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November-December 2007
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< previous | 1 | 2 | 3 | 4 “But then in 1990,” says Kolter, “scientists showed that the DNA complexity in a typical soil sample meant that there had to be thousands of times more diversity than was being plated.” Norman Pace, a professor at the University of Colorado, began to wonder if scientists simply lacked the basic knowledge needed to grow most of the bacteria on the planet—if perhaps we were so ignorant of these bacteria that we could not culture them. He hit on the idea that he could instead analyze a sample of soil or water for its DNA content in order to ascertain how many species it contained. “Pace went to Yellowstone National Park, to some of its famous hot springs, where the water was nearly boiling, and collected a sample of sediment,” Kolter explains. “He extracted the DNA, cloned it, and put it into a little bacterium that he knew how to grow.” Then he sequenced the genes. “In that one little gram of sediment,” Kolter notes, “Pace discovered more diversity than we ever knew existed before, when using our traditional, century-old techniques for cultivating bacteria.” The world of animals—from elephants to ants—is divided into 13 phyla (vertebrates are one phylum, insects another). In the microbial world, their equivalents are called, for the time being, “deep-rooting branches.” In 1987, 13 of these big divisions were known in the bacterial domain: Woese sampled these to create his tree of life. But by 1997, there were almost three times as many: 36 in all. “Twelve of them we had never cultivated, and the others we began to learn how to cultivate,” Kolter explains. “By 2003, there were 53 divisions, but the more we discovered, the more we found representative microbes that we could not cultivate. We learned how to cultivate microbes from only two of these in six years and by 2004 we had found 80 such divisions from which we couldn’t cultivate even a single representative.” Each of these deep-branching divisions is thought to represent millions, if not hundreds of millions, of species. “That means there are lots of genes out there, and we have no clue what they are doing,” Kolter says. “So when you think about biodiversity, and the extent of diversity on the planet, you really get a sense of how little we know about this undiscovered world. We are at the stage of discovery where, everywhere we look, we see new species. “The reason we are so excited about preserving diversity,” he continues, “is because that is how we preserve the richness of the planet.” Where does that richness lie? Traditionally, taxonomists classified life according to morphology (by appearance, form, or pattern): one finch’s beak resembles that of another, but is nothing like that of hummingbird. Bats fly, but are more like mice with wings than like birds. Microbes, on the other hand, often appear similar to one another to the human eye. Plants, animals, and fungi may seem wildly diverse from a morphological perspective, but in fact, Kolter explains, there is “far less genetic difference between [a human being] and a potato” than there is between, say, “the bacterium that causes tuberculosis and the one that causes cholera. So as magnificent as is the diversity of the tropical rainforest, it pales by comparison to the microbial world.” Microbial MedicinesIn the interests of human health, researchers have begun to hunt among all this newfound microbial diversity for new antibiotics. Says Richard Losick, “I have many wonderful colleagues in the chemistry department, but without question the champion synthetic organic chemists on the planet are the microbes.” “The majority of our antibiotics and many anticancer compounds come from soil-dwelling bacteria,” notes Jon Clardy, a professor of biological chemistry and molecular pharmacology at HMS. Unfortunately, he says, due to the spread of antibiotic resistance, “we are running out of effective antibiotics.” For economic reasons, pharmaceutical companies are not investing as much as they used to in the development of new ones, which has left physicians looking for an antibiotic of last resort to be held in reserve for the one patient once a year who has a resistant strain of tuberculosis or pneumonia, explains Kolter. “That drug would be the billion-dollar blockbuster that nobody buys, because doctors shouldn’t be prescribing it widely. Nobody in the corporate world will develop it.” He believes that “there may be a role for the University in research that is not going to be as profitable for corporations to pursue: the development of targeted, ecologically sound antibiotics.”
Two colonies of Bacillus subtilis growing on top of another bacterium, Streptomyces coelicolor. The Bacillus colonies “talk” to the Streptomyces using small molecules. The strain of Bacillus subtilis at left makes compounds for turning production of a small pigmented molecule in Streptomyces on and off. But in a mutant strain of Bacillus that cannot produce the “off switch,” Streptomyces produces the pigmented red ring. This led to the discovery of the molecule that acts as the off switch: bacillaene. Below, the molecule’s chemical structure.
Whole-genome studies of microbes suggest that only a small fraction of the natural products that come from even well-known bacteria have been discovered. A prime example is Streptomyces avermitilis, the bacterium from which researchers derived a drug called Ivermectin. “Ivermectin,” says Clardy, “is grown on the ton scale because it is used against river blindness in Africa, for treating almond trees in California, and for getting rid of all kinds of parasites” (equine worms, for example). “Here you have a ‘bug’ that is producing a useful molecule grown on a huge scale, and intensively so.” Once the genome was sequenced, he says, “Just looking at it casually, you could see where Ivermectin is made, but you could also see [other gene sequences]—over 30 of these clusters—that it seems should each make a small molecule. We know only three molecules that come from that bug,” he adds—one of them the source of Ivermectin. “That means we know only 10 percent of what it can potentially make.” But probing those secrets is far from easy. When grown in a lab in pure culture, microbes apparently don’t need to activate all their genetic machinery to survive. In their natural setting, by contrast, microbes live in a complex ecology: they interact with their environment and with other microbes by using a vast array of virtually unknown small-molecule products. These organic compounds often play multiple roles: a small molecule used for signaling among bacteria engaged in mutually beneficial metabolite exchange (one microbe’s metabolic waste is another’s meal) might also be used to kill competitors trying to gain a foothold in the same ecological niche. Such compounds, if researchers could identify them, produce them, and figure out how they work, might form the next generation of medical antibiotics. At Harvard, an MSI-facilitated collaboration between Kolter and Clardy uses creative methods for prompting even unculturable microbes to yield such genetic secrets. Clardy was among the earliest researchers to use “metagenomics,” which involves sampling the environment—your gut, a lake, or in his case, the soil—and collecting the “metagenome,” or composite genome, of all the individual organisms dwelling there. After extracting the many strands of DNA, Clardy screens for sequences that make compounds. He can isolate these sequences and transfer them into E. coli (or another “tame” bacterium that he knows how to cultivate) in order to get them to express the target compounds. Kolter, meanwhile, has been creating controlled ecological settings, exploring whether certain bacteria behave differently in the context of other species, as they do in their natural environment. He has given Clardy a tool that indicates when a bug responds to a microbial signal, and Clardy has developed other tools for figuring out what that signaling compound is. These organic molecules, often antibiotic, frequently “turn out to be something new that nobody has ever seen before,” says Kolter, “often with properties that are chemically very interesting.”
Photographs by Ann Pearson Left: The double membranes of Gemmata obscuriglobus may be durable enough to produce “molecular fossils”: traces of early life on Earth that have been preserved for billions of years. Right: Bacillus subtilis forms spores (in green) with lipid membranes that help protect the spore from oxygen damage. These tough cellular membranes appear in the fossil record at about same time Earth’s atmosphere became enriched with oxygen, and may help trace changes in the planet’s early atmosphere. To show that their method works in principle, the team in 2006 published an early discovery using the bacterium Bacillus subtilis, which has been studied for more than 100 years. The compound they identified, bacillaene, may or may not turn out to be useful in medicine, they caution. But it is a highly complex, resource-intensive structure for any single-celled organism to make, and that suggests that it plays an important biological role. Two new MSI postdoctoral fellows arriving at the medical school this year will use the collaborative techniques Kolter and Clardy have developed to facilitate further discoveries. They will enter a universe of seemingly endless therapeutic possibility, between the small-molecule products of bacteria yet undiscovered, and the 90 percent of untapped coding sequences from the bugs we thought we knew. Bacterial Biomarkers of Ancient EarthWithin FAS, Ann Pearson, Cabot associate professor of earth and planetary sciences, also became interested in bacteria for what they might reveal about Earth’s early history. Pearson, a biogeochemist, has collaborated with Richard Losick, who during the last 35 years has elucidated in great detail the molecular mechanisms that control life cycle and differentiation in the bacterium Bacillus subtilis. She “does wonderful research,” Losick says, “on [the origins of] biological molecules that can be recovered from hundreds of millions, if not billions, of years ago, reflecting the earliest evidence for life on Earth.”
Roseobacteria, which form pinkish colonies, aid in the formation of clouds by producing gases that nucleate water droplets (detail below).
“If you have very highly preserved, organic-rich, sedimentary rocks that haven’t been deeply buried and heated in their geologic history,” Pearson says, “you can extract measurable amounts of lipids or fats that make up some cell membranes. We call these ‘molecular fossils’ because they have structure, they can be identified, and occasionally you can tell what type of organism they might have come from—for example, there are certain molecules that are produced only by archaea.” 1 | 2 | 3 | 4 | continued >  
 
 
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