Pathways for Life

blue cells
Photo of a live culture of Methanosarcina acetivorans cells courtesy of Everly Conway de Macario and Alberto J. L. Macario

Among the archaea, only the Methanosarcineae can form multicellular structures, usually in response to en-vironmental change.

Microbiologist J. Greg Ferry is surprisingly calm when he talks about the most exciting scientific experience he's ever had.

It was spring 2002, and Ferry was in Cambridge, Massachusetts, gathered with "a tight-knit group" of about two dozen researchers to discuss an obscure microbe. "The bug," as he calls it, also known as Methanosarcina acetivorans, is Ferry's baby. He discovered it 20 years ago living in a mass of kelp in an underwater trench off the coast of southern California. He even got to name it. "Acetivorans means voracious for acetate," Ferry explains. Among other things, M. acetivorans eats acetate—a salt derived from acetic acid—and expels methane. It eschews oxygen. It excels in harsh environments, like sludge pools, animal intestines, and bogs. And it's been around for billions of years—long enough to make it a key player in the evolution of life on Earth.

In 2001, as the Human Genome Project neared completion, Ferry had urged colleagues at MIT's Whitehead Institute—a well-funded locus of genetics research—to sequence M. acetivorans. Surely they would recognize "its importance in all of biology," thought Ferry. They did. The sequencing took less than a year.

Computers got first crack at making sense of the sequence—culling databases to match strings of code parsed into genes with known proteins and enzymes. Then the data were sent to a handful of devotees, who with Ferry had been studying M. acetivorans for years. "For the first time we were seeing the details of how the bug works, and it was like, 'Wow!'" Ferry says in a quiet voice. He pauses. "It was almost overwhelming, actually."

But the work was just beginning. The experts needed to verify the computer's translation of the code. "The computer could misidentify a protein," says Ferry. "The information in the databases could be wrong." The ultimate test is to run experiments in the lab to test the gene-protein pair, he explains. "But we're talking about a huge number of genes!" So the team selected certain proteins that have the most impact on the organism's functioning and split up the work, with each research group assigned different parts of the sequence to verify. Ferry's group was asked to test and comment on a gene that codes for a key enzyme in the process of converting acetate to methane—a pathway involving several proteins and enzymes that carry out chemical reactions in steps.

The collaborators communicated via email, sharing their observations, and marveling at what the genome was revealing to them. Then came the best part. They all came together at Whitehead for what amounted to a M. acetivorans summit. For two days they asked questions of each other, presented findings from their respective labs, and formulated plans for further research.

"The meeting was extremely fascinating, enormously fun," says Ferry. "Getting that many scientists together is a true testament to the uniqueness of M. acetivorans."

One surprise that Ferry and his colleagues uncovered was the sheer size of the M. acetivorans genome: With 4,500 genes, the amount of genetic information it contained seemed huge for a one-celled organism. (The human genome, by comparison, is only about 30,000 genes.) "The size of the genome is a manifestation of the organism's diversity," Ferry explains. "It can adapt to its environment better than any other organism in the archaea domain because it has the information to produce proteins and enzymes that allow it to respond to environmental changes."

scuba diver underwater
© Phillip Colla, all rights reserved worldwide

Science diving: individual species of Methanosarcina have been found in freshwater and marine environments, such as kelp beds (above), and in decaying organic matter, among other places.

The archaea—one of the three main branches of life—have only recently been recognized as a distinct life form. For years, scientists thought these tiny organisms were merely an offshoot of bacteria, distant cousins, perhaps, of familiar disease-causing bugs like E. coli and Streptococcus. Then, in the late 1970s, Carol Woese, a biologist at the University of Illinois, discovered that these microbes had genes that were fundamentally different from those of other bacteria. In fact, genetically-speaking, the archaea are more closely related to the third branch of life, the eukarya—multicellular organisms including fungi, plants, and animals. Scientists now believe that the archaea and the bacteria evolved separately from a common ancestor, and that the eukarya branched off from archaea at a later point.

Still, archaea resemble bacteria in many ways: they are one-celled organisms with a coil of genetic material, and some of them exist alongside bacteria in seawater or soil. But most archaea are more exotic in their choice of habitat. One group prefers extremely cold environments like Antarctic ice. Another likes things hot: the boiling springs of Yellowstone. Yet another subset of archaea, called the methanogens, eke out a living in oxygen-poor environments and produce methane as a waste product. M. acetivorans is a methanogen. So toxic is oxygen to this class of organisms that microbiologists like Ferry must grow them in special sealed chambers accessible only by a pair of built-in rubber gloves.

M. acetivorans is the largest of the archaea, and its genome harbors many of the tricks and tools that archaea have developed over the millenia to survive. Unraveling its genome, the research team uncovered a slew of interesting properties. It is the only species in its domain, for example, that has three different ways of converting its food to methane. (The acetate-to-methane pathway is only one of its choices.) "These pathways are ancient, perhaps the first pathways to obtain energy for life," Ferry explains. Three to four billion years ago, at the time of the origin of life, acetate and carbon monoxide, another food source for M. acetivorans, were in abundance in the environment.

Another interesting feature: Although the organism is a strict anaerobe, meaning it lives without oxygen, it possesses a gene that codes for an enzyme that helps to break down oxygen. Its genetic code also contains instructions for flagella—those whiplike structures that help cells move —and for chemotaxis, the ability to maneuver toward or away from a specific chemical. Curiously, no one has ever observed such purposeful movement in M. acetivorans.

The organism also exhibits several examples of multiple genes coding for the same protein, "which may seem wasteful," Ferry explains, "but is actually an indication of the bug's amazing ability to adapt to changing environments." These may be only the beginning of M. acetivorans's important qualities, he adds. The functions of over 35 percent of the organism's genes remain a mystery.

I would say that 75 percent of the research taking place in my lab now is based on questions that came out of the meeting at MIT," says Ferry. Indeed, in the summer of 2002, with a grant from the National Science Foundation, Ferry spearheaded the formation of the Consortium for Archaeal Genomics and Proteomics—a collaboration among several universities based at Penn State—using M. acetivorans as its model organism. By studying the genes and proteins of this single microbe, the members of the consortium hope to advance the understanding of the entire archaea domain.

M. acetivorans makes a good model because of its size, Ferry says, and also because researchers have developed a procedure for inserting random genetic information into Methanosarcina cells, something that can't easily be done with every type of cell. "Inserting nonsense information into the sequence for a given gene can inactivate or 'knock-out' the gene," Ferry explains. "Then you can look at how the organism has been crippled. Which pathway was knocked out? That can tell you which enzyme in which pathway a particular gene codes for."

Identifying these enzymes could lead to the discovery of novel proteins and enzymes useful for drug design, or to clean up chemical spills, Ferry says. For example, M. acetivorans can make an enzyme to break down kepone—a toxic compound once used as an insecticide—into hydrochloric acid and methane. M. acetivorans' ability to break down so many different food sources into methane is interesting to scientists because methane is a potential alternative energy source.

Ferry himself is particularly interested in finding out how M. acetivorans uses redundancy—multiple genes coding for the same proteins— to respond quickly to changes in its environment, such as the presence or absence of certain kinds of food.

In the lab, he says, if M. acetivorans is given acetate to eat and nothing else, it will "turn on" genes that code for the enzymes that process acetate. But it will also turn on one of three duplicate genes that code for the enzyme that processes methanol, another food source, as a just-in-case. That way, Ferry explains, if methanol suddenly became available, M. acetivorans could begin to metabolize it immediately. Where the situation is reversed, he adds, and it only has methanol to eat, all three genes for the processing of methanol are turned on, and just one of the acetate enzymes is turned on. "Now we have a reason why they may have duplicated genes—it's a new way of adapting to their environment," says Ferry. "Usually there's one gene and it gets turned on and off in response to an environmental condition.

"In nature, a slow-growing organism like M. acetivorans would want to switch pathways rapidly so that it could quickly take advantage of a new food source." If it can manage that without much lag time—the time required to turn on a new gene —"it would have a leg up on competing organisms," he says.

Ferry pauses. "They're thinking," he muses. "It's kind of scary, isn't it? They're enormously adaptive, extremely intelligent."

Ferry is also interested in how M. acetivorans responded to the rise of oxygen in Earth's early environment. "This is an interesting story," he says, smiling. "Did you know that oxygen is the worst pollutant ever produced in the history of life?" Although many forms of life have come to depend on oxygen for respiration, he explains, it's still toxic to most cells. Oxygen can form highly reactive free radicals that attack enzymes. To protect itself, the human body has evolved enzymes that protect us from those free radicals. Still, oxygen wears down cells, and plays a major role in aging.

Ferry paints the scene: For the first billion years of life on Earth, microbes thrived without oxygen. Then plants came along, and photosynthesis. The oxygen produced attacked many of life's molecules, combined with them, changed their structures. "It was a doozy, the biggest environmental change that life had encountered. Organisms had to invent new pathways—new proteins and enzymes—for dealing with toxic oxygen," he says. Fortunately, the rise of oxygen was slow enough that many organisms had time to adjust instead of perishing. To help their chances, some anaerobes hid themselves in places where no oxygen could reach, such as deep in the mud at the bottom of a swamp.

The kelp-filled trench where Ferry and a graduate student discovered M. acetivorans back in 1983 was just that sort of place. Fishermen call the areas above the trenches "bubble holes" because the organisms below release small bubbles of methane gas—their waste product—that expand as they rise and then burst when they reach the surface.

It was here, in this imperfect hiding place, that M. acetivorans might have developed its unique characteristics. "The organisms probably encounter trace amounts of oxygen in the kelp beds," Ferry explains. The water layer above the kelp bed contains dissolved oxygen, and during a storm, trace amounts of oxygen can be mixed into the kelp layer. "Oxygen is very poisonous to it so it must have developed mechanisms to cope," says Ferry, including, perhaps, a pathway to convert free radicals to less toxic compounds. Researchershave already discovered in M. acetivorans a gene that codes for oxidase, an enzyme that partially breaks down oxygen, he notes. A full pathway for metabolizing oxygen has yet to be uncovered, however.

"If we can understand how Methanosarcina deals with oxygen now, Ferry suggests, we'll have a clue as to how life early on developed mechanisms to survive this incredible insult." That could lead to "a fundamental advance in our understanding of evolution, and of how humans deal with oxidative stress.

"Methanosarcina and other anaerobes are our ancestors," he says. "They laid down all the fundamental metabolism for life as we know it today."

J. Greg Ferry, Ph.D., is Person professor of biochemistry and molecular biology in the Eberly College of Science, 205 S. Frear Building, University Park, PA 16802; 814-863-5721; jgf3@psu.edu. His research is funded by the National Science Foundation and the NASA Astrobiology Institute. For more information about the genome of Methanosarcina acetivorans visit www.broad.mit.edu/annotation/microbes/methanosarcina/.

Last Updated May 01, 2004