Focus: Missing Pieces

24 Harvard Science Review • fall 2009

By Jue Wang

biology’s first and final frontier

In 1676, Antonie van Leeuwenhoek looked through the lens of a home-

made microscope and became the first human to lay eyes on a live microor-ganism. With a penchant for making detailed and colorful descriptions of the tiny rod-like creatures tumbling through water (1), he launched the field of microbiology and effectively paved the way for everything from cell theory to the techniques that revealed the structure of DNA.

An Obselete Science?Now, three centuries after Leeuwen-

hoek, you might expect bacteria and other microbes to have exhausted their store of secrets. After all, cutting-edge biology in the late 20th century has mostly focused on higher organisms like mice and monkeys, using sophis-ticated molecular techniques that are a far cry from streaking out dirt on a Petri dish and waiting blindly to see what grew.

A glance, at the news, however, would

quickly assure you that the microbial world is as strange as ever and still full of surprises. In the year 2008 alone, researchers reported the first observa-tions of virophages (2)—viruses that infect other viruses—and of super-re-sistant bacteria that actually metabolize the antibiotics designed to kill them (3). In the last decade, molecular genomics techniques have discovered millions of previously undiscovered microbes in nature (4), and high-throughput cul-turing methods have revealed blatantly unintuitive—yet completely logical—features of bacterial evolution in the presence of antibiotics (5). Instead of becoming irrelevant over time, the study of microorganisms has become a focal point for basic questions about evolution and ecology. And if viruses are considered a special class of mi-crobes—as they sometimes are—then questions in microbiology begin to encompass the debate over the defini-tion of life itself.

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The Microbial World

“Instead of becoming irrelevant over

time, the study of microorganisms has

become a focal point for basic questions about evolution and

ecology.”

ABOVE: Colonies of soil bacteria, viewed by a phase-contrast mi-croscope. The different growth morphologies visible here is just a hint of the sheer variety of different microbes living in the soil.

Focus: Missing Pieces

fall 2009 • Harvard Science Review 25

to be such a fertile source of scientific insight, despite having been studied for so long? Their diversity is part of the answer; there are more microbial spe-cies, living in more varied habitats, than any other form of life on the planet. Another reason is their simplicity. For scientists trying to study the evolution and behavior of living things residing together in complex systems, having well-characterized and easy-to-maintain model systems like E. coli is a major saving grace. This is why modern microbiology is a smorgasbord of discoveries spanning many sub-fields, and influencing many more. Here are a few of the tastiest and most recent morsels, along with their broader bio-logical implications.

The Great Plate Count AnomalyOne of the most important open

questions in microbiology today is ironically also one of the oldest. In 1918, while comparing the number of microbial colonies cultured on agar plates with the number of cells from the same sample that could be counted under the microscope, Conn (6) noticed that the microscopic count was 10 to 70 times greater than the plate count. In other words, only one in ten bacterial cells was able to grow on the nutrients supplied by laboratory growth media. Recent estimates of this ratio, using the latest fluorescent stains, show an even larger gap: only one in ten thousand soil bacteria form colonies on a Petri dish (7).

The “Great Plate Count Anomaly,” as the discrepancy came to be known among microbial ecologists (8), would not be half as “great” if the few cells that did grow on a plate were fair rep-resentatives of their more stubborn and numerous cousins in the soil. However, this idea is false. Direct sequencing of DNA from the soil has found evidence that entire classes of microbes have eluded the myopic reach of traditional culture-based methods (9). Given that just one bacterial genus, Streptomyces, was enough to provide us with more

than half of the naturally occurring antibiotics used in medicine, what un-told riches await us in that uncultured ninety-nine percent? More importantly, what is the best way to study these or-ganisms, and to put our knowledge of them to use?

An experimental approach called metagenomics has provided a partial answer to the problem of studying unculturable microbes. By collecting and sequencing DNA directly from soil or seawater, microbiologists can bypass the traditional step of plating for colonies. Whereas Petri culture, by necessity, studies only microbes that grow to colonies of millions of cells, metagenomics can detect DNA from strains that are represented by just a few cells in a habitat (4). This provides the perfect tool for sampling microbial diversity. One massive metagenomic study of the Sargasso sea has turned up 148 previously unknown micro-bial strains and 1.2 million new genes, including 782 genes—10 times more than the currently known number—for rhodopsin-like photoreceptors, an evolutionary precursor to the proteins responsible for our sense of sight (10). Another study discovered several new genes for resistance to aminoglycoside antibiotics, out of a soil DNA library of 4 Gb (four billion base pairs, or cr

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ABOVE: Seawater stained with SYBR Green dye to reveal bacterial cells and viruses. Less than one percent of these cells are currently known to produce colonies in the lab.

Given that just one bacterial genus,

Streptomyces, was enough to provide us

with more than half of the naturally occurring

antibiotics used in medicine, what untold riches await us in that uncultured ninety-nine

percent?

Focus: Missing Pieces

26 Harvard Science Review • fall 2009

about one million genes) (11). This is an enormous amount of informa-tion, and would be all but impossible to access without the high-throughput sequencing and analysis methods of modern biology.

Of course, the sequence information collected by these methods is not from a single species, but a community of many different organisms—hence the “meta” in metagenomics. If the goal is to learn more about how mi-crobes g row and interact at the individual level, in other words, about microbial phenotypes, then something more—or perhaps less—than DNA sequences is needed.

What do antibiotics really do?Fortunately, the magic technique

for studying microbial phenotypes has already been invented—300 years ago, in fact. Using the time-tested method of agar culture and adding a modern twist here and there, researchers have continued to make an astonishing array of discoveries. One particularly fruitful field of recent research has been the study of antibiotic resistance.

Antibiotics, roughly defined as any chemical that can kill or stop the growth of microbes, have been an important weapon in the fight against infectious disease. Unfortunately there are an increasing number of microbial strains that seem resistant to these drugs. In some cases, it might seem that our use of antibiotics as a “weapon” is downright backwards. One group of researchers, while looking through the soil in cornfields, uncovered bacteria that happily grow at 50 times the con-centration of antibiotics that kills most other bugs (3)! To add insult to injury, these “super-resistant” bacteria actually seemed to metabolize the drug mol-ecules to obtain useful carbon atoms. Imagine a person being fed poison pills, and instead of getting sick or dying, he

hungrily wolfs down the pills and comes back stronger the next day!

Antibiotic resistance also helps us make sense of the way bacteria evolve in response to their environment. In a deceptively simple experiment, re-searchers at Harvard Medical School mixed together two different strains of E. coli, one resistant and one sensitive to an antibiotic called doxycycline, and cultured them both in growth media

containing the drug (5). Over time, the doxycycline killed the sensitive bacteria but left the resistant ones to repopulate the culture. No surprises so far—this is a textbook example of natural se-lection. It is the same principle that explains drug resistance in HIV and cancer, and the reason that hospital operating rooms—where antibiotics are used constantly—are especially vulner-able to colonization by drug-resistant bacteria (citation).

Next, the researchers did what hospi-tals do to combat resistance—use com-binations of antibiotics. For example, it is known that doxycycline and another antibiotic called erythromycin interact synergistically, meaning that the com-bined antibacterial power of the two drugs is greater than the sum of their effects acting alone. In the synergistic environment, the doxycycline-resistant bacteria had an even bigger relative ad-vantage than in the single-drug environ-ment, although in absolute terms, both strains grew more slowly due to the presence of erythromycin (citation).

The surprise came when a different drug combination, doxycycline and cip-rofloxacin, was used. Here, instead of synergy, ciprofloxacin behaves chemi-cally in a way that causes suppression—it partially cancels out the effects of doxycycline and weakens the drug combination. Could this effect also

cancel out the evolutionary advantage enjoyed by resistant bacteria? Surely enough, when the growth experiment was done, it was found that certain con-centrations of the two drugs actually favored the sensitive bacteria over the resistant ones—all while still inhibiting the growth of both! This suggests that suppressive drug combinations, which have long been ignored clinically be-cause of their apparent weakness, might

in fact have long term benefits in selecting against the evolution of resistance (cita-tion).

The discovery of antibiotic-eating bacteria reveals a natural world that almost seems to delight in being capricious, unintui-tive, and paradoxical. But as the drug combination experiments show, the strangest finding is often the most com-pelling testimony to the unimpeachable logic of natural selection. After all, the word “antibiotic” is nothing but a name given to chemicals that happen to be useful to humans who need to kill microbes. But who knows if this is the chemicals’ most important feature for their original makers? Recent evidence even suggests that, through millions of years of evolution, chemicals that we call antibiotics may have evolved to fill a rather modest role in the soil—as signaling compounds, subtly modulat-ing the levels of gene expression in microbial communities (12). It is not a stretch to think that antibiotics could be useful as a food source for bacteria as well. We are used to thinking of antibiotics as chemical weapons used by microbes locked against one another in a battle for survival. As in any kind of battle, however, it seems that killing your opponent is not the only way to come out on top.

A mama of a virusPerhaps nowhere is the contrast be-

tween intuitively shocking and deeply unsurprising more pronounced than in

After all, the word “antibiotic” is nothing but a name given to chemicals that happen to be useful to humans...who

knows if this is the chemicals’ most important feature for their original makers?

Focus: Missing Pieces

fall 2009 • Harvard Science Review 27

dustry to produce useful chemicals, and in medicine, where they serve both as pathogens to be fought as well as sources of antibiotics for fighting them. In the search for alternative sources of energy, microbes are spearheading the way in the industrial conversion of plant matter to ethanol (14). Bacteria might even become a direct source of electrical power, if fuel cells can be designed that take full advantage of the specialized electron-transport mechanisms built into strains such as Shewanella and Clostridium (15).

The study of microbes launched the revolution of modern biology, and now it is unlocking yet more fascinating fields of research. From the deepest puzzles in evolution to the most per-tinent challenges in public health and medicine, microbiology has much to offer, and much more potential to be tapped. It is doubtful that Leeuwen-hoek would even recognize the field today as the one that he set in motion 300 years ago, but he would surely find it every bit as fascinating as the first time he peered across the threshold of his microscope and saw a mysterious world brimming with possibilities.

References1. W. Bulloch, The History of Bacteriology. (Oxford University Press, New York, N.Y., 1938).2. B. L. Scola et al., Nature 455, 100 (2008).3. G. Dantas, M. O. A. Sommer, R. D. Oluwasegun, G. M. Church, Science 320, 100 (2008).4. J. Handelsman, Microbiology and Molecular Biology Reviews 68, 669 (2004).5. R. Chait, A. Craney, R. Kishony, Nature 446, 668 (2007).6. H. J. Conn, N.Y. Agr. Exp. Sta. Tech. Bull. 64, 3 (1918).7. V. Torsvik, L. Øvreås, Current Opinion in Microbiol-ogy 5, 240 (2002).8. J. T. Staley, A. Konopka, Annula Review of Micro-biology 29, 321 (1985).9. P. H. Janssen, Applied Environmental Microbiology 72, 1719 (2006).10. J. C. Venter, K. Remington, J. F. Heidelberg, A. L. Halpern, e. al., Science 304, 66 (2004).11. C. S. Riesenfeld, R. M. Goodman, J. Handelsman, Environmental Microbiology 6, 981 (2004).12. J. F. Linares, I. Gustafsson, F. Baquero, J. L. Martinez, PNAS 103, 19484 (2006).13. C. M. Fauquet, M. A. Mayo, J. Maniloff, in Virus Taxonomy (Eighth Report of the International Com-mittee on Taxonomy of Viruses), U. Desselberger, L. A. Ball, Eds. (Elsevier, London, 2005), pp. 1163–1169.14. T. W. Jeffries, Y.-S. Jin, Applied Microbiology and Biotechnology 63, 495 (2004).15. D. R. Lovley, Nature Reviews 4, 497 (2006).

—Jue Wang ‘09 is a Chemical and Physical Biology concentrator in Mather House.

the field of virology. Viruses are small pieces of protein-coated genetic infor-mation, in the form of either DNA or RNA. They are not strictly considered alive, if a living organism is defined as something that can independently propagate itself. However, viruses have an undeniably life-like ability. They reproduce and spread by hijacking the biochemical machinery of other organisms, often causing disease and cell death along the way.

Now we know that even viruses themselves are not immune to this take-over. In 2006, researchers studying wild bacterial isolates reported what they believed was a new strain of a particu-larly small and simple bacterium from the surface of a water-cooling tower. It was not until a year later that another group of scientists realized that these tiny things weren’t bacteria after all, but rather, a very large virus that specializes in infecting amoeba (2).

These giant viruses, which come in two flavors named “mimivirus” and the larger “mamavirus,” were unprec-edented and by themselves already

constituted an astonishing discovery. Imagine the shock, then, when the researchers discovered something even smaller growing inside the ma-maviruses, and affecting their ability to reproduce. These tiny particles, which were co-opting the mamaviruses (which in turn, lest we forget, lived inside the amoebas), were named “virophages” after their similarity to bacteria-eating viruses called phages. In fact, the vi-rophages bore a striking resemblance to another class of microscopic particles called satellites, virus-like objects which have been implicated in certain plant diseases (13). The biology of satel-lites, which are simpler than viruses but more complicated than self-replicating proteins called prions, provided us with a tantalizing clue as to what the earliest forms of life might have looked like.

The microscopic futureIn describing interesting basic discov-

eries, this article has not even touched on all the ways that applications of microbiology have shaped our lives. Microbes have long been used in in- cr

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ABOVE: An electron micrograph of a mimivirus, a smaller cousin of the mamavirus. The mimivirus was first discovered in 1992, but was mistakenly thought to be a bacterium until 2003.

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