Tiny Wars-1

Tiny Wars We Barely Understand

November 2, 2012 | by Karl Leif Bates

Microbes are ancient creatures doing some of the world’s oldest jobs. Making oxygen, for example. But little is known about these microscopic life forms that battle each other constantly and evolve at breakneck speeds. Two LSA professors have braved the bottoms of icy lakes and the insides of sweltering caves to study the teeniest building blocks comprising life on Earth.

Two miles out and 70 feet down there’s a spot on the bottom of Lake Huron covered with an undulant rubbery skin of royal purple. It’s a Dr. Seuss-ian landscape, punctuated by little patches of something else that’s white.

Then, as the sun sets behind Alpena and the last of the dappled light fades from this submarine garden, one of the world’s most ancient battles begins in earnest. The white patches start to expand, spreading outward over the top of the purple skin. Within a few hours, the bottom will appear white with purple patches. And at dawn, when sunlight arrives, the battle will reverse.

Despite its resemblance to blotches of spilled latex paint, this carpet is a living mat made of vast, uncounted armies of cyanobacteria, single-celled microbes that thrive around what’s known as the Middle Island Sinkhole. The mat is a community of closely related species, doing subtly different jobs and communicating with each other. The purple and the white are each a sort of superorganism, allowing the individual bacteria within to communicate and coordinate their actions in the competition for resources. They’re  here because of groundwater that seeps   into the lake from the earth below, rich in sulfate and low in oxygen. The purple mat harvests sunlight by photosynthesis and exhales oxygen; the white community of microbes, called chemosynthesizers, eat hydrogen sulfide contentedly in total darkness, and burp sulfur.

Though their charisma falls far short of the tropical rainforest, bacterial communities like this have at least as much claim to being the lungs of the planet, performing up to a third of the planet’s oxygen-producing photosynthesis. (Free-floating microbes on the surface of lakes and oceans are so numerous they can be seen from space and may outnumber the grains of sand on all the world’s beaches by at least a factor of ten.) Life on Earth isn’t so much about soaring raptors and endless herds of grass-eaters on the savannah; it’s really about the microbes. These simple creatures have been doing this work for three and a half billion years, long before the first plants. Lake Huron’s purple mat is believed to be a close approximation of the creatures that first created oxygen on Earth.

The paradox of the microbes is that they’re both very ancient and very new, capable of evolving at unimaginable rates with a new generation every 18 hours. But despite being literally everywhere, they are so mind-bogglingly diverse—and so tiny—that they’ve been very hard to study.

“We know next to nothing about them,” says Gregory Dick, an LSA assistant professor of Earth and environmental sciences and of ecology and evolutionary biology, who studies the bugs in Lake Huron. When researchers run these simple organisms through the latest DNA sequencing machines to see what they’re made of, “30 or 40 percent of the genes we see are totally new to us.”

In the cold depths of Lake Huron at the Middle Island Sinkhole, a location low in oxygen and high in sulfur, researcher Russ Green collects a microbial mat structure built by cyanobacteria. The tiny microbes comprise an ecosystem that may approximate the world's earliest oceans and life forms.

The fortunes of each kind of bacterial mat battling it out in the Middle Island Sinkhole wax and wane as environmental conditions shift. In this case, the rising and setting of the sun happens to be the oldest and most predictable environmental change of all, but what about microbes in a system that isn’t so old and steady. How do they adapt?

Vincent Denef, an LSA assistant professor of ecology and evolutionary biology, had a petri dish for asking this question that might have been designed by Dante. It’s a dark tunnel underground, dripping with acids strong enough to burn flesh. Carbon monoxide levels approach the human safety threshold and the humidity is 100 percent. The temperature holds steady at a nearly unbearable 118 degrees Fahrenheit. All of these conditions—the acid, the air quality, and the beastly temperature—are created by the action of iron-eating microbial biofilms that line the cave. Denef spent six years as part of a team exploring the northern California cave’s tunnels for four or five hours at a time, clad head to toe in hot protective gear and dipping samples out of pools of greenish water matted with bright pink biofilms. “Ambient conditions are often close to the limit of human endurance,” he and a U-C Berkeley colleague wrote in a recent paper appearing in the journal Science.

Though hostile to humans, the abandoned Richmond Mine in California provides an ideal model system for studying how microbial communities survive, adapt, and swap useful genes in their never-ending struggle for primacy and survival. The cave has relatively few microbe species and relatively little contact with the outside world that might introduce new microbes and their genes. Denef wanted to learn how biofilm populations adapt to a shifting environment. Is there a diversity of species with different abilities lined up for succession, like in the Lake Huron sinkhole, or do the microbes evolve in response to changing conditions? The answer turned out to be a bit of both.

Seeing Life in a New Way

The cave’s ecology provided a glimpse of a complete kind of rainforest of unseen creatures eating other creatures. We often think of the base of the food chain as these microbes, but really it’s elements like iron and sulfur, the stuff the microbes eat. They make energy by breaking molecules apart, starting the whole process in motion. “Once the biofilm builds this system up, other organisms can come in and either use this biofilm or supplement it with their own iron oxidation,” Denef says. Acid-loving fungi move in to feed on byproducts of microbial chemistry, and little protists graze on the bacteria like tiny cattle. “We produced genomic information for 20 to 25 bacteria, archaea and eurkaryotes—all three domains of life—as well as viruses,” Denef says.

Denef’s painstaking assembly of complete genomes from the Richmond Mine revealed six distinct varieties of an iron-eating bacterium called Leptospirillium thriving in the isolated hothouse. Reading small changes in the genomes carefully over five years, he was able to estimate the rate at which a given letter of DNA might be changed by mutations, trading genes with other strains, and adaptation—that is, the evolutionary rate of just one species. This has been done in labs, but not in nature, where bacteria are hardly ever singular about anything.

Running that clock backwards indicates that the six varieties had sprung from a single ancestor in a matter of decades. Not eons or millennia, just decades. They could also see the rise and fall of some strains’ fortunes. In some instances, a hybrid species that struggled to hang on in one condition suddenly became predominant when the environment changed.

The genomic vision required to see subtle differences across a microbial community and variations as small as a single letter of DNA distinguishing closely related strains didn’t exist until about five years ago. This kind of science relies on something called “next-generation sequencing,” sophisticated and very expensive robotic laboratory machines about the size of a dorm-room refrigerator. They can do as much DNA sequencing in a day as it took the Human Genome Project 13 years and $3 billion to achieve.

Rather than teasing out individual microbes, stretching out their DNA and carefully reading each letter of code individually, next-gen machines can tear through a slurry of many species at once, chopping them into short segments of DNA for reading in a massively parallel fashion. The magic is in “bioinformatics,” sophisticated math and statistics that put billions of chunks of completed DNA sequence back together into a coherent picture of individual species like a giant word jumble. Without computers, it simply wouldn’t be possible, and Dick’s lab includes several people who just do bioinformatics.

This makes it possible to see, with great precision, that two microbes appearing very similar outwardly and sharing huge swaths of their genomes may be doing different chemistry to make a living, something Denef calls “microbial behavior.” For the first time, next-generation sequencing is giving biologists the ability to see microbial behavior and really understand what tools a microbe has and how it may be using them.

Jillian Banfield, a professor at the University of California, Berkeley, explores California's Richmond Mine—a hot, humid, acidic locale hostile to humans but perfect for very particular microbes. Denef has worked with Banfield to map changes in microbial genomes, documenting how subtle differences in DNA give these miniscule creatures distinct abilities, and change how they interact with the environment.

The microbes in a biofilm form an interdependent community, Denef says. And even though a given microbe may only be able to do a single job, it is surrounded by microbes doing other jobs that help it. For Denef, who started with an interest in using custom-tailored bugs to perform chemical cleanups, this helps explain why the engineered bugs don’t always perform as they should. “They generally lose out because they’re just not that good at competing with the indigenous microbes that were there,” Denef says. “We put them in there without their natural partners.”

Figuring out what makes each part of a microbial community tick has become a hot research topic. Like explorers of a vast new continent, biologists are fanning out to look at the microbes battling in the lungs of cystic fibrosis patients, in the guts of newborn babies, and on your skin. A Colorado researcher has found different microbes living on the human face than on the ears (because the ears are a little cooler), as well as communities on the left hand that share only 20 percent of their species with the right hand. It’s been known for some time that the microbes thriving in your gut are essential to your digestion, but now the communities in your lungs, on your skin, and inside your nose are being examined with new tools, revealing them as co-dependents that evolved with us, and probably shaped our evolution too. “Even soil researchers are starting to assemble genomes from soil, and that is probably the most complex system of all,” Denef says.

Fortunately, next-generation sequencing is able to see not just the spelling of the genes, but which genes are active at a given snapshot in time. This truer picture of microbial behavior—what tools are in use in response to given environmental conditions—comes from catching a real-time picture of its RNA, the molecule that translates the recipe of genes into specific actions. But with RNA’s half-life of only minutes, and an organism that can change its stripes almost as quickly, microbial researchers sometimes need to work very fast to get an accurate picture of what RNA was doing when the microbe was just chugging along minding its own business. When Dick works in the deep sea, his team uses diving robots that can administer an RNA fixative to fresh samples while still on the bottom. In the Great Lakes, where he and Denef will be working now, it just requires some human hustle.

During the summers, weather permitting, Denef will drop a plastic sampling tube they call “Big Bertha” overboard from a research ship to capture 30 liters of water at a given depth. Working fast to catch the RNA in its native state, he’ll run about 100 liters of water through progressively finer filters, precipitating out the viruses and then fixing a 10-liter sample with a high-salt solution to stop the RNA in action. “We just want their RNA to be the way it was in their environment. They’re fast, so we have to be fast too.” Some samples will be fast-frozen for reanimation and culturing later in the lab. Other samples will be set aside for microscopy, literally to see what’s there.

Denef, a Belgian who fell in love with the Great Lakes while at Michigan State for his doctorate, will be working with Dick on characterizing the food web of the Great Lakes starting from its microbial base in cooperation with the National Oceanic and Atmospheric Administration’s Ann Arbor-based Great Lakes Environmental Laboratory, the U.S. Environmental Protection Agency, and the U.S. Geological Survey. They’ll sequence and characterize bacterial, archaeal, and viral communities in water columns under day and night conditions, at varying depths, inshore and offshore. “There’s incredible diversity, and we’ve barely scratched the surface,” Denef says.

Not only do we not know what microbial communities are there now, we have no idea how they may have changed or will be changing under the constant assault of new invasive species, Dick says. Regional climate change may also alter the communities, he adds.

This past summer, Denef also enlisted inland lake homeowners in the northwestern Lower Peninsula to measure water quality and zebra mussel infestation, in cooperation with the Tip of the Mitt Watershed Council. Remarkably, some of the inland lakes still haven’t been invaded, giving Denef an opportunity to see microbial communities before, during, and presumably after the dramatic changes brought on by mussel invasion.

Microbes form ancient living communities, Dick says, doing some of the world’s oldest jobs—like making oxygen. But what happens when their conditions change very rapidly? With the temperature and acidity of lakes and oceans changing at an unprecedented rate and scale, the bigger question isn’t just what these microbes are contributing to life, but how their contributions may change. They’ll adapt; they always do. But how?

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