Resurrected organisms reveal life’s bare essentials
by Colin Barras / 17 May 2017

“Wind the clock back 5 million years, to a blustery day in the dry season somewhere in Africa. The winds whip dust high up into the atmosphere, then blow it south and dump it in Antarctica. As fresh snow falls, a small piece of prehistoric Africa gets locked away in the ice. It carries an invisible cargo: thousands of tiny microbes have survived the journey. When their burial chamber is opened, it’s because humans have arrived, keen to probe the continent’s secrets. They drill into the ice, extract a dirt-speckled layer, melt it and incubate the water in a darkened corner of their lab. Months later, something amazing happens: the ancient microbes begin to grow.

The man who led this astonishing feat of resurrection was Paul Falkowski at Rutgers University in New Jersey. His microbes were frozen at a time when our earliest ancestors had barely separated from those of chimpanzees. It isn’t the first story of its kind. The oldest “resurrection” claim so far came in 2000, when a team said they had revived microbes found inside a 250-million-year-old salt crystal from the Salado formation in New Mexico. Lazarus microbes aren’t just fascinating oddities. By inhabiting a twilight zone between life and death, they offer a unique opportunity to probe the very nature of life itself. At first glance, such claims seem to defy scientific logic. Many biologists say it stretches credulity to imagine that anything can survive being locked away for millions of years.

Ancient bacteria have been found in halite crystals

Take the Salado bacteria that Russell Vreeland at the West Chester University of Pennsylvania and his colleagues say they revived in the lab. When times are tough, bacterial cells grow a tough shell and become dormant spores. Their metabolism slows to the point where they might as well be dead – meaning they have no way to repair the damage that millions of years of exposure to environmental radiation will do to their DNA.

“Simulated space trip made E. coli tougher”

And that creates a fundamental problem. Under ideal, cold and dry conditions, DNA has a half-life of about 160,000 years, which means half of it is degraded during that time. After about 7 million years, there should be essentially none left. “To think of completely intact chromosomes after many millions of years sounds a bit like science fiction to me,” says Morten Allentoft at the University of Copenhagen, Denmark, who led the calculations of DNA’s half-life. Arguments like this lead sceptics to insist that so-called resurrected species are merely modern bacteria that have contaminated ancient samples. Not so fast, say resurrection biologists. Maybe dormant spores can’t stay viable for millions of years – but what if the cells never actually go to sleep? As long as they sustain basic metabolic functions, they could continue repairing DNA damage even if they do little else.

“Atacama Desert in North Chile with inset showing microbes found beneath it”

Work led by Brent Christner at the University of Florida supports this idea. His team began by placing Psychrobacter arcticus – a bacteria that had been resurrected from 20,000-year-old Siberian permafrost – into a deep freeze at -15°C, then blasted it with DNA-destroying radiation. For hundreds of days nothing seemed to happen. Then, slowly, some cells began to stick the pieces of their genome back together again. Even locked in ice, they could keep their genome functional.

Other studies suggest a bit of clever sharing might help some cells get the energy to do this. In 2009, a team led by Tim Lowenstein at Binghamton University in New York reported that they had resurrected microbes from 30,000-year-old salt crystals. Crucially, the resuscitation only worked in cells that had been trapped together with algal cells from the ancient lake. They stayed active by eating glycerol, an alcohol that leaches out of dead algal cells. Lowenstein’s team estimated the glycerol from a single alga could sustain one cell for 12 million years.

Even though they had packed away food stores to survive the long winter, the microbes probably faced a gruelling existence – for a cell, at least. When Lowenstein and Yaicha Winters, also at Binghamton, starved the revived cultures for several months, the rod-like cells broke up into smaller, spherical forms. This is probably a survival strategy – smaller cells need less energy and their larger surface-area-to-volume ratio makes it easier to absorb any nutrients around. Significantly, the 30,000-year-old cells were also small and spherical, suggesting they had changed shape to survive on their poorly stocked pantry.

Life may not be any easier for microbes trapped in ice, although the special way that ice forms offers some reprieve. When water freezes, any impurities – including nutrients – are squeezed out of the ice crystals into tiny pockets of brine that stay liquid down to -15°C. It’s in those pockets that trapped microbes eke out a living, says Christner. Just like algae in salt crystals, the concentrated nutrients could allow cells in ice to survive on the edge of starvation.

“Morphological changes in two populations of Hbt. salinarum strain 670 during 56 days of starvation. Rod-shaped cells dominated the populations at the start of starvation, but pleomorphic properties emerged as starvation progressed.”

Doing so involves paring down some pretty fundamental activities. Things that are generally thought to be essential for life get squashed or eliminated in zombie organisms. For instance, “within these tiny environments you could absolutely not have large-scale reproduction,” says Vreeland. “You would quickly use up critical supplies.” On this point everybody agrees. Christner has simulated the conditions bacteria buried in ice at -15°C would experience. The tiny pockets of water between the crystals are so salty that most cells just sit there. Few can afford the luxury of growing and reproducing, he says: “It’s like a microbial rest home.”

Steven D’Hondt at the University of Rhode Island has looked at conditions in the mud below the sea floor, where cells have survived being buried for millions of years. He says the energy available is barely sufficient to allow them to carry out basic repairs, let alone anything else. “There’s probably not much reproduction going on,” he says. This implies individual bacteria must live unusually long lives. “It’s conceivable,” says D’Hondt, “that cells in sub-sea floor sediment live for millions of years without reproducing.”

As a rule of thumb, bacteria tend to live fast, reproduce often and die young. Resurrected microbes do exactly the opposite. One explanation is that they are bizarre rule-breakers, but there is an alternative. Could exceptionally long, frugal existences be what most microbes adopt if conditions demand it? After all, says D’Hondt, the microbes buried beneath the sea were once part of the regular surface community. Christner has found that even garden-variety bacteria like E. coli can stay active to some degree when frozen. “Perhaps this is a capacity that evolution has hard-baked into microbes,” he says.

Vreeland suspects as much. He thinks biologists may be reading too much into the behaviour of the few microbial species they can grow in the lab. Out in the wild, things behave differently. “Laboratory strains are growing maybe 1000 times faster than they would grow in nature,” he says. Resurrected bugs may reveal what real microbial life is like – slow, steady and able to persist under seemingly impossible conditions. There’s another reason to be excited about Lazarus microbes. We think of evolution as an inevitable fact of life: with each passing generation, organisms accumulate the tiny mutations that drive evolution. But if some cells don’t reproduce, do they actually evolve?

Andreas Schramm at Aarhus University, Denmark, and his colleagues analysed microbial DNA collected from below the sea floor off the Danish coast, where some bacteria have been buried for more than 5000 years. The little bacterial DNA they could recover from the 5000-year-old mud was very similar to DNA found at the surface. In other words, there was no evidence the bacteria had picked up mutations and adapted to subsurface living. That’s particularly surprising given the environmental changes these communities had experienced over millennia, from living on the sea floor to gradually being buried deep in the mud. “An environment without adaptive evolution seems to go against biology’s rules as we understand them,” says Schramm.

And that gives biologists an extraordinary new opportunity to study evolution itself. These resurrected cells are living time capsules, snapshots of how things looked hundreds or even thousands of years ago. Resuscitating them is, crudely speaking, equivalent to bringing our hominid ancestors back to life. Lawrence Weider at the University of Oklahoma is a leading figure in the field of resurrection ecology, which compares ancient live organisms with their modern descendants to understand how they evolved, and maybe even predict future changes. The organisms he studies aren’t microbes, but tiny planktonic crustaceans called Daphnia, present in many freshwater lakes. Their eggs settle on the lake floor, where any that fail to hatch are gradually buried in the mud.

Since the late 1980s, Weider has been recovering these eggs from sediments and encouraging them to hatch in the lab. More often than not, the hatchlings come from eggs laid a few decades ago. In 2011, Weider and his colleagues applied the same approach to eggs recovered from the sediment at the bottom of South Center Lake in Minnesota. Only after they hatched did the team realize that some of them came from sediment that was 600 to 700 years old.

Daphnia are, to date, the oldest animals to be resurrected, but Weider thinks their significance is greater than that. “I was never excited about having the oldest of anything – I just wanted to use the animals to look at evolutionary change through time.” The hatchlings grew into healthy adults and, because Daphnia can reproduce asexually, they were in fact natural clones, populations that look and behave like Daphnia that existed before Christopher Columbus reached the Americas. “That’s the beauty of resurrection ecology,” says Weider. These days, biologists can learn a lot about the past by extracting ancient DNA from the remains of dead animals, but resurrected organisms take that research one step further.

Already the team have learned how Daphnia changed when European settlers began farming land near the lake. Run-off from fertiliser-laden fields led to a surge in lake nutrients, and the crustaceans evolved in response. In a 2014 study, Weider and his colleagues discovered that the 700-year-old Daphnia use the phosphorus they ingest sparingly, probably because the nutrient was rare before the settlers arrived. In contrast, Daphnia growing in South Center today have lost the ability to squirrel away the nutrient for later use.

Some of Weider’s recent studies have thrown up more puzzling results. The team ran experiments pitting old and modern Daphnia against each other in water that was either low in nutrients to mimic the bygone lake, or high in nutrients to mimic modern conditions. Unexpectedly, the modern Daphnia always outcompeted their ancestral counterparts – even in low-nutrient water. “It will take more work to explain that,” says Weider. “Perhaps there are genetic defects in the ancient lineages that don’t allow them to be as successful.” But skeptics, he concedes, put it down to the ancient eggs being poor-quality: after all, they did fail to hatch all those years ago.

While Weider and his colleagues remain cautious about drawing conclusions for now, they are testing new ideas. Their latest push is to cross-breed Daphnia strains. “We’ve actually got one of our ancient clones to cross with the modern Daphnia,” says Weider. Ancient-modern hybrids may exist in the wild too. Earth is geologically active, so what goes down usually comes up. As old rocks are brought back to the surface, organisms that are buried alive can eventually rejoin the surface biosphere. “Earth is this incredible machine where everything cycles,” says Vreeland.

Falkowski thinks it’s plausible that bacteria locked in ice might swap genes with other microbes when the ice finally melts. He says some researchers think that the rate of microbial evolution accelerates following an ice age, and that access to a freshly thawed bank of genetic material could be part of the explanation. More generally, it could even be that as ice melts, it naturally resurrects organisms that go on to influence the modern biosphere. Christner cautions that the idea still needs to be fleshed out by evidence, but Weider is open to it. “We’re already seeing stories about pathogen release,” he says. In 2016, an anthrax outbreak in Siberia claimed at least one human life. The bacterium responsible is thought to have come from the remains of a reindeer that succumbed to anthrax 70 years ago. For decades the carcass was preserved in permafrost, until a heatwave thawed it and released the deadly cargo. With the world in a state of climate flux, natural resurrection may soon be occurring fairly regularly. “There are a lot of dynamics to the resurrection process,” says Weider. “We are only now starting to appreciate the complexities.”

“This article appeared in print under the headline “Wakey, wakey”




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