Bacteria: Survival in Siberia

A cross-section of permafrost shows an ice wedge hiding just below the surface. This sample is in the Arctic National Wildlife Refuge in northeast Alaska. Credit: U.S. Fish and Wildlife Service

The concept of suspended animation supports the plots of dozens of science fiction books and movies. If such a procedure could ever work with humans, it's surely many decades away. But exobiologists count on suspended animation as one of the best chances of finding life on other planets, Mars specifically. This spring, Gene D. McDonald and colleagues gave them some solid reasons for hope: evidence that single-celled organisms such as bacteria, archaeans and fungi repair cellular damage for tens of thousands of years - and perhaps many times longer - after being frozen solid.

Scientists have known for nearly a century that microorganisms can survive in the Siberian permafrost. How they do this, however, is not fully understood. McDonald's discovery hinges on the fact that, even in permafrost, animation turns out to be not quite completely suspended.

Even when all life processes appear to have stopped, processes that affect life do not. Organisms frozen in soil continue to be bombarded by radiation from elements within the soil itself. And at any temperature above absolute zero, all molecules vibrate a little. Thus, cells' DNA and other important molecules continue to sustain life-threatening damage. For organisms to remain viable for long periods of time, they must somehow maintain a minimal level of molecular repair.

"One of the reasons we did this work is because it had been believed that the organisms that had been buried in permafrost for tens of thousands or hundreds of thousands of years were basically inactive metabolically," McDonald says. But, he wondered, if they remained completely inactive, how much radiation damage could the cells sustain?

To answer that question, McDonald and his colleagues, Karen Brinton and Alexandre Tsapin, both working with him at Jet Propulsion Laboratory in Pasadena, and David Gilichinsky, of the Russian Academy of Sciences in Pushchino, needed a convenient indicator of the molecular-repair activity of cells embedded in Siberian permafrost, where they obtained their samples.

One such indicator is the rate at which certain building blocks of proteins undergo a normal molecular change. Because they're asymmetrical molecules, amino acids come in both right-handed and left-handed forms, labeled D and L respectively. Left alone at any temperature above absolute zero, any individual amino acid molecule occasionally will switch from the right-handed configuration to the left-handed one and vice versa in a process called racemization. A bowl of any given amino acid, regardless of its initial balance of left- and right-handed molecules, eventually will reach an equilibrium state, with roughly equal numbers molecules of each configuration at any moment.

The speed of this process differs among the amino acids and depends on temperature and other parameters of the environment, McDonald says. "It could literally range from hours in boiling acidic water to billions of years in a cold dry sediment."

This process, while normal to chemistry, is inimical to biology. Proteins in living organisms don't work if they contain right-handed amino acids. But the right-to-left and left-to-right reactions go on continuously even in living cells. As a result, McDonald explains, organisms have evolved "enzymes that basically go around and scavenge the D amino acids, the right-handed ones, and get rid of them. Because if the D amino acids build up to too high a level, they can poison protein synthesis and essentially kill the cell."

L aspartic acid molecule. Credit: biology.clc.uc.edu

These enzymes prove so efficient that a living cell maintains a near-zero ratio of right-handed to left-handed amino acids, or D/L ratio. This process provides a sort of clock to determine the age of dead cells or cells with suspended molecular activity. Once the repair process stops, the clock starts ticking, and the level of right-handed amino acids slowly rises.

"If you know what the temperature of the environment is, and if you've measured the rate of racemization [molecular switching] at that temperature, you can then predict what the amount of racemization would be for a given age," McDonald says.

Amino acids undergo this right-to-left-to-right switch at different rates, so McDonald chose to study the fastest amino acid, one called aspartic acid. To calibrate the clock, the team studied the rate at which aspartic acid switched from left to right at different temperatures and ran a carbon-14 dating technique on the samples. Organisms build all their carbon-containing molecules using carbon from the environment. That carbon is a mixture of stable and radioactive forms of carbon. The amount of carbon-14, a radioactive form, continuously decreases after a cell stops building new molecules with atmospheric carbon-when it's frozen or dies, for example. By measuring the total amount of carbon-14 in a sample, researchers have an independent way to determine the sample's age.

The researchers can express the amino acid clock's results in a variety of ways. Because the process depends on temperature, McDonald compared the temperature predicted by the measured amount of right-handed amino acid in a sample with the actual average temperature of the permafrost at the depth of the sample.

The amino acid clock suggested that the samples had been continuously cooled to a temperature of minus 19 degrees Celsius (minus 2 degrees Fahrenheit). But the measured temperature of the permafrost is 6 to 8 C (11 to 14 F) warmer. When the clock's results are expressed as a temperature, colder means less measured right-handed aspartic acid.

What could account for the discrepancy?

"If there's less of the right-handed form" than would be expected if no biological activity had been taking place, McDonald says, "then the only real explanation for that is that the organisms are scavenging these D amino acids and getting rid of them." Enzymes convert D amino acids to the L form or break down the D amino acids, recycling the parts of the molecule.

McDonald and colleagues suggest two possible ways the soil organisms could have continued this molecular maintenance. First, the permafrost may have warmed periodically, thawing the frozen organisms. But independent research shows only very low levels of molecular activity in permafrost samples.

D aspartic acid molecule. Credit: bioscience.org

The second possibility is that the organisms continue to scavenge right-handed aspartic acid even at permafrost temperatures. This process would be slow but steady. And if the organisms are performing maintenance on aspartic acid, McDonald argues, they may also be maintaining DNA and other essential biomolecules (although there is, as yet, no direct evidence of this). McDonald's results suggest that permafrost organisms can continue this molecular maintenance for at least 30,000 years.

"We can't absolutely prove it, but the assumption is that there's essentially no cell division. So we're talking about a cell population that's basically been there since soon after permafrost was deposited," McDonald says.

On to Mars

"A lot of papers are being published in exobiology but very few have really direct relevance to the goals of exobiology," says E. Imre Friedmann, a microbial ecologist and astrobiologist at NASA Ames Research Center.

"This is one of those which does have a direct relevance to the goals of exobiology. This is a possible method to document microbial activity in Martian permafrost."

While Mars experts have gathered evidence of ice on Mars for some time, results in May from the Odyssey spacecraft showed large amounts of subsurface ice.

Friedmann says the amino acid clock could help determine if life once existed on Mars-or even if living organism still reside in the Martian permafrost.

"Now having said that, this is not something that we will do tomorrow. Because in order to use this method, we will have to go to Mars, drill into Mars to reach the permafrost and bring permafrost back. It is not something which will be done next year. But [the technique] is a very important preparation for a more advanced stage of exploration of Mars."

Drilling would be an essential first step because the surface of Mars cannot sustain liquid water. "Conditions in considerable depth below the surface may be more suitable for life," Friedmann says, "but of course this is also uncertain.

This artist's representation shows a standing pool of water on Mars -- impossible today, but what of the future? Scientists consider the possibilities in "Once Upon a Water Planet." Image Credit: Duane Hilton

Friedmann calls Martian permafrost the most likely place to find life on the red planet. But for life to have survived, even in almost suspended animation, organisms would have to have survived far longer on Mars than they have so far on Earth.

"The oldest Siberian permafrost is about three million years old," Friedmann says. "On Mars, life, if there was any, probably stopped over three billion years ago. Billion. So it is an enormous difference between the Earth conditions and the Martian conditions. Still it is not impossible that we can find living bacteria, not near the surface, but maybe quite deep."

What's Next?

McDonald pictures uses for the amino acid clock on Earth and on Mars. Here at home, he plans to obtain samples from permafrost in Alaska and deeper in the Siberian subsurface. As for Mars, he says, "There are several instruments under development that would measure amino acid D/L ratios on Mars, from a lander or rover." And the Mars lander scheduled for 2009 may well have a drill capable of obtaining permafrost samples-if a lander can reach the parts of Mars suspected of having permafrost, not an easy feat. If such instruments work, they would save the considerable trouble of transporting samples of Martian permafrost back to Earth.

In the lab, McDonald hopes to examine the enzymes organisms use to maintain a viable amino-acid ratio. "I'm interested in how these organisms go back and forth, for instance, between the enzymes that require oxygen and other enzymes that don't in order to keep doing this repair," he says. "We don't really know what the enzymes involved are. It could be the same enzyme that they would use at the higher temperatures or they could have evolved a different enzyme or a different way of dealing with it. We don't really know that. That's one of the things we'd like to look at."

Related Web Pages

Aspartic Acid Racemization and Age-Depth Relationships for Organic Carbon in Siberian Permafrost
Found it! Ice on Mars
Racemization of Meteoritic Amino Acids
Stereochemistry