Most microbial researchers grow their microbes in petri-dishes to study how they grow and how they respond to damaging conditions. But researchers in Louisiana State University’s Department of Biological Sciences are doing something almost unheard of: studying microbes under freezing conditions to understand how organisms could survive for hundreds of thousands of years in deep Antarctic permafrost, or perhaps even buried in ice on Mars.

“I could take you down the hallway into a common room we have, full of freezers,” said Brent Christner, an associate professor in LSU’s Department of Biological Sciences and Antarctic microbe explorer extraordinaire, leaning excitedly over his desk as he shows me microscope images of cells encased in ice. “And in those freezers are cells and tissues. We put them there, because that is actually a way to preserve them.”

Christner’s lab keeps thousands of different strains of microbes stored at -80 degrees Celsius. These microbes, he explains, are actually quite happily revived if taken out of the freezer and placed in nutrient-rich media. “But what if those freezers could run for a million years?” Christner asked. “We’d actually expect a fraction of them to die off every year, because they are made up of molecules that, even if frozen, are subject to decay.”

Christner and colleagues at LSU, postdoctoral researcher Markus Dieser and Mary Lou Applewhite Professor of biology John Battista, recently had a paper on DNA repair mechanisms in frozen microbes accepted in the journal of Applied and Environmental Microbiology. To understand how microbes survive in frozen conditions, Christner and colleagues focused on DNA, the hereditary molecule that encodes the genetic instructions used in the development and function of living organisms from humans to microbes. Microbe DNA is typically millions of base pairs, or units, long, often arranged into a single circular structure that encodes thousands of genes.

“We know that there are spontaneous reactions that occur that result in that molecule being damaged,” Christner said. The worst kind of damage is known as a double-stranded break, when the microbe’s single circular “chromosome” breaks into two separate pieces that need to be put back together to make the chromosome functional again.

“This kind of damage is what we know has to happen when the cells are sitting there frozen for thousands of years,” he said. “Imagine that a microbe is in ice for these extended periods of time, and its DNA is getting sheared into pieces. There is actually some point in time when the microbe’s DNA becomes so damaged that it’s basically not a useful informational storage molecule anymore. So now you no longer have a microbe, you basically have a corpse.”

The situation would seem dire for the longevity of microbes in ice. But curiously, researchers have been able to revive microbes buried in ice for hundreds of thousands of years. In fact, Christner managed to revive several different types of bacteria from near the bottom of the Guliya ice cap on the Qinghan-Tibetan plateau in Western China – ice that is 750,000 years old, from long before the age of humans.

“Not long ago, frozen environments which cover a significant portion of the earth were considered devoid of life deserts,” said Vanya Miteva, Penn State professor and author of a chapter on microorganisms associated with glaciers in the Encyclopedia of Snow, Ice and Glaciers. “During the last two decades this view changed, and now we talk about the cryosphere as a major Earth habitable ecosystem.”

But how is it possible to revive a microbe from 750,000-year-old ice? The survival of microorganisms in anacient glacial ice and permafrost has traditionally been ascribed to their ability to persist in a dormant, metabolically inert state. But even this explanation does not account for the background amounts of ionizing radiation that almost surely cause damage to these microbes’ DNA, frozen at the bottom of a glacier or not.

“In order to survive that long, different studies for instance point towards dormancy or 'slow motion metabolism', but regardless of the physiological state, without active DNA repair an organism will accumulate DNA damage to an extent that will lead to cell death,” Dieser said.

But today, there is a research-supported alternative explanation for the curious longevity of microbes in ice: “worker” enzymes that go about fixing damage to DNA, even under freezing conditions.

“In your cells right now, there are actually DNA repair enzymes that are active, even though you aren’t being exposed to high levels of radiation,” Christner said. He is referring to the background levels of ionizing radiation coming from both natural and artificial sources all around us, including radon sources, cosmic radiation from space and terrestrial radiation from the ground. Although the level of background radiation on Earth is typically very small at any given time, you might imagine that microbes surviving for hundreds of thousands of years accumulate a large amount of damage even from these small background levels.

In 2007, after finding intact microbial DNA preserved in 600,000-year-old permafrost samples, researchers suggested that active DNA repair mechanisms might be at play. But unlike Christer’s group, they lacked any specific knowledge of how microbes might be fixing their DNA under such unfavorable frozen conditions. “Everything changes if the microbe in question is not just sitting there in a state of suspended animation, but actually has an active metabolism,” Christner said. “Now that sounds really hard to imagine in ice, right?”

NASA has a mantra of “follow the water,” based on the general idea that where there is water, there is life, and that cells require water to grow and metabolize. But it’s important to remember, Christner says, that even in ice there is nearly always a small amount of liquid water. As I sit in his office, he pulls up a microscope image of ice crystals on his computer monitor. The ice crystal structure looks like an opaque jigsaw puzzle made up of large hexagonal pieces. But Christner points out clear, narrow gaps between the edges and points of the ice puzzle-pieces.

“That is liquid water,” he said. “There is water in this ice.”

Even more surprising, Christner shows an image of the same ice crystals that have been treated with a green dye that stains only living cells. The gaps between the ice crystals are bright with little green microbes.

“The organisms in this frozen sample are actually migrating to these water environments within the ice,” Christner said. “And we actually know from other experiments we’ve done that these microbes can metabolize and actually create new DNA under frozen conditions.” Research has shown that microbes can continue metabolic activity in ice even at subfreezing temperatures of -40oC and below.

Christner’s newest paper is taking these results to another level, showing that frozen microbes can not only make new DNA under frozen conditions, but can repair DNA that is damaged over time – even those tough double-stranded breaks. Christner and colleagues took frozen (-15oC) samples of Psychrobacter arcticus, a recently-discovered model organism of cold-adapted microbes isolated from Siberian permafrost, and exposed them to a dose of DNA-damaging ionizing radiation equivalent to an amount the microbes would have experienced over a span of 225,000 years in permafrost. As a reference, this dose of radiation is 45 times higher than the lethal dose for humans. The researchers then let the microbes sit under freezing conditions for a period of two years, periodically checking the microbes’ DNA.

As expected for a bacteria not resistant to radiation, the high dose had greatly damaged the single circular microbial chromosome of P. arcticus, transforming it into a slurry of smaller pieces due to double-stranded breaks in the molecule. What surprised the researchers was that, over the course of two years in the freezer, the microbes’ DNA pieces began to slowly come back together in their proper order.

“This can’t be a random process,” Christner said. “If a chromosome is chopped up into a bunch of pieces, and a cell goes to put it back together, it can’t put it back together willy-nilly. The chromosome is in an order, and for the organism to survive, it has to be put back in the correct order. This tells us that the cells are repairing this DNA.”

According to the data, P. arcticus appeared to be able to repair between 7 and 10 double-stranded breaks per year in the freezer. The findings are important, Christner says, because we don’t usually think of freezing temperatures as being conditions under which complex biological processes can occur.

“The results demonstrate that despite low temperatures of -15oC and high salinity of the liquid vein network in ice crystals, certain organisms can repair their DNA, thus enhancing their long term ability to survive in icy environments,” said Mark Skidmore, associate professor in the Department of Earth Sciences at Montana State University. The results support previous research by Skidmore and Christner that the liquid gaps in ice are viable microbial habitats.

“This study provides convincing experimental evidence that under frozen conditions cells can successfully repair irradiation DNA damages, thus delaying their lethal effect and significantly extending cell survival,” Miteva said. “Overall, this well-written paper will definitely have positive implications improving our understanding of microbial longevity on Earth and possibly elsewhere.”

While there has been evidence before Christner’s study that microbes can have very low levels of activity under freezing conditions, specifically what they could be doing to improve their survival in these conditions has previously only been a matter of speculation.

These findings make it reasonable to speculate that if life ever evolved on Mars, and microbes are still frozen somewhere in the subsurface, they might still be viable if given the right conditions.

“This is clearly a pathway that would be relevant to an organism that is surviving in a place like Antarctica, or in deep permafrost, even in a place like Mars,” Christner said. “This is relevant in an astro-biological sense, because if these mechanism are working in our cryobiosphere, microbes might be using this survival mechanism in icy environments on other moons and bodies in the solar system. We are very excited about these results.”

But the research on cold-loving bacteria is far from over. While Christner and colleagues have addressed the DNA repair problem of these long-living microbes, the question still remains of how these bacteria get the energy to maintain DNA metabolic activities for hundreds of thousands of years. Future research will need to address this question, and look into other survival mechanisms used by bacteria living in extreme environments.

References:

Christner, B. C., Mosley‐Thompson, E., Thompson, L. G., & Reeve, J. N. (2003). Bacterial recovery from ancient glacial ice. Environmental Microbiology, 5(5), 433-436.

Dieser, M., Battista, J.R., Christner, B.C. (Published ahead of print 27 September 2013). Double-strand DNA break repair at -15°C. Appl. Environ. Microbiol., AEM.02845-13, doi:10.1128/AEM.02845-13