Undiscovered Planet

44 November - December 2007

opernicus, kepler, galileo, newton—these arefamiliar names. During a 150-year span in the sixteenthand seventeenth centuries, they led the scientific revo-lution that placed the sun, rather than the earth, at thecenter of things astronomical. But have you ever heardof Carl Woese? He set in motion a scientific revolutionin biology that, in its repudiation of anthropocentric

views of life, is proving no less profound. In 1977, Woese (pronounced “woes”), a professor at the Univer-

sity of Illinois at Urbana-Champaign, drew a terrestrial familytree that showed the genetic relatedness of all living things onthis planet. Using modern tools of molecular biology, he sampledthe known single-celled, microscopic organisms we call mi-crobes, and also the denizens of the human-scale world withwhich we are familiar: the flowers, trees, and shrubs; the animals;and the fungi. His map of all this new information revealed thattaxonomists of ages past had been looking at the world throughthe wrong end of a telescope. The formerly great “kingdoms” ofPlantae, Animalia, and Fungi almost disappeared, shrinking to fiton a small, trifurcating branch of his tree. In their place werethree vast “domains”: Bacteria (single-celled microorganisms thatlack a distinct nucleus and organelles); Archaea, or Archaebacte-ria (similar in appearance and simplicity to bacteria, but with no-tably di≠erent molecular organization); and Eukarya (all organ-isms whose cells have a distinct nucleus—or, simply put,everything else). Life on Earth, Woese’s model showed, is over-whelmingly microbial. In fact, the extent of microbial diversity isso great that scientists have di∞culties estimating its actual size.Some estimates place the number of microbial species in the

range of billions, exceeding the number of species of “large” or-ganisms by several orders of magnitude.

In light of this new understanding of life, scientists with ad-vanced research tools are focusing anew on microbes, which, fol-lowing the great discoveries of penicillin and other antibiotics inthe mid twentieth century, had largely been consigned to theconfines of pharmaceutical research.

“Our planet has been shaped by an invisible world,” saysRoberto Kolter, a professor of microbiology and molecular genet-ics at Harvard Medical School (HMS). He and Je≠rey professor ofbiology Colleen Cavanaugh of the Faculty of Arts and Sciences(FAS) together co-direct Harvard’s Microbial Sciences Initiative,which serves as a focal point for researchers in the field from allover the University. “Microbes mediate all the important elementcycles on Earth, and have played a defining role in the develop-ment of the planet,” says Kolter. They form clouds, break downrocks, deposit minerals, fertilize plants, condition soils, and cleanup toxic waste. Among their ranks, explains Cavanaugh, are thephotosynthetic “primary producers” that use sunlight, water, andcarbon dioxide to form the broad base of the food chain, and to-gether with plants make up the earth’s largest source of biomass.The earliest life on our planet was entirely microbial, and if lifeexists on other planets, it is surely microbial there as well.

In the realm of human health, microbes help us digest food andproduce vitamins, protect us against infection, and are the mainsource of antibiotic medicines. The human cells in your body num-ber 10 trillion, but that pales by comparison to the estimated 100trillion microbial cells that live in and on you. “Without them, youwould be in trouble,” Kolter says: animals experience abnormal

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The Undiscovered

PlanetMicrobial science illuminates a world of astounding diversity.

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Harvard Magazine 45

growth and become sick if deprived of their microflora during de-velopment. Although a few microbes are known to cause disease,the precise role played by the vast majority is essentially unknown.

The same could be said for microbes around the planet. Thereare a billion of them in a gram of soil, and a billion per liter ofseawater, but we know neitherwhat they are nor what they do.

In the poetic conclusion to his1994 autobiography, Naturalist, thegreat sociobiologist and PellegrinoUniversity Professor emeritus E. O.Wilson mused on what he woulddo, “[i]f I could do it all over againand relive my vision in the twenty-first century. I would be a microbialecologist...,” he wrote. “Into thatworld I would go with the aid ofmodern microscopy and molecu-lar analysis. I would cut my waythrough clonal forests sprawledacross grains of sand, travel in animagined submarine through dropsof water proportionately the size oflakes, and track predators and preyin order to discover new life waysand alien food webs. All this, and Ineed venture no farther than tenpaces outside my laboratory build-ing. The jaguars, ants, and orchidswould still occupy distant forests inall their splendor, but now theywould be joined by an even strangerand vastly more complex livingworld virtually without end.”

The Limits of CulturesAs a practical matter, the Mi-crobial Sciences Initiative (MSI)began in 2002 as a grass-roots e≠ortamong faculty members who recog-nized the unexplored ecology andpotential of these organisms andwanted to share information aboutmicrobial research across diversedisciplines: biology, medicine, chem-istry, engineering, geology, astron-omy, and evolutionary and plane-tary history. The group held informal“chalk-talks” weekly, and in 2004staged a day-long symposium withspeakers from around the world.When President Lawrence H. Sum-mers issued a call that year for ini-tiatives that would bring people to-gether from across the science andengineering disciplines, MSI was a perfect candidate, says Cabotprofessor of biology Richard Losick, a member of its steeringcommittee. “I think there are few disciplines that lend them-selves better to cross-disciplinary approaches,” he says, “and few

subjects that have implications across a wider spectrum of sci-ences than is true for microbiology.” As a result, in 2006 MSI re-ceived four years of support, totaling $2.7 million, from theprovost’s o∞ce.

“It kills me that people think only that bacteria are disease-causing,” says Cavanaugh, whostudies the chemosynthetic symbi-otic bacteria that make life possiblefor giant clams and tubewormsdwelling near deep ocean hy-drothermal vents. Far from sun-light, they operate by mechanismsboth similar to and much di≠erentfrom the photosynthetic organismswe see every day. “Although intra-cellular, these bacteria are helpfulto their animal hosts,” she adds.“Like chloroplasts in plants [whichevolved from symbiotic photosyn-thetic bacteria], the chemosyn-

thetic symbionts turn carbon diox-ide into sugars and proteins, feed-ing their hosts internally.”

But most people do associate mi-crobes with disease. “Antibacteri-als” have been incorporated into allkinds of consumer products: soaps,sponges, toilet paper, towels, andcutting boards—even clothing. Kol-ter traces the origins of this “ludi-crous” antimicrobial “scorched-earthpolicy” to the time of Louis Pasteur,

who formulated germ theory, and Robert Koch, who developedmethods for culturing bacteria. “Medical microbiology for al-most 150 years has been driven by the idea that germs are thecausative agents of disease. And there is no doubt that Koch and

1: In terms of gene content, humansand potatoes are more closely related than these two bacteria areto each other—one measure ofbacterial diversity. On the left, Vibrio cholerae; on the right,Mycobacterium tuberculosis.2: From the billions of bacteria in asoil sample, these few cells landedon a nutrient medium where theycould grow and form colonies.“There is so much beauty every-where we look,” says microbiologistRoberto Kolter. 3: A strain ofStreptomyces that produces severalantibiotics widely used in medicineand agriculture. When starved,these bacteria take on a “hairy”appearance as they initiate growthof aerial structures where protectivespores develop. The pigmented areasconsist of small molecules, oftenwith antibiotic properties. 4: In thepresence of the antibiotic strepto-mycin, colorful antibiotic-resistantmutants of Streptomyces coelicolorspring up and form colonies.

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Pasteur were right, that Mycobacterium tuberculosiscauses tuberculosis and Vibrio cholerae causescholera,” says Kolter. But microbes have also ledto most of our antibiotics, a development thatKolter calls “the most important advance inmedical history.”

The formerly limited view of the microbialworld arose from what has turned out to be aninherently constrained approach to the study ofbacteria: the practice of culturing them. Formore than a hundred years, scientists had beenmystified by what was called the “plate countparadox.” Whenever they tried to grow a sam-ple of bacteria from the environment on a nutri-ent medium in a petri dish (an agar plate), only afew microorganisms grew and multiplied toform colonies, when there should have been at aminimum thousands of such colonies (based onthe number of di≠erent species discernible just by lookingthrough a microscope). Various explanations were o≠ered—that99.9 percent of the bacteria in the sample were dead, or that they

must all be the same bacterium because they looked similar.“But then in 1990,” says Kolter, “scientists showed that the

DNA complexity in a typical soil sample meant that there had tobe thousands of times more diversity than was being plated.”Norman Pace, a professor at the University of Colorado, began towonder if scientists simply lacked the basic knowledge neededto grow most of the bacteria on the planet—if perhaps we wereso ignorant of these bacteria that we could not culture them. Hehit on the idea that he could instead analyze a sample of soil orwater for its DNA content in order to ascertain how manyspecies it contained. “Pace went to Yellowstone National Park, tosome of its famous hot springs, where the water was nearly boil-

ing, and collected a sample of sediment,” Kolterexplains. “He extracted the DNA, cloned it, andput it into a little bacterium that he knew howto grow.” Then he sequenced the genes. “In thatone little gram of sediment,” Kolter notes, “Pacediscovered more diversity than we ever knewexisted before, when using our traditional, cen-tury-old techniques for cultivating bacteria.”

Scientists had known that there are more mi-crobes in an ounce of soil than humans alive onEarth, but that was just a measure of abundance.

Pace’s discovery demonstrated something new, a previously un-fathomed repository of biodiversity. Scientists began sequencingDNA from all sorts of environments. After looking at human gut

microflora, they learned that each individual has his or her owncharacteristic set of a thousand species. “These represent threemillion genes that you carry,” points out Kolter, “as compared to theestimated 18,000 genes of the human genome. So you are livingand exchanging [metabolites] constantly with a diverse pool ofsome three million genes.” Microbiologists continue to find newtaxonomic divisions of microbes far faster than they can figureout how to culture them.

The world of animals—from elephants to ants—is divided into13 phyla (vertebrates are one phylum, insects another). In the mi-crobial world, their equivalents are called, for the time being,“deep-rooting branches.” In 1987, 13 of these big divisions were

Plantae

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“Old” World-View of Biodiversity The Three-Domain Tree of Life

BacteriaArchaea

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“We are at the stage of discovery where, everywhere we look, we see new species.”

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Not much can be gleaned about the differencesbetween these two microorganisms just by look-ing at them, but genetic analysis tells us that theyare not even in the same domain. 1: Soil-dwellingBacillus anthracis is classified as a bacterium. 2: Heat-loving Thermoproteus tenax is anarchaean that lives in hot springs.

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The modern “tree oflife,” based on geneticanalysis, shows that the bulk of Earth’s bio-diversity resides amongthe Archaea, Bacteria,and that portion ofthe Eukarya that doesnot include plants, animals, and fungi.

The anthropocentricfive-kingdom systemclassified all unicellularorganisms lacking nuclei(archaea and bacteria)as Monera. The nucleat-ed eukaryotes compris-ing plants, animals, andfungi were thought to represent the bulk of biological diversity. All other nucleatedeukaryotes weregrouped in a grab-bagclassification known as Protista.

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known in the bacterialdomain: Woese sam-pled these to create histree of life. But by 1997,there were almost threetimes as many: 36 in all. “Twelve of them we had never cultivat-ed, and the others webegan to learn how tocultivate,” Kolter ex-plains. “By 2003, therewere 53 divisions, butthe more we discovered,the more we found rep-resentative microbesthat we could not culti-vate. We learned howto cultivate microbesfrom only two of thesein six years and by 2004we had found 80 suchdivisions from which wecouldn’t cultivate evena single representative.” Each of these deep-branching divisions isthought to represent millions, if not hundreds of millions, ofspecies. “That means there are lots of genes out there, and wehave no clue what they are doing,” Kolter says. “So when youthink about biodiversity, and the extent of diversity on the planet,you really get a sense of how little we know about this undiscov-ered world. We are at the stage of discovery where, everywherewe look, we see new species.

“The reason we are so excited about preserving diversity,” hecontinues, “is because that is how we preserve the richness ofthe planet.” Where does that richness lie? Traditionally, taxon-omists 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 morelike mice with wings than like birds. Microbes, on the otherhand, often appear similar to one another to the human eye.Plants, animals, and fungi may seem wildly diverse from a mor-phological perspective, but in fact, Kolter explains, there is “far

less genetic di≠erence be-tween [a human being] anda potato” than there is be-tween, say, “the bacteriumthat causes tuberculosis andthe one that causes cholera.So as magnificent as is thediversity of the tropical rainforest, it pales by comparison tothe microbial world.”

Microbial Medicines In the interests of human health, researchers have begunto hunt among all this newfound microbial diversity for new an-tibiotics. Says Richard Losick, “I have many wonderful colleaguesin the chemistry department, but without question the championsynthetic organic chemists on the planet are the microbes.”

“The majority of our antibiotics and many anticancer com-pounds come from soil-dwelling bacteria,” notes Jon Clardy, a pro-

1: Their cell membranes outlined in red, individualBacillus subtilis bacteriacontain the biosyntheticmachinery—shown as greendots—for making bacillaene,a newly discovered small molecule. 2: Colonies formedby the wild strain of Bacillussubtilis generate complexstructures, as seen at left,while the domesticatedstrain—presumably selected for uniformity bygenerations of researcherswho have used it in laborato-ry experiments for many decades—has lost near-ly all colony architecture. 3:Two colonies of Bacillus sub-tilis growing on top of another bacterium,Streptomyces coelicolor.The Bacillus colonies “talk”to the Streptomyces usingsmall molecules. The strain of Bacillus subtilis at leftmakes compounds for turningproduction of a small pigmented molecule inStreptomyces on and off. But in a mutant strain ofBacillus that cannot produce the “off switch,”Streptomyces produces thepigmented red ring. This ledto the discovery of the molecule that acts as the off switch: bacillaene. Below,the molecule’s chemical structure.

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Methanol, Cheeseburgers, MetalsInterdisciplinary science like that supported by the Microbial Sciences Initia-tive depends heavily on training the next generation of researchers: junior fac-ulty members who use unconventional tools to probe new questions. “The realfuture of the place,” says Fisher professor of natural history Andrew Knoll, “isin these people.” Here are portraits of three such young scientists at Harvard.

Evolution in Action“Nature on the small scale,” says assistant professor of or-ganismic and evolutionary biology Christopher Marx, “is just asinteresting as nature on the savanna that you grew up watchingon TV.” Marx studies evolution in the lab, using bacteria thatgrow on methanol.

With a population of a billion bacteria, one that doubles insize every eight hours, he can perform true experiments in evolu-tion. In contrast, he notes that “even in a herd of 100 cows, inwhich you select, for example, the fattest in each generation,there is such a small amount of genetic variability that you arereally selecting for new combinations of alleles,* and not for newalleles occurring, because the chance of a new, useful gene arisingin 100 cows is relatively low per generation. Here [in the lab], alot happens every day. One out of every thousand bacterial ‘ba-bies’ has a mutation somewhere, which is not much”—but with abillion new bacteria in each generation, that is a million new mu-tations every day. “The genome size of [the bacterium I work on]is about seven million base pairs,” he says, “so in seven genera-tions you will have tested, on average, every single site, so youcan quickly generate many di≠erent variants for selection to actupon.”

“Organisms that eat methanol are sprinkled all over the bacte-rial tree of life,” Marx continues. “But if you look at the genes in-volved, the sequences are in almost all cases highly related.”What has happened is called “horizontal gene transfer,” the ex-change of genetic material between di≠erent kinds of bacteria: inthis case, an entire “cassette” of perhaps a hundred genes thatcontrol metabolism. Marx wants to see what physiological stepstake place after a gene transfer like this, in hope of extrapolatingprinciples that might allow him to predict the probability of anyparticular beneficial mutation taking place before another.

One of the byproducts of metabolizing methanol is formalde-hyde, which kills most living things. Because Marx’s “bugs” neu-tralize formaldehyde by oxidizing it, he decided to see whatwould happen if he swapped the genes that gen-erate their formaldehyde oxidation pathway fora completely di≠erent set of genes from an unre-lated bug. “It is as if you ripped a gasoline engineout of a car and replaced it with an electricmotor,” he says. “This is di≠erent chemistry—di≠erent pathways, and di≠erent intermedi-ates—but it gets you from the same point A topoint B on the metabolic map.” There was noguarantee that the experiment would work—

but luckily, it did. “With this new pathway, these bugs can stillgrow on methanol,” Marx reports. “But what’s most interestingis that they do so three times more slowly and are only 40 per-cent as e∞cient.” But as the bugs evolved using the new pathway,their fitness improved. After 120 generations, fitness had dou-bled. At present, after 600 generations, they reproduce almost asquickly as the original strain does with the original pathway.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 thephysiological changes that led to their improved fitness.

Fueling Deep-sea DiscoveryAssistant professor of organismic and evolutionary biologyPeter Girguis studies “ecological physiology,” the physiology ofgroups of uncultured microbes that live at hydrothermal vents2,500 meters deep in the ocean, where the pressure—about 4,000pounds per square inch—is “the equivalent of a small car beingbalanced on your thumbnail.”

“If you go to the sea floor, you can measure changes in methaneconcentration or butane, or nitrate or sulfate, but we often don’tknow exactly which organisms are responsible,” he says. Becausegroups of microbes living in complex ecologies can be di∞cult tounderstand, Girguis sometimes brings samples back to the lab,where he recreates marine environments with clever engineer-ing: simulating the pumping action of tides on sediments, for ex-ample. Then he perturbs the system—perhaps by adding oxy-gen—to see how the microbes respond. “We are focusing mainlyon natural perturbations,” he says, “but anthropogenic ones areequally important.” He notes that there has been a lot of talkabout managing the greenhouse-gas problem by sequesteringcarbon in the deep ocean (by capturing carbon-dioxide emis-sions from fossil-fuel-burning power plants and storing them inliquid form atop the ocean floor 3,000 meters or more below thesurface, where the waste could remain for millions of years; see“Fueling Our Future,” May-June 2006, page 40). “That makes menervous,” he says, “because I think the response of the microbesmay be quite di≠erent from what we assume.”

Many of Girguis’s experiments take place in the field, but run-ning the necessary equipment in the deep ocean, with its highpressure and extreme cold, can be very expensive. It takes 125size “D” alkaline batteries to provide power for a year to a half-watt device like a bike lamp, so Girguis has enlisted the aid ofmicrobes to solve his on-site fuel problem. A microbial fuel cell isless a≠ected by the temperature and pressure, and can run as

long as 15 years without needing a“charge” of fresh organic matter.(Girguis runs a pair of demonstra-tion cells in his o∞ce on cow ma-nure.)

He compares what the microbesare doing—generating electric-ity—to what we do when we eat a


At the bottom of the sea, life for these giant tubeworms ismade possible by symbiotic bacteria that convert chemicalsejected from hydrothermal vents into food, in a processcalled chemosynthesis.

______________*Alleles are gene variants in a particular location on achromosome that may, for example, cause brown eyes, be-cause they are coded by the dominant allele of the genefor eye color, rather than blue eyes, which are coded bythe recessive version.

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cheeseburger. “All life on Earth,” he explains, “is pretty muchabout moving electrons.” When you eat, “what you are doing istaking all the electrons that are tied up in that organic matter—in the burger and the cheeseand the bun and the secretsauce—and moving thoseelectrons through your respi-ratory pathways,” harnessingtheir energy in the process.“Then you are going to dumpthe electrons onto oxygen—you need that oxygen becauseit 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 dumpthe electrons onto sulfates, nitrates, and even solids. Andthis is where it gets crazy. They are capable of shuttlingelectrons from reduced organic matter through their bio-chemical pathways, generating energy, and then stickingthose electrons onto a chemical that they shuttle outside theirbodies and then dumping [the ‘shuttle,’ thereby depositing] theelectrons onto iron or another solid-phase oxidant.” Some mi-crobes don’t need the chemical shuttle at all, he reports: “Theyjust dump their electrons through little conductive nanowires.That is like you eating a cheeseburger and being able to respire itwithout oxygen. It is like holding your breath and dumping yourelectrons onto my filing cabinet. That is the premise behind mi-crobial fuel cells. You can set up a microbial fuel cell, encouragethe growth of microbes that can shuttle electrons and breakdown the organic matter in an environment, and then have themdump those electrons onto the fuel cell and harness that energy.”

Girguis’s fuel cells generate enough electricity to run a lightbulb, power a wireless network, or charge a cell phone. His aspi-ration to power lights in the developing world has led to a col-laboration with two Harvard Business School graduates whohope to bring a product to market. Says Girguis with a grin, “Mi-crobes really run everything.”

The Clean-up ConundrumOne of the things that attracted Colleen Hansel to Harvardlast January was the energy she saw going into the Microbial Sci-ences Initiative. An assistant professor of environmental micro-biology in the School of Engineering and Applied Sciences, shestudies microbial interactions with metals. The work is impor-tant for cleaning up toxic waste, for improving agricultural prac-tices and human health, and even for understanding planetaryhistory.

Hansel has worked on cleaning up sites contaminated withmetals such as arsenic, chromium, and technetium. More andmore research, she says, is showing “an interesting correlation”between antibiotic resistance and microbial resistance to metals.Microbes that have developed genes enabling them to resist an-tibiotics can withstand higher metal concentrations and there-fore may no longer detoxify them. Such resistance is growingrapidly, because antibiotics are becoming common in the envi-ronment, streaming from animal feed lots and even householdsewage. They can even arise spontaneously as a response totoxic-waste cleanup e≠orts.

“The strategy we use to clean up a site,”Hansel explains, is to provide additional foodfor the microbes that are capable of immobi-lizing metals. “That,” she says, “will stimu-late the microbes to increase their metabo-

lism and clean up the soils and sediments.” Clean-up crewspump a carbon compound such as glucose or lactate into the soil.The organisms respond by achieving “higher densities and highergrowth yields,” she says. But they also “start producing antibi-otics, because they want to kill o≠ their competitors.” Stimulat-ing the production of antibiotics has the unfortunate conse-quence of providing selective pressure for antibiotic-resistantgenes, which move between microorganisms much faster thanother kinds of genes.

“Are we doing some harm when we are trying to do somegood?” she wonders. Microbes reduce or oxidize metals in orderto gain energy or detoxify themselves, she explains. But if the mi-crobes become resistant to the metals, as they do when they ac-quire antibiotic resistance genes, they no longer do this usefulwork. Meanwhile, they are also introducing many more antibi-otic-resistant genes (potentially of danger to humans) into theenvironment.

Hansel’s research into the oxidation of iron and manganese(primarily for use in cleaning contaminated sites) may also helpanswer geological questions. Banded iron formations—sedimen-tary deposits of rock billions of years old in which the iron andmanganese layers have been oxidized—are often cited as evi-dence of oxygen in earth’s early atmosphere. “But the big ques-tion,” says Hansel, “is whether or not oxygen is needed to formthese iron oxides. Is the process abiotic, or were microbes in-volved?”

Unlike iron, manganese is very hard to oxidize, she says. Butcertain organisms that she has isolated from contaminated sitesproduce a molecule during their growth that oxidizes the metalwhen exposed to light. “The bacterium is not oxidizing themetal itself,” she emphasizes, it is just producing a photoreactiveorganic molecule. “Could there have been organics present twobillion years ago, during the Pre-Cambrian when the bandediron formations appeared, that were photoexcited and oxidizedmanganese?” It is the sort of exciting question—bearing on theorigins of life and the creation of the biosphere—that she hopesto explore with her new colleagues. But she acknowledges, “Ifind microbes are humbling, too. The more you understand, themore you realize just how diverse they are.”

Harvard Magazine 49

These 2.5 billion-year-old sedimentary rocks in KarijiniNational Park, northwesternAustralia, contain layers ofiron that has been oxidized.Such “banded iron formations” may allow geologists to determine whenoxygen first appeared inEarth’s atmosphere.

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fessor of biological chemistryand molecular pharmacologyat HMS. Unfortunately, hesays, due to the spread of an-tibiotic resistance, “we arerunning out of e≠ective an-tibiotics.” For economic rea-

sons, pharmaceutical companies are not investing as much as theyused to in the development of new ones, which has left physicianslooking for an antibiotic of last resort to be held in reserve for theone patient once a year who has a resistant strain of tuberculosis orpneumonia, explains Kolter. “That drug would be the billion-dollarblockbuster that nobody buys, because doctors shouldn’t be pre-scribing it widely. Nobody in the corporate world will develop it.”He believes that “there may be a role for the University in researchthat is not going to be as profitable for corporations to pursue: thedevelopment of targeted, ecologically sound antibiotics.”

Whole-genome studies of microbes suggest that only a smallfraction of the natural products that come from even well-

known bacteria have been discovered. A prime example is Strep-tomyces avermitilis, the bacterium from which researchers derived adrug called Ivermectin. “Ivermectin,” says Clardy, “is grown onthe ton scale because it is used against river blindness in Africa,for treating almond trees in California, and for getting rid of allkinds of parasites” (equine worms, for example). “Here you havea ‘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 ismade, but you could also see [other gene sequences]—over 30 ofthese clusters—that it seems should each make a small molecule.We know only three molecules that come from that bug,” headds—one of them the source of Ivermectin. “That means weknow only 10 percent of what it can potentially make.”

But probing those secrets is far from easy. When grown in alab in pure culture, microbes apparently don’t need to activate alltheir genetic machinery to survive. In their natural setting, bycontrast, microbes live in a complex ecology: they interact withtheir environment and with other microbes by using a vast arrayof virtually unknown small-molecule products. These organiccompounds often play multiple roles: a small molecule used forsignaling among bacteria engaged in mutually beneficial metabo-lite exchange (one microbe’s metabolic waste is another’s meal)might also be used to kill competitors trying to gain a foothold inthe same ecological niche. Such compounds, if researchers couldidentify them, produce them, and figure out how they work,might form the next generation of medical antibiotics.

At Harvard, an MSI-facilitated collaboration between Kolterand Clardy uses creative methods for prompting even uncultur-able microbes to yield such genetic secrets. Clardy was amongthe earliest researchers to use “metagenomics,” which involvessampling the environment—your gut, a lake, or in his case, thesoil—and collecting the “metagenome,” or composite genome, ofall the individual organisms dwelling there. After extracting themany strands of DNA, Clardy screens for sequences that makecompounds. He can isolate these sequences and transfer them

Glossary for an Invisible WorldArchaea: One of three major domains, or classifications, of life,archaea are morphologically similar to bacteria, but have adi≠erent molecular transcription machinery, indicating an an-cient evolutionary divergence.Bacteria: One of three major domains of life. Bacteria are single-celled microorganisms with neither a nucleus nor organelles(specialized intracellular structures; see below).Cyanobacteria: A division of bacteria that produce their energythrough photosynthesis. Formerly called blue-green algae(hence the name “cyano,” or blue, bacteria), they are not “true”algae. Algae are eukaryotes, while the cyanobacteria areprokaryotes.Eukarya: One of three major domains of life, the eukarya includeall single and multicellular organisms in which the cells have adistinct nucleus. Examples are plants, animals, and fungi. Theclassification includes everything that is not a bacterium or anarchaean.Microbe: Generally considered to include all life that cannot beseen with the unaided eye, microbes are found in all three

major divisions of life and include all the bacteria, all the ar-chaea, and some of the eukarya. Some scientists say that onlyprokaryotes (see below) should be considered microbes,thereby excluding even single-celled fungi. Others argue thateven multicellular organisms can be considered microbes.Mushrooms, for example, are the multicellular fruiting bodiesof fungi that may start as unicellular organisms in the soil. Fur-thermore, both unicellular bacteria and archaea form aggre-gates (“colonies”) on surfaces, often referred to as biofilms, thatexhibit distinctive multicellular patterning.Organelles: Structures within a cell that perform specializedfunctions, such as energy production. Mitochondria andchloroplasts are examples of energy-producing organelles thatare thought to have evolved from autonomous bacteria. Eventu-ally they became dependent on their hosts—and vice versa. Primary production: Production is the creation of new organicmatter. Primary production refers to the creation of new or-ganic matter by photosynthetic microbes.Prokaryotes: An inclusive term that refers to the bacteria and ar-chaea, single-celled organisms that, as distinct from eukary-otes, lack a nucleus.

1: The double membranesof Gemmata obscuriglobusmay be durable enough to produce “molecular fossils”: traces of early lifeon Earth that have beenpreserved for billions ofyears. 2: Bacillus subtilisforms spores (in green)with lipid membranes thathelp protect the spore fromoxygen damage. Thesetough cellular membranesappear in the fossil recordat about same time Earth’s atmosphere becameenriched with oxygen, and may help trace changes in the planet’s early atmosphere.

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into E. coli (or another “tame” bacterium that heknows how to cultivate) in order to get them toexpress the target compounds. Kolter, meanwhile,has been creating controlled ecological settings,exploring whether certain bacteria behave di≠er-ently in the context of other species, as they do intheir natural environment. He has given Clardy atool that indicates when a bug responds to a mi-crobial signal, and Clardy has developed othertools for figuring out what that signaling com-pound is. These organic molecules, often antibi-otic, frequently “turn out to be something newthat nobody has ever seen before,” says Kolter,“often with properties that are chemically very in-teresting.”

To show that their method works in principle, the team in 2006published an early discovery using the bacterium Bacillus subtilis,which has been studied for more than 100 years. The compoundthey identified, bacillaene, may or may not turn out to be useful inmedicine, they caution. But it is a highly complex, resource-inten-sive structure for any single-celled organism to make, and thatsuggests that it plays an important biological role. Two new MSIpostdoctoral fellows arriving at the medical school this year willuse the collaborative techniques Kolter and Clardy have developedto facilitate further discoveries. They will enter a universe of seem-ingly endless therapeutic possibility, between the small-moleculeproducts of bacteria yet undiscovered, and the 90 percent of un-tapped coding sequences from the bugs we thought we knew.

Bacterial Biomarkers of Ancient Earth Within FAS, Ann Pearson, Cabot associate professor of earth andplanetary sciences, also became interested in bacteria for whatthey might reveal about Earth’s early history. Pearson, a biogeo-chemist, has collaborated with Richard Losick, who during the

last 35 years has eluci-dated in great detailthe molecular mecha-nisms that control lifecycle and di≠erentia-tion in the bacteriumBacillus subtilis. She “does wonderful research,” Losick says, “on [theorigins of] biological molecules that can be recovered from hun-dreds of millions, if not billions, of years ago, reflecting the earliestevidence for life on Earth.”

“If you have very highly preserved, organic-rich, sedimentaryrocks that haven’t been deeply buried and heated in their geo-logic history,” Pearson says, “you can extract measurableamounts of lipids or fats that make up some cell membranes. Wecall these ‘molecular fossils’ because they have structure, theycan be identified, and occasionally you can tell what type of or-ganism they might have come from—for example, there are cer-tain molecules that are produced only by archaea.”

“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.

1: Roseobacteria, which form pinkish colonies, aid in the forma-tion of clouds by producing gasesthat nucleate water droplets. 2: Bacteria fertilize plants throughthe formation of specialized symbiotic “tissues” on the roots,known as “root nodules.” Bacteriain these nodules form “factories”able to capture atmospheric nitrogen gas and convert it intoammonia-containing compoundsthat the plants can use for growth.By themselves, plants cannot effectthis transformation, which is why they need “fixed” nitrogen(not gas) as fertilizer.

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something from similar molecules found in contemporary mi-crobes.” It turned out that the bacterium that Losick’s lab studieshas genes similar to those known to produce a certain type ofthese molecular fossils. Their shared MSI-sponsored postdoctoralfellow, Tanja Bosak (now an assistant professor at MIT), learnedthe microbial genetics techniques Losick’s lab uses and obtainedevidence that in the contemporary bacterium, these moleculeshelp to protect its spores from damage by oxygen. (Although manylife forms need oxygen, it can also cause a lot of damage.) Bacillussubtilis, Losick explains, creates a “particularly macho type ofspore, maybe the most sturdy kind of dormant cell on the planet.It can survive extremes of time and temperature and radiation.”What might this molecule have been doing two billion years ago?“The exciting implication,” Losick replies, “is that these moleculesmay have appeared as a response to the rise of oxygen in the at-mosphere, so the timing of their appearance in the rock recordmay bear on the issue of when oxygen levels first rose.”

“The planet is about 4.5 billion years old,” elaborates another ofPearson’s collaborators, paleontologist Andrew Knoll, Fisher pro-fessor of natural history and professor of earth and planetary sci-ences. “The oldest rocks we can look at are 3.8 billion years old.”Chemical evidence suggests that life was already present then,but certainly “by 3.5 billion years ago there were active microbialcommunities on the earth’s surface.” These microbes were a littledi≠erent from those we know today because there was still nooxygen. (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 surfaceyou are down to anaerobic microbialecosystems, which remain critical for mostof the biologically important element cy-cles on the earth’s surface.”) By 2.4 billionyears ago, the chemistry of sedimentaryrocks suggests that there was “at least a lit-tle bit of oxygen in the atmosphere andsurface ocean.” Then, “for a period of abouteighteen hundred million years, oxygenlevels hovered at maybe a couple of percentof today’s levels. Only in the last 600 mil-lion years or so do we have environmentswith enough oxygen to support the biol-ogy of large animals, and only in that inter-val do large animals actually appear,” Knollsays. That is also when the algae first be-come ecologically important, joining andeventually displacing photosynthetic bac-teria as the basis of early food chains in theworld’s oceans. “So there is a time coinci-dence,” he says, “between major events inthe history of life and major events in thehistory of the planet.” Following thosethreads might also suggest how life could

arise, and what it might look like, elsewhere in the universe.“Everybody is interested in finding the perfect molecule to

trace cyanobacteria,” Pearson reports, in order to trace the ori-gins of oxygenic primary production (cyanobacteria use a type ofphotosynthesis that releases oxygen, and this process is respon-sible for our oxygen-rich atmosphere). More broadly, she hopesto “figure out how to relate the incredible diversity of microbes

Moving Microbial Science ForwardToday, operating out of borrowed space in Har-vard’s Center for the Environment, the Microbial SciencesInitiative (www.msi.harvard.edu) funds six postdoctoralfellows who typically bridge two labs in di≠erent depart-ments, often across schools; sponsors 12 undergraduatesummer fellowships; arranges monthly seminars thatbring in speakers from around the world; and hosts an an-nual symposium. This past summer, MSI held a workshopfor high-school teachers designed to help them incorpo-rate microbial education into their curricula. In the cur-rent academic year, the initiative will introduce two newundergraduate courses: Life Sciences 190hf, “Diverse Mi-crobial Strategies for Metabolism, Pathogenesis andChemical Signaling,” taught by Harvard Medical School(HMS) professor of genetics Gary Ruvkun, and Life Sci-ences 110, “A Microbial World,” designed for studentspursuing microbial science as a secondary field and co-taught by professor of microbiology and molecular genet-ics Roberto Kolter and professor of biological chemistryand molecular pharmacology Jon Clardy, both of HMS,and Cabot associate professor of earth and planetary sci-ences Ann Pearson, a biogeochemist in the Faculty of Artsand Sciences.

1: These cave mineral deposits were difficult to explain throughpurely geologic processes. Spelunker scientists in the 1970s discovered that microbes play a role in their formation. 2: The recognition that microbes can respire insoluble metals hasled to a complete reassessment of processes, such as rusting, thatused to be considered largely abiotic (nonliving) and are nowknown to have a microbial component. Scientists refer to this as“microbial-assisted corrosion.”

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that are out there to the kinds of preservable or-ganic molecules—or biomarkers—that theymake...and that we can find in the sedimentaryrecord.” Because individual species of microbes aremutable (“They evolve quickly and we have noidea if there is phylogenetic integrity over billionsof years”), she is trying to relate particular pre-served molecules to their functions in the environ-ment, rather than to species, in order to try to getan idea of what the ecosystem looked like as lifeevolved on Earth and, in turn, shaped Earth’s envi-ronment—oxygenating the atmosphere, detoxify-ing the oceans, breaking down rock and organic matter to formsoils. “MSI has been absolutely essential to building the bridgesthat I have needed to other research groups,” Pearson says. “Al-though my questions are geology- and earth-history-driven, thework that I do in order to answer those questions is all biologyand chemistry. I don’t practice field geology,” she says, “so build-ing bridges with other parts of the University has been critical.”

Historically, says Knoll, “there have been a few of us in FASwho have been interested in microorganisms, but there wasn’tanything as tangible as a Museum of Comparative Zoology,which has provided a focal point for people interested in ani-mals for 150 years, [or] the Herbarium, which has done the samething for people interested in plants” (which Knoll calls, half-joking, “green algae with a graduate degree”). He continues, “Be-cause of Harvard’s good fortune in the nineteenth and earlytwentieth centuries, we built up these really quite remarkablefacilities for studying plants and animals...and, not surprisingly,we then populated our faculty with people who study [them].

These areas are not unimportant now,” he explains, “but somany horizons have opened up for studying microorganismsthat the simple identification through the MSI of a communityof people concerned about microbiology has been tremendouslyimportant. Many of the basic biological processes that underliethe nature of changes in the environment are microbial, so in aworld where the environment may be changing faster than ourability to understand it, having a better process-oriented idea ofwhat microbial communities are actually doing in nature has

practical impor-tance as well.”

For his part,geochemist DanielSchrag, professorof earth and plan-etary sciences and a member of the MSI steering committee, empha-sizes that “almost all the reactions on the earth’s surface are catalyzedby microbes—in soils, in waters, in swamps. If you don’t know whatmicrobes are doing, you don’t really know what is going on.”

“It is clear that this field will not reach its maturity, in thesense that it becomes less exciting, during my research lifetimeor probably the research lifetimes of my students,” Knoll says.“We have whole new horizons in the nature and treatment ofdisease; whole new horizons in simply understanding the diver-sity of life as it actually exists—not what we thought existed be-cause we could see it; whole new horizons in the nature of therelationship between organisms and the environment both

today and in the future, and on the timescale of the whole devel-opment of the planet. These are wonderfully large, excitingquestions and MSI gives us a forum to discuss them. You dofind, every once in a while, someone who has actually thoughtabout the same problem in a very di≠erent way”—and that canbe the most important sort of catalyst: the kind that leads tonew discoveries.

Jonathan Shaw ’89 is managing editor of this magazine.

“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.”

1: Photosynthetic cyanobacte-ria form colorful mats in thewarm springs at YellowstoneNational Park, a place thatplays a central role in microbial-diversity studies. 2: Other kinds of “extremo-phile” microbes can live without light or oxygen nearhydrothermal vent chimneys.The ecosystems around thesevents are entirely supportedby both the free-living and thesymbiotic bacteria.

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Undiscovered Planet

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