbial biology program at both the undergraduate and graduate levels to serve increased needs for microbial biologists in many sectors of society. "Microbes are the workhorses of biotechnology," Buchanan explains. "Biotech firms need scientists with expertise in microbial physiology to keep the microbes alive. At the same time, the resurgence of resistant microbial infections worldwide has created a need to find new targets for antibiotics." Controlling crop diseases also relies on better knowledge about pathogenic bacteria. Finally, Buchanan says, bioremediation efforts depend on our ability to identify microbes that can thrive in the extreme environments of sludge or toxic waste. Postponing Frost with Microbes Microbial ecologist Steve Lindow has been scrutinizing the interplay between bacteria and plants since his college days. While still a graduate student, he discovered that a bacterium found in great numbers worldwide is what makes frost normally form on plant leaves. Take away the microbe-Pseudomonas syringae-and temperatures can drop five or six degrees below "freezing" before dust or other material triggers water to turn into ice. At Berkeley Lindow determined that a single gene in the microbe is responsible for this freezing trait. He then used recombinant DNA techniques to develop a strain of the bacteria in which the ice gene was disabled but the bacteria still thrived-a strain he called "ice-minus." He devised a plan to coat potato seed pieces with the modified microbe before planting, hoping that if the altered bacterium thrived on the sprouts, it would keep out the wild-type, frost-causing form ("ice-plus"). The potatoes would gain five degrees of frost protection. Lindow, now a professor in the division, became the first scientist approved by the federal government to run a small field experiment using genetically modified bacteria. Through his experiment, Lindow was able to show that the genetic approach protected the plants. The strategy since has been developed commercially. Lindow's lab also has found that a related common bacteria species, Pseudomonas florescens, can be genetically modified to protect plants from both frost damage and fire blight, the major disease of pears and apples worldwide. Blightban, a commercial spray containing the microbes, is now used on about 100,000 acres of apples and pears across the country. Defending Against Plant Disease Genetically A different gene-based approach to protect crops has been the focus of Brian Staskawicz' research for the past 15 years. A professor in the Division of Plant Biology with a secondary affiliation in Microbial Biology, he and his lab team are drawing from both fields to enhance plants' natural genetic defenses against pathogenic microbes. The Staskawicz lab was the first in the world to isolate a bacterial gene that triggers a defensive response in plants, and it has since played a key role in discoveries of disease-resistance genes, plant genes that counter the attacks. In the past few years, more than a dozen disease-resistance genes have been identified in plants. "We are genetically dissecting disease resistance in plants," Staskawicz says. "Once we understand how these genes function, we hope to directly and precisely introduce disease-resistance genes to crops to increase productivity. This has been a dream of plant molecular biology for many years." Professor Loy Volkman is studying viruses as a means of plant protection. Her focus is a virus that infects moth larvae-some of which are serious crop pests. Last year she and colleagues discovered that insects are capable of mounting an immune response to viruses, a fact not previously known. This suggests a new strategy against crop pests: identify a gene from the virus of another organism that can suppress the larval immune response; then link this gene to one harmful to the larvae and expose the voracious caterpillar to the genetically modified virus. The insect's immune system would be unable to fight off the virus, allowing the second introduced gene to do its deadly work. Says Volkman, "We're trying to understand the benchmarks of infection, so we can learn what kinds of additional genes the virus needs to attack the host more effectively." Saving a Bacterial Factory Through Manipulating Microbe Environments At first glance, Sydney Kustu's lab appears to pursue only basic science. For 20 years, Kustu has explored the regulation of nitrogen metabolism in bacteria, a process essential for the microbes to create nucleic acids and proteins. The bacteria store nitrogen as two intermediate products: glutamate and glutamine. They use glutamate as an osmotic regulator (to maintain a balance of salts inside and outside the cell) and both glutamate and glutamine as the source of nitrogen for all cell growth, including production of DNA, RNA and proteins. Among other problems, Kustu and her colleagues study how the cell regulates its growth rate in the face of changing supplies of nitrogen. How is the cell informed of the current size of its nitrogen bank balance? "This is very basic," says Kustu. "There aren't many people who think about how bacterial cells grow. But this is what biotech needs to keep microbes functioning at optimal levels." She recalls how she was asked to consult with one biotech firm when its bacterial factory was failing. "The bacteria had suddenly started converting glucose-their food source-directly into glutamate, instead of using the sugar to produce the desired product, in this case indigo dye,'' she says. "The firm's engineers were very smart. They had tried a lot of things, but they were not bacterial physiologists.'' Toward the end of the day, Kustu realized that if the microbes were producing lots of glutamate (their means of controlling osmosis), they probably were suffering from osmotic stress. She recommended adding an improved osmotic regulator to the microbes' environment, a strategy that could relieve both the bacteria and the firm's stockholders. Professor John Taylor had his ''15 minutes of fame'' when his lab revealed the sex life of Coccidioides immitis. (The story made The New York Times). Coccidioides is the microscopic fungus that causes Valley Fever, which infects up to 100,000 people a year and kills up to 100. Dogma in Taylor's field held that a full 25 percent of fungi reproduce entirely asexually. But Taylor was among the researchers who doubted so many species had remained viable without the benefits of genetic recombination that sexual reproduction provides. He and his lab colleagues are seasoned practitioners of the technique revolutionizing microbial studies: retrieving snippets of DNA from organisms, amplifying them and comparing the sequences between individuals, species and even kingdoms. Pursuing this approach with Coccidioides, the researchers found that the genetic material showed evidence of a high degree of recombination that must come from sex. "We can't see them breed, but we can deduce their activity from the DNA," Taylor says. "We also found great genetic variation in the genomes, maybe too much for members of the same species." Post-doc Vasso Kaufopanou compared the DNA sequences from different geographic regions and concluded that the Texas and Arizona Valley Fever fungi have been genetically isolated from their California relatives for at least eight million years, long enough for the two groups to become separate species. Such genetic differences offer a potential clinical benefit. Fungi are notoriously difficult to treat, and new drugs are in high demand. Now that clinicians understand that fungi from different regions have a different genetic makeup, Taylor says, they can include fungi with different genotypes in their drug and vaccine trials. It's difficult to overstate the power of the newfound ability to retrieve, through genetic analysis, what would otherwise remain unknown and unknowable. Recently, off the southeastern U.S. coast, a research team discovered vast deposits of waxy, methane-containing compounds called clathrates in ocean sediments 500 meters below sea level. Based on unique DNA signatures, archaeal microbes appear to be producing the deposits. The clathrates contain enough hydrocarbons to power the United States for centuries. "And no one knew they were there before," Pace says. Some microbial prospecting holds more immediate utility. Efforts to break down oil spills and other hydrocarbon pollution have relied on a familiar bacteria species genetically modified to metabolize the hydrocarbons. But the bacteria have been ineffective, because they don't normally live in a hydrocarbon-rich environment and can't compete with the native microbes. "We should use the microbes that live in hydrocarbon-rich regions," Pace stresses. "We have to find out about these natural microbial communities. They're running the planet. It's fundamental to our understanding of what makes the biosphere function." Basic research and its application: bound together as the fruit to the
tree. Reprinted from College of Natural Resources "Breakthroughs,"
Fall 1997 issue. Used with permission.
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