The Undiscovered Planet Microbial science illuminates a world of astounding diversity.

by JONATHAN SHAW

opernicus, kepler, galileo, newton—these are range of billions, exceeding the number of of “large” or- familiar names. During a 150-year span in the sixteenth ganisms by several orders of magnitude. and seventeenth centuries, they led the scientific revo- In light of this new understanding of , scientists with ad- lution that placed the sun, rather than the earth, at the vanced research tools are focusing anew on microbes, which, fol- center of things astronomical. But have you ever heard lowing the great discoveries of penicillin and other in of Carl Woese? He set in motion a scientific revolution the mid twentieth century, had largely been consigned to the in that, in its repudiation of anthropocentric confines of pharmaceutical research. Cviews of life, is proving no less profound. “Our planet has been shaped by an invisible world,” says In 1977, Woese (pronounced “woes”), a professor at the Univer- Roberto Kolter, a professor of and molecular genet- sity of Illinois at Urbana-Champaign, drew a terrestrial family ics at Harvard Medical School (HMS). He and Je≠rey professor of tree that showed the genetic relatedness of all living things on biology Colleen Cavanaugh of the Faculty of Arts and Sciences this planet. Using modern tools of , he sampled (FAS) together co-direct Harvard’s Microbial Sciences Initiative, the known single-celled, microscopic we call mi- which serves as a focal point for researchers in the field from all crobes, and also the denizens of the human-scale world with over the University. “Microbes mediate all the important element which we are familiar: the flowers, trees, and shrubs; the animals; cycles on Earth, and have played a defining role in the develop- and the fungi. His map of all this new information revealed that ment of the planet,” says Kolter. They form clouds, break down taxonomists of ages past had been looking at the world through rocks, deposit minerals, fertilize plants, condition soils, and clean the wrong end of a telescope. The formerly great “kingdoms” of up toxic waste. Among their ranks, explains Cavanaugh, are the Plantae, Animalia, and Fungi almost disappeared, shrinking to fit photosynthetic “primary producers” that use , water, and on a small, trifurcating branch of his tree. In their place were carbon dioxide to form the broad base of the food chain, and to- three vast “domains”: (single-celled that gether with plants make up the earth’s largest source of biomass. lack a distinct nucleus and organelles); Archaea, or Archaebacte- The earliest life on our planet was entirely microbial, and if life ria (similar in appearance and simplicity to bacteria, but with no- exists on other planets, it is surely microbial there as well. tably di≠erent molecular organization); and Eukarya (all organ- In the realm of human health, microbes help us digest food and isms whose cells have a distinct nucleus—or, simply put, produce vitamins, protect us against infection, and are the main everything else). Life on Earth, Woese’s model showed, is over- source of . The human cells in your body num- whelmingly microbial. In fact, the extent of microbial diversity is ber 10 trillion, but that pales by comparison to the estimated 100 so great that scientists have di∞culties estimating its actual size. trillion microbial cells that live in and on you. “Without them, you

Some estimates place the number of microbial species in the would be in trouble,” Kolter says: animals experience abnormal EURELIOS/PHOTO RESEARCHERS, INC.

44 November - December 2007 growth and become sick if deprived of their microflora during de- subjects that have implications across a wider spectrum of sci- velopment. Although a few microbes are known to cause disease, ences than is true for microbiology.” As a result, in 2006 MSI re- the precise role played by the vast majority is essentially unknown. ceived four years of support, totaling $2.7 million, from the The same could be said for microbes around the planet. There provost’s o∞ce. are a billion of them in a gram of soil, and a billion per liter of “It kills me that people think only that bacteria are disease- seawater, but we know neither causing,” says Cavanaugh, who what they are nor what they do. studies the chemosynthetic symbi- In the poetic conclusion to his otic bacteria that make life possible 1994 autobiography, Naturalist, the for giant clams and tubeworms great sociobiologist and Pellegrino dwelling near deep ocean hy- University Professor emeritus E.O. drothermal vents. Far from sun- 1 Wilson mused on what he would light, they operate by mechanisms SCIMAT/ PHOTO RESEARCHERS, INC. do, “[i]f I could do it all over again both similar to and much di≠erent and relive my vision in the twenty- from the photosynthetic organisms first century. I would be a microbial we see every day. “Although intra- ecologist...,” he wrote. “Into that cellular, these bacteria are helpful world I would go with the aid of to their animal hosts,” she adds. modern microscopy and molecu- “Like chloroplasts in plants [which lar analysis. I would cut my way evolved from symbiotic photosyn- through clonal forests sprawled thetic bacteria], the chemosyn- across grains of sand, travel in an imagined through drops 1: In terms of gene content, humans of water proportionately the size of and potatoes are more closely 2 related than these two bacteria are lakes, and track predators and prey to each other—one measure of in order to discover new life ways bacterial diversity. On the left, and alien food webs. All this, and I ; on the right, need venture no farther than ten Mycobacterium tuberculosis. 2: From the billions of bacteria in a paces outside my laboratory build- soil sample, these few cells landed ing. The jaguars, ants, and orchids on a nutrient medium where they would still occupy distant forests in could grow and form colonies. “There is so much beauty every- all their splendor, but now they where we look,” says microbiologist would be joined by an even stranger Roberto Kolter. 3: A strain of and vastly more complex living Streptomyces that produces several world virtually without end.” antibiotics widely used in and agriculture. When starved, these bacteria take on a “hairy” The Limits of Cultures 3 appearance as they initiate growth As a practical matter, the Mi- of aerial structures where protective crobial Sciences Initiative (MSI) develop. The pigmented areas consist of small molecules, often began in 2002 as a grass-roots e≠ort with antibiotic properties. 4: In the among faculty members who recog- presence of the antibiotic strepto- nized the unexplored ecology and mycin, colorful antibiotic-resistant mutants of Streptomyces coelicolor potential of these organisms and spring up and form colonies. wanted to share information about microbial research across diverse thetic symbionts turn carbon diox- disciplines: biology, medicine, chem- ide into sugars and proteins, feed- istry, engineering, , astron- ing their hosts internally.” omy, and evolutionary and plane- But most people do associate mi- tary history. The group held informal crobes with disease. “Antibacteri- “chalk-talks” weekly, and in 2004 4 als” have been incorporated into all staged a day-long symposium with kinds of consumer products: soaps, speakers from around the world. sponges, toilet paper, towels, and When President Lawrence H. Sum- cutting boards—even clothing. Kol- mers issued a call that year for ini- ter traces the origins of this “ludi- tiatives that would bring people to- crous” antimicrobial “scorched-earth gether from across the science and policy” to the time of Louis Pasteur, engineering disciplines, MSI was a perfect candidate, says Cabot who formulated germ theory, and Robert Koch, who developed professor of biology , a member of its steering methods for culturing bacteria. “Medical microbiology for al- committee. “I think there are few disciplines that lend them- most 150 years has been driven by the idea that germs are the selves better to cross-disciplinary approaches,” he says, “and few causative agents of disease. And there is no doubt that Koch and

All images courtesy of Roberto Kolter, Harvard Magazine 45 unless otherwise noted Pasteur were right, that Mycobacterium tuberculosis Not much can be gleaned about the differences causes tuberculosis and Vibrio cholerae causes between these two microorganisms just by look- cholera,” says Kolter. But microbes have also led ing at them, but genetic analysis tells us that they are not even in the same domain. 1: Soil-dwelling to most of our antibiotics, a development that Bacillus anthracis is classified as a bacterium. 1 Kolter calls “the most important advance in 2: Heat-loving Thermoproteus tenax is an medical history.” archaean that in hot springs. The formerly limited view of the microbial world arose from what has turned out to be an ing, and collected a sample of sediment,” Kolter inherently constrained approach to the study of explains. “He extracted the DNA, cloned it, and bacteria: the practice of culturing them. For put it into a little bacterium that he knew how more than a hundred years, scientists had been to grow.” Then he sequenced the genes. “In that mystified by what was called the “plate count one little gram of sediment,” Kolter notes, “Pace paradox.” Whenever they tried to grow a sam- discovered more diversity than we ever knew 2 ple of bacteria from the environment on a nutri- existed before, when using our traditional, cen- ent medium in a petri dish (an agar plate), only a tury-old techniques for cultivating bacteria.” few microorganisms grew and multiplied to Scientists had known that there are more mi- form colonies, when there should have been at a crobes in an ounce of soil than humans alive on minimum thousands of such colonies (based on Earth, but that was just a measure of abundance. the number of di≠erent species discernible just by looking Pace’s discovery demonstrated something new, a previously un- through a microscope). Various explanations were o≠ered—that fathomed repository of biodiversity. Scientists began sequencing

99.9 percent of the bacteria in the sample were dead, or that they DNA from all sorts of environments. After looking at human gut WOLFGANG BAUMEISTER/PHOTO RESEARCHERS, INC; DR. GARY GAUGLER /PHOTO INC “We are at the stage of discovery where, everywhere we look, we see new species.”

must all be the same bacterium because they looked similar. microflora, they learned that each individual has his or her own “But then in 1990,” says Kolter, “scientists showed that the characteristic set of a thousand species. “These represent three DNA complexity in a typical soil sample meant that there had to million genes that you carry,” points out Kolter, “as compared to the be thousands of times more diversity than was being plated.” estimated 18,000 genes of the human genome. So you are living Norman Pace, a professor at the University of Colorado, began to and exchanging [metabolites] constantly with a diverse pool of wonder if scientists simply lacked the basic knowledge needed some three million genes.” Microbiologists continue to find new to grow most of the bacteria on the planet—if perhaps we were taxonomic divisions of microbes far faster than they can figure so ignorant of these bacteria that we could not culture them. He out how to culture them. hit on the idea that he could instead analyze a sample of soil or The world of animals—from elephants to ants—is divided into water for its DNA content in order to ascertain how many 13 phyla (vertebrates are one phylum, insects another). In the mi- species it contained. “Pace went to Yellowstone National Park, to crobial world, their equivalents are called, for the time being, some of its famous hot springs, where the water was nearly boil- “deep-rooting branches.” In 1987, 13 of these big divisions were

The anthropocentric five-kingdom system classified all unicellular “Old” World-View of Biodiversity organisms lacking nuclei The Three-Domain Tree of Life (archaea and bacteria) Fungi as Monera. The nucleat- ed eukaryotes compris- Plantae Animalia ing plants, animals, and Archaea fungi were thought Bacteria to represent the bulk of biological diversity. All other nucleated eukaryotes were grouped in a grab-bag classification known as Protista. Fungi Animalia The modern “tree of Plantae } life,” based on genetic analysis, shows that the bulk of Earth’s bio- Protista diversity resides among the Archaea, Bacteria, and that portion of the Eukarya that does Monera not include plants, animals, and fungi. Eukarya

46 November - December 2007 1

1: Their membranes 2 outlined in red, individual bacteria known in the bacterial contain the biosynthetic domain: Woese sam- machinery—shown as green dots—for making bacillaene, pled these to create his a newly discovered small tree of life. But by 1997, molecule. 2: Colonies formed there were almost three by the wild strain of Bacillus subtilis generate complex times as many: 36 in structures, as seen at left, all. “Twelve of them while the domesticated we had never cultivat- strain—presumably ed, and the others we selected for uniformity by generations of researchers began to learn how to 3 who have used it in laborato- cultivate,” Kolter ex- ry experiments for plains. “By 2003, there many decades—has lost near- were 53 divisions, but ly all colony architecture. 3: Two colonies of Bacillus sub- the more we discovered, tilis growing on the more we found rep- top of another bacterium, resentative microbes Streptomyces coelicolor. The Bacillus colonies “talk” that we could not culti- to the Streptomyces using vate. We learned how small molecules. The strain to cultivate microbes of Bacillus subtilis at left from only two of these makes compounds for turning production of a small in six years and by 2004 pigmented molecule in we had found 80 such Streptomyces on and off. divisions from which we But in a mutant strain of Bacillus that cannot couldn’t cultivate even produce the “off switch,” a single representative.” Each of these deep-branching divisions is less genetic di≠erence be- Streptomyces produces the thought to represent millions, if not hundreds of millions, of tween [a human being] and pigmented red ring. This led species. “That means there are lots of genes out there, and we a potato” than there is be- to the discovery of the molecule that acts as the have no clue what they are doing,” Kolter says. “So when you tween, say, “the bacterium off switch: bacillaene. Below, think about biodiversity, and the extent of diversity on the planet, that causes tuberculosis and the molecule’s chemical you really get a sense of how little we know about this undiscov- the one that causes cholera. structure. ered world. We are at the stage of discovery where, everywhere So as magnificent as is the we look, we see new species. diversity of the tropical rainforest, it pales by comparison to “The reason we are so excited about preserving diversity,” he the microbial world.” continues, “is because that is how we preserve the richness of the planet.” Where does that richness lie? Traditionally, taxon- Microbial Medicines omists classified life according to morphology (by appearance, In the interests of human health, researchers have begun form, or pattern): one finch’s beak resembles that of another, to hunt among all this newfound microbial diversity for new an- but is nothing like that of hummingbird. Bats fly, but are more tibiotics. Says Richard Losick, “I have many wonderful colleagues like mice with wings than like birds. Microbes, on the other in the chemistry department, but without question the champion hand, often appear similar to one another to the human eye. synthetic organic chemists on the planet are the microbes.” Plants, animals, and fungi may seem wildly diverse from a mor- “The majority of our antibiotics and many anticancer com- phological perspective, but in fact, Kolter explains, there is “far pounds come from soil-dwelling bacteria,” notes Jon Clardy, a pro-

Harvard Magazine 47 but luckily, it did. “With this new pathway, these bugs can still Methanol, Cheeseburgers, Metals grow on methanol,” Marx reports. “But what’s most interesting is that they do so three times more slowly and are only 40 per- Interdisciplinary science like that supported by the Microbial Sciences Initia- cent as e∞cient.” But as the bugs evolved using the new pathway, tive depends heavily on training the next generation of researchers: junior fac- their fitness improved. After 120 generations, fitness had dou- ulty members who use unconventional tools to probe new questions. “The real bled. At present, after 600 generations, they reproduce almost as future of the place,” says Fisher professor of natural history Andrew Knoll, “is quickly as the original strain does with the original pathway. in these people.” Here are portraits of three such young scientists at Harvard. Marx freezes samples of the bugs at di≠erent stages of their evo- lution so that later he can analyze the sequence and timing of the in Action physiological changes that led to their improved fitness. “ on the small scale,” says assistant professor of or- ganismic and evolutionary biology Christopher Marx, “is just as Fueling Deep-sea Discovery interesting as nature on the savanna that you grew up watching Assistant professor of organismic and evolutionary biology on TV.” Marx studies evolution in the lab, using bacteria that Peter Girguis studies “ecological ,” the physiology of grow on methanol. groups of uncultured microbes that live at hydrothermal vents With a population of a billion bacteria, one that doubles in 2,500 meters deep in the ocean, where the pressure—about 4,000 size every eight hours, he can perform true experiments in evolu- pounds per square inch—is “the equivalent of a small car being tion. In contrast, he notes that “even in a herd of 100 cows, in balanced on your thumbnail.” which you select, for example, the fattest in each generation, “If you go to the sea floor, you can measure changes in there is such a small amount of genetic variability that you are concentration or butane, or nitrate or sulfate, but we often don’t really selecting for new combinations of alleles,* and not for new know exactly which organisms are responsible,” he says. Because alleles occurring, because the chance of a new, useful gene arising groups of microbes living in complex ecologies can be di∞cult to in 100 cows is relatively low per generation. Here [in the lab], a understand, Girguis sometimes brings samples back to the lab, lot happens every day. One out of every thousand bacterial ‘ba- where he recreates marine environments with clever engineer- bies’ has a mutation somewhere, which is not much”—but with a ing: simulating the pumping action of tides on sediments, for ex- billion new bacteria in each generation, that is a million new mu- ample. Then he perturbs the system—perhaps by adding oxy- tations every day. “The genome size of [the bacterium I work on] gen—to see how the microbes respond. “We are focusing mainly is about seven million base pairs,” he says, “so in seven genera- on natural perturbations,” he says, “but anthropogenic ones are tions you will have tested, on average, every single site, so you equally important.” He notes that there has been a lot of talk can quickly generate many di≠erent variants for selection to act about managing the greenhouse-gas problem by sequestering upon.” carbon in the deep ocean (by capturing carbon-dioxide emis- “Organisms that eat methanol are sprinkled all over the bacte- sions from fossil-fuel-burning power plants and storing them in rial tree of life,” Marx continues. “But if you look at the genes in- liquid form atop the ocean floor 3,000 meters or more below the volved, the sequences are in almost all cases highly related.” surface, where the waste could remain for millions of years; see What has happened is called “horizontal gene transfer,” the ex- “Fueling Our Future,” May-June 2006, page 40). “That makes me change of genetic material between di≠erent kinds of bacteria: in nervous,” he says, “because I think the response of the microbes this case, an entire “cassette” of perhaps a hundred genes that may be quite di≠erent from what we assume.” control . Marx wants to see what physiological steps Many of Girguis’s experiments take place in the field, but run- take place after a gene transfer like this, in hope of extrapolating ning the necessary equipment in the deep ocean, with its high principles that might allow him to predict the probability of any pressure and extreme cold, can be very expensive. It takes 125 particular beneficial mutation taking place before another. size “D” alkaline batteries to provide power for a year to a half- One of the byproducts of metabolizing methanol is formalde- watt device like a bike lamp, so Girguis has enlisted the aid of hyde, which kills most living things. Because Marx’s “bugs” neu- microbes to solve his on-site fuel problem. A microbial fuel cell is tralize formaldehyde by oxidizing it, he decided to see what less a≠ected by the temperature and pressure, and can run as would happen if he swapped the genes that gen- long as 15 years without needing a erate their formaldehyde oxidation pathway for “charge” of fresh organic matter. a completely di≠erent set of genes from an unre- (Girguis runs a pair of demonstra- lated bug. “It is as if you ripped a gasoline engine tion cells in his o∞ce on cow ma- out of a car and replaced it with an electric nure.) motor,” he says. “This is di≠erent chemistry— He compares what the microbes di≠erent pathways, and di≠erent intermedi- are doing—generating electric- ates—but it gets you from the same point A to ity—to what we do when we eat a point B on the metabolic map.” There was no At the bottom of the sea, guarantee that the experiment would work— life for these giant tubeworms is ______made possible by symbiotic *Alleles are gene variants in a particular location on a bacteria that convert chemicals chromosome that may, for example, cause brown eyes, be- ejected from hydrothermal cause they are coded by the dominant allele of the gene vents into food, in a process for eye color, rather than blue eyes, which are coded by called . the recessive version. PETER GIRGUIS

48 November - December 2007 cheeseburger. “All life on Earth,” he explains, “is pretty much about moving electrons.” When you eat, “what you are doing is taking all the electrons that are tied up in that organic matter— in the burger and the cheese These 2.5 billion-year-old and the bun and the secret sedimentary rocks in Karijini sauce—and moving those National Park, northwestern electrons through your respi- Australia, contain layers of ratory pathways,” harnessing iron that has been oxidized. Such “banded iron their energy in the process. formations” may allow “Then you are going to dump geologists to determine when the electrons onto oxygen— oxygen first appeared in you need that oxygen because Earth’s atmosphere. it is like an electron magnet— and you ditch the oxygen, electrons, and some carbon [as carbon dioxide]. “Microbes do the same thing, but they can also dump “The strategy we use to clean up a site,” the electrons onto sulfates, nitrates, and even solids. And Hansel explains, is to provide additional food

this is where it gets crazy. They are capable of shuttling ANDREW KNOLL for the microbes that are capable of immobi- electrons from reduced organic matter through their bio- lizing metals. “That,” she says, “will stimu- chemical pathways, generating energy, and then sticking late the microbes to increase their metabo- those electrons onto a chemical that they shuttle outside their lism and clean up the soils and sediments.” Clean-up crews bodies and then dumping [the ‘shuttle,’ thereby depositing] the pump a carbon compound such as glucose or lactate into the soil. electrons onto iron or another solid-phase oxidant.” Some mi- The organisms respond by achieving “higher densities and higher crobes don’t need the chemical shuttle at all, he reports: “They growth yields,” she says. But they also “start producing antibi- just dump their electrons through little conductive nanowires. otics, because they want to kill o≠ their competitors.” Stimulat- That is like you eating a cheeseburger and being able to respire it ing the production of antibiotics has the unfortunate conse- without oxygen. It is like holding your breath and dumping your quence of providing selective pressure for antibiotic-resistant electrons onto my filing cabinet. That is the premise behind mi- genes, which move between microorganisms much faster than crobial fuel cells. You can set up a microbial fuel cell, encourage other kinds of genes. the growth of microbes that can shuttle electrons and break “Are we doing some harm when we are trying to do some down the organic matter in an environment, and then have them good?” she wonders. Microbes reduce or oxidize metals in order dump those electrons onto the fuel cell and harness that energy.” to gain energy or detoxify themselves, she explains. But if the mi- Girguis’s fuel cells generate enough electricity to run a light crobes become resistant to the metals, as they do when they ac- bulb, power a wireless network, or charge a cell phone. His aspi- quire antibiotic resistance genes, they no longer do this useful ration to power lights in the developing world has led to a col- work. Meanwhile, they are also introducing many more antibi- laboration with two Harvard Business School graduates who otic-resistant genes (potentially of danger to humans) into the hope to bring a product to market. Says Girguis with a grin, “Mi- environment. crobes really run everything.” Hansel’s research into the oxidation of iron and manganese (primarily for use in cleaning contaminated sites) may also help The Clean-up Conundrum answer geological questions. Banded iron formations—sedimen- One of the things that attracted Colleen Hansel to Harvard tary deposits of rock billions of years old in which the iron and last January was the energy she saw going into the Microbial Sci- manganese layers have been oxidized—are often cited as evi- ences Initiative. An assistant professor of environmental micro- dence of oxygen in earth’s early atmosphere. “But the big ques- biology in the School of Engineering and Applied Sciences, she tion,” says Hansel, “is whether or not oxygen is needed to form studies microbial interactions with metals. The work is impor- these iron oxides. Is the process abiotic, or were microbes in- tant for cleaning up toxic waste, for improving agricultural prac- volved?” tices and human health, and even for understanding planetary Unlike iron, manganese is very hard to oxidize, she says. But history. certain organisms that she has isolated from contaminated sites Hansel has worked on cleaning up sites contaminated with produce a molecule during their growth that oxidizes the metal metals such as arsenic, chromium, and technetium. More and when exposed to light. “The bacterium is not oxidizing the more research, she says, is showing “an interesting correlation” metal itself,” she emphasizes, it is just producing a photoreactive between antibiotic resistance and microbial resistance to metals. organic molecule. “Could there have been organics present two Microbes that have developed genes enabling them to resist an- billion years ago, during the Pre-Cambrian when the banded tibiotics can withstand higher metal concentrations and there- iron formations appeared, that were photoexcited and oxidized fore may no longer detoxify them. Such resistance is growing manganese?” It is the sort of exciting question—bearing on the rapidly, because antibiotics are becoming common in the envi- origins of life and the creation of the biosphere—that she hopes ronment, streaming from animal feed lots and even household to explore with her new colleagues. But she acknowledges, “I sewage. They can even arise spontaneously as a response to find microbes are humbling, too. The more you understand, the toxic-waste cleanup e≠orts. more you realize just how diverse they are.”

Harvard Magazine 49 1: The double membranes known bacteria have been discovered. A prime example is Strep- of Gemmata obscuriglobus tomyces avermitilis, the bacterium from which researchers derived a may be durable enough drug called Ivermectin. “Ivermectin,” says Clardy, “is grown on to produce “molecular fossils”: traces of early life the ton scale because it is used against river blindness in Africa, on Earth that have been for treating almond trees in California, and for getting rid of all preserved for billions of 1 kinds of parasites” (equine worms, for example). “Here you have years. 2: Bacillus subtilis a ‘bug’ that is producing a useful molecule grown on a huge scale, forms spores (in green) with lipid membranes that and intensively so.” Once the genome was sequenced, he says, help protect the from “Just looking at it casually, you could see where Ivermectin is oxygen damage. These made, but you could also see [other gene sequences]—over 30 of tough cellular membranes appear in the fossil record these clusters—that it seems should each make a small molecule.

ANN PEARSON at about same time We know only three molecules that come from that bug,” he Earth’s atmosphere became adds—one of them the source of Ivermectin. “That means we enriched with oxygen, know only 10 percent of what it can potentially make.” 2 and may help trace changes in the planet’s early But probing those secrets is far from easy. When grown in a atmosphere. lab in pure culture, microbes apparently don’t need to activate all their genetic machinery to survive. In their natural setting, by fessor of biological chemistry contrast, microbes live in a complex ecology: they interact with and molecular pharmacology their environment and with other microbes by using a vast array at HMS. Unfortunately, he of virtually unknown small-molecule products. These organic says, due to the spread of an- compounds often play multiple roles: a small molecule used for tibiotic resistance, “we are signaling among bacteria engaged in mutually beneficial metabo- running out of e≠ective an- lite exchange (one microbe’s metabolic waste is another’s meal)

ANN PEARSON tibiotics.” For economic rea- might also be used to kill competitors trying to gain a foothold in sons, pharmaceutical companies are not investing as much as they the same ecological niche. Such compounds, if researchers could used to in the development of new ones, which has left physicians identify them, produce them, and figure out how they work, looking for an antibiotic of last resort to be held in reserve for the might form the next generation of medical antibiotics. one patient once a year who has a resistant strain of tuberculosis or At Harvard, an MSI-facilitated collaboration between Kolter pneumonia, explains Kolter. “That drug would be the billion-dollar and Clardy uses creative methods for prompting even uncultur- blockbuster that nobody buys, because doctors shouldn’t be pre- able microbes to yield such genetic secrets. Clardy was among scribing it widely. Nobody in the corporate world will develop it.” the earliest researchers to use “metagenomics,” which involves He believes that “there may be a role for the University in research sampling the environment—your gut, a lake, or in his case, the that is not going to be as profitable for corporations to pursue: the soil—and collecting the “metagenome,” or composite genome, of development of targeted, ecologically sound antibiotics.” all the individual organisms dwelling there. After extracting the Whole-genome studies of microbes suggest that only a small many strands of DNA, Clardy screens for sequences that make fraction of the natural products that come from even well- compounds. He can isolate these sequences and transfer them Glossary for an Invisible World major divisions of life and include all the bacteria, all the ar- chaea, and some of the eukarya. Some scientists say that only Archaea: One of three major domains, or classifications, of life, prokaryotes (see below) should be considered microbes, archaea are morphologically similar to bacteria, but have a thereby excluding even single-celled fungi. Others argue that di≠erent molecular transcription machinery, indicating an an- even multicellular organisms can be considered microbes. cient evolutionary divergence. Mushrooms, for example, are the multicellular fruiting bodies Bacteria: One of three major domains of life. Bacteria are single- of fungi that may start as unicellular organisms in the soil. Fur- celled microorganisms with neither a nucleus nor organelles thermore, both unicellular bacteria and archaea form aggre- (specialized intracellular structures; see below). gates (“colonies”) on surfaces, often referred to as biofilms, that Cyanobacteria: A division of bacteria that produce their energy exhibit distinctive multicellular patterning. through photosynthesis. Formerly called blue-green algae Organelles: Structures within a cell that perform specialized (hence the name “cyano,” or blue, bacteria), they are not “true” functions, such as energy production. Mitochondria and algae. Algae are eukaryotes, while the cyanobacteria are chloroplasts are examples of energy-producing organelles that prokaryotes. are thought to have evolved from autonomous bacteria. Eventu- Eukarya: One of three major domains of life, the eukarya include ally they became dependent on their hosts—and vice versa. all single and multicellular organisms in which the cells have a Primary production: Production is the creation of new organic distinct nucleus. Examples are plants, animals, and fungi. The matter. Primary production refers to the creation of new or- classification includes everything that is not a bacterium or an ganic matter by photosynthetic microbes. archaean. Prokaryotes: An inclusive term that refers to the bacteria and ar- Microbe: Generally considered to include all life that cannot be chaea, single-celled organisms that, as distinct from eukary- seen with the unaided eye, microbes are found in all three otes, lack a nucleus.

50 November - December 2007 1 into E. coli (or another “tame” bacterium that he 1: Roseobacteria, which form knows how to cultivate) in order to get them to pinkish colonies, aid in the forma- express the target compounds. Kolter, meanwhile, tion of clouds by producing gases that nucleate water droplets. has been creating controlled ecological settings, 2: Bacteria fertilize plants through exploring whether certain bacteria behave di≠er- the formation of specialized ently in the context of other species, as they do in symbiotic “tissues” on the roots, their natural environment. He has given Clardy a known as “root nodules.” Bacteria in these nodules form “factories” 2 tool that indicates when a bug responds to a mi- able to capture atmospheric crobial signal, and Clardy has developed other nitrogen gas and convert it into tools for figuring out what that signaling com- ammonia-containing compounds that the plants can use for growth. pound is. These organic molecules, often antibi- By themselves, plants cannot effect otic, frequently “turn out to be something new this transformation, which is that nobody has ever seen before,” says Kolter, why they need “fixed” nitrogen “often with properties that are chemically very in- (not gas) as fertilizer. teresting.” To show that their method works in principle, the team in 2006 last 35 years has eluci- published an early discovery using the bacterium Bacillus subtilis, dated in great detail which has been studied for more than 100 years. The compound the molecular mecha- they identified, bacillaene, may or may not turn out to be useful in nisms that control life medicine, they caution. But it is a highly complex, resource-inten- cycle and di≠erentia- sive structure for any single-celled to make, and that tion in the bacterium suggests that it plays an important biological role. Two new MSI Bacillus subtilis. She “does wonderful research,” Losick says, “on [the postdoctoral fellows arriving at the medical school this year will origins of] biological molecules that can be recovered from hun- use the collaborative techniques Kolter and Clardy have developed dreds of millions, if not billions, of years ago, reflecting the earliest to facilitate further discoveries. They will enter a universe of seem- evidence for life on Earth.” ingly endless therapeutic possibility, between the small-molecule “If you have very highly preserved, organic-rich, sedimentary products of bacteria yet undiscovered, and the 90 percent of un- rocks that haven’t been deeply buried and heated in their geo- tapped coding sequences from the bugs we thought we knew. logic history,” Pearson says, “you can extract measurable amounts of lipids or fats that make up some cell membranes. We Bacterial Biomarkers of Ancient Earth call these ‘molecular fossils’ because they have structure, they Within FAS, Ann Pearson, Cabot associate professor of earth and can be identified, and occasionally you can tell what type of or- planetary sciences, also became interested in bacteria for what ganism they might have come from—for example, there are cer- they might reveal about Earth’s early history. Pearson, a biogeo- tain molecules that are produced only by archaea.” chemist, has collaborated with Richard Losick, who during the “Ann had the idea,” Losick explains, “that maybe we could learn 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.

Harvard Magazine 51 something from similar molecules found in contemporary mi- crobes.” It turned out that the bacterium that Losick’s lab studies Moving Microbial Science Forward has genes similar to those known to produce a certain type of Today, operating out of borrowed space in Har- these molecular fossils. Their shared MSI-sponsored postdoctoral vard’s Center for the Environment, the Microbial Sciences fellow, Tanja Bosak (now an assistant professor at MIT), learned Initiative (www.msi.harvard.edu) funds six postdoctoral the microbial techniques Losick’s lab uses and obtained fellows who typically bridge two labs in di≠erent depart- evidence that in the contemporary bacterium, these molecules ments, often across schools; sponsors 12 undergraduate help to protect its spores from damage by oxygen. (Although many summer fellowships; arranges monthly seminars that life forms need oxygen, it can also cause a lot of damage.) Bacillus bring in speakers from around the world; and hosts an an- subtilis, Losick explains, creates a “particularly macho type of nual symposium. This past summer, MSI held a workshop spore, maybe the most sturdy kind of dormant cell on the planet. for high-school teachers designed to help them incorpo- It can survive extremes of time and temperature and radiation.” rate microbial education into their curricula. In the cur- What might this molecule have been doing two billion years ago? rent academic year, the initiative will introduce two new “The exciting implication,” Losick replies, “is that these molecules undergraduate courses: Life Sciences 190hf, “Diverse Mi- may have appeared as a response to the rise of oxygen in the at- crobial Strategies for Metabolism, Pathogenesis and mosphere, so the timing of their appearance in the rock record Chemical Signaling,” taught by Harvard Medical School may bear on the issue of when oxygen levels first rose.” (HMS) professor of genetics Gary Ruvkun, and Life Sci- “The planet is about 4.5 billion years old,” elaborates another of ences 110, “A Microbial World,” designed for students Pearson’s collaborators, paleontologist Andrew Knoll, Fisher pro- pursuing microbial science as a secondary field and co- fessor of natural history and professor of earth and planetary sci- taught by professor of microbiology and molecular genet- ences. “The oldest rocks we can look at are 3.8 billion years old.” ics Roberto Kolter and professor of biological chemistry Chemical evidence suggests that life was already present then, and molecular pharmacology Jon Clardy, both of HMS, but certainly “by 3.5 billion years ago there were active microbial and Cabot associate professor of earth and planetary sci- communities on the earth’s surface.” These microbes were a little ences Ann Pearson, a biogeochemist in the Faculty of Arts di≠erent from those we know today because there was still no and Sciences. oxygen. (Even today, he says, oxygen is just a “veneer on the sur- face of the planet. If you put your shovel in the mud of a marsh and dig a centimeter beneath the surface you are down to anaerobic microbial , which remain critical for most of the biologically important element cy- cles on the earth’s surface.”) By 2.4 billion years ago, the chemistry of sedimentary rocks suggests that there was “at least a lit- tle bit of oxygen in the atmosphere and surface ocean.” Then, “for a period of about eighteen hundred million years, oxygen levels hovered at maybe a couple of percent of today’s levels. Only in the last 600 mil- lion years or so do we have environments with enough oxygen to support the biol- ogy of large animals, and only in that inter- val do large animals actually appear,” Knoll says. That is also when the algae first be- come ecologically important, joining and eventually displacing photosynthetic bac- teria as the basis of early food chains in the world’s oceans. “So there is a time coinci- dence,” he says, “between major events in 1 2 the history of life and major events in the history of the planet.” Following those threads might also suggest how life could 1: These cave mineral deposits were difficult to explain through arise, and what it might look like, elsewhere in the universe. purely geologic processes. Spelunker scientists in the 1970s “Everybody is interested in finding the perfect molecule to discovered that microbes play a role in their formation. 2: The recognition that microbes can respire insoluble metals has trace cyanobacteria,” Pearson reports, in order to trace the ori- led to a complete reassessment of processes, such as rusting, that gins of oxygenic primary production (cyanobacteria use a type of used to be considered largely abiotic (nonliving) and are now photosynthesis that releases oxygen, and this process is respon- known to have a microbial component. Scientists refer to this as sible for our oxygen-rich atmosphere). More broadly, she hopes “microbial-assisted corrosion.” to “figure out how to relate the incredible diversity of microbes

52 November - December 2007 1 that are out there to the kinds of preservable or- 1: Photosynthetic cyanobacte- ganic molecules—or biomarkers—that they ria form colorful mats in the make...and that we can find in the sedimentary warm springs at Yellowstone record.” Because individual species of microbes are National Park, a place that plays a central role in mutable (“They evolve quickly and we have no microbial-diversity studies. idea if there is phylogenetic integrity over billions 2: Other kinds of “extremo- 2 of years”), she is trying to relate particular pre- phile” microbes can live served molecules to their in the environ- without light or oxygen near functions chimneys. ment, rather than to species, in order to try to get The ecosystems around these an idea of what the looked like as life vents are entirely supported evolved on Earth and, in turn, shaped Earth’s envi- by both the free-living and the ronment—oxygenating the atmosphere, detoxify- symbiotic bacteria. ing the oceans, breaking down rock and organic matter to form practical impor- soils. “MSI has been absolutely essential to building the bridges tance as well.” that I have needed to other research groups,” Pearson says. “Al- For his part, though my questions are geology- and earth-history-driven, the geochemist Daniel work that I do in order to answer those questions is all biology Schrag, professor

and chemistry. I don’t practice field geology,” she says, “so build- of earth and plan- COLLEEN CAVANAUGH ing bridges with other parts of the University has been critical.” etary sciences and a member of the MSI steering committee, empha- Historically, says Knoll, “there have been a few of us in FAS sizes that “almost all the reactions on the earth’s surface are catalyzed who have been interested in microorganisms, but there wasn’t by microbes—in soils, in waters, in swamps. If you don’t know what anything as tangible as a Museum of Comparative Zoology, microbes are doing, you don’t really know what is going on.” which has provided a focal point for people interested in ani- “It is clear that this field will not reach its maturity, in the mals for 150 years, [or] the Herbarium, which has done the same sense that it becomes less exciting, during my research lifetime thing for people interested in plants” (which Knoll calls, half- or probably the research lifetimes of my students,” Knoll says. joking, “green algae with a graduate degree”). He continues, “Be- “We have whole new horizons in the nature and treatment of cause of Harvard’s good fortune in the nineteenth and early disease; whole new horizons in simply understanding the diver- twentieth centuries, we built up these really quite remarkable sity of life as it actually exists—not what we thought existed be- facilities for studying plants and animals...and, not surprisingly, cause we could see it; whole new horizons in the nature of the we then populated our faculty with people who study [them]. relationship between organisms and the environment both “Almost all the reactions on the earth’s surface are catalyzed by microbes—in soils, in waters, in swamps. If you don't know what microbes are doing, you don't really know what is going on.”

These areas are not unimportant now,” he explains, “but so today and in the future, and on the timescale of the whole devel- many horizons have opened up for studying microorganisms opment of the planet. These are wonderfully large, exciting that the simple identification through the MSI of a community questions and MSI gives us a forum to discuss them. You do of people concerned about microbiology has been tremendously find, every once in a while, someone who has actually thought important. Many of the basic biological processes that underlie about the same problem in a very di≠erent way”—and that can the nature of changes in the environment are microbial, so in a be the most important sort of catalyst: the kind that leads to world where the environment may be changing faster than our new discoveries. ability to understand it, having a better process-oriented idea of what microbial communities are actually doing in nature has Jonathan Shaw ’89 is managing editor of this magazine.

Harvard Magazine 53