Ultrasmall bacteria from Antarctic Lake raise questions about the limits of life

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Credit: Wikimedia Commons

Credit: Wikimedia Commons

Imagine you were forced to live in perpetually subzero temperatures, with no oxygen, no light, and way more salt than your system could handle. How would you manage? One way might be to get extremely small. At least, that seems to be what’s happening in a frozen Antarctic lake that’s cut off from the rest of the world by 27 meters of perennial ice.

Lake Vida, Antarctica, has come under biological scrutiny recently. It’s an fascinating environment for a number of reasons. For one, it represents a unique combination of extreme conditions. Vida’s high salt concentrations keep the lake’s water liquid at -13.4ºC, or 7.9 ºF. And, even more intriguing, this super-chilled salt bath has been cut off from the outside world for nearly 3,000 years.

The microbial inhabitants of Lake Vida have had a unique opportunity to evolve in complete isolation. For microbial ecologists, this means a potential goldmine of novel adaptations and genetically unique organisms.

Approximate location of Lake Vida, Antarctica. Credit: Wikimedia Commons

Approximate location of Lake Vida, Antarctica. Credit: Wikimedia Commons

So far, Lake Vida’s microbes have lived up to expectations. In a study published recently in the journal Applied and Environmental Microbiology, Dr. Alison Murrary and colleagues find Lake Vida’s brine is teeming with some very tiny critters. These ultrasmall microbes, or ultramicrocells, are roughly 200 nanometers in diameter, just undercutting the theoretical “lower size limit” for a single-celled organism. In addition, these tiny critters display some fascinating adaptations for handling the stress of life in cold, salty brine.

Murray and colleagues used several techniques to characterize the ecology of Lake Vida brine samples collected in 2010, including scanning electron microscopy, spectroscopy, and x-ray diffraction.

In their recent study, the scientists observed two cell populations in Lake Vida’s brine. One population of rod-shaped bacteria ranged in size from ~0.4-1.5 µm, while a smaller class of spherical bacteria were approximately ~0.2 µm, or 200 nanometers, in diameter. This second class, designated the “ultrasmalls”, was 100 times more abundant than their larger counterparts. Even smaller particles that ranged in size from 20-140 nanometers were also abundant.

Further analysis using x-ray spectroscopy indicated that both ultrasmalls and nanoparticles had granular, iron-rich surface coatings. Interestingly, these coatings resemble iron oxide minerals found in old, weathered soils. It was also common for ultrasmalls to possess exopolysaccharides– long, filamentous proteins- connecting them to the nanoparticles.

Exopolysaccharides can serve many functions for microorganisms. In this case, the scientists speculate exopolysaccharides act as a nucleation site for iron particles- that is, a surface to which iron particles can precipitate in solid form. The resultant “iron exoskeleton” may be a unique adaptation for protection against extreme cold.

The nanoparticles remain something of a mystery, but the scientists hypothesize these may also be a part of an elaborate ultrasmall survival strategy. The size and morphology of the nanoparticles suggests they may, in fact, be extracellular membrane vesicles– pieces of cells that have popped off their parent and become self-contained storage units . Other scientists have found that microbes produce such vesicles in response to temperature stress. Like a storage unit, vesicles allow microbes to sweep their house clean, removing unnecessary clutter. One sort of unwanted baggage for the Lake Vida ultrasmalls may be misfolded proteins. Protein misfolding is a common problem in subzero environments. Harboring useless misfolded proteins represents a drain on valuable cellular resources.

Europa, Jupiter's icy moon, has excited astrobiologists as a potential site for extraterrestrial life in our solar system. Credit: Wikimedia Commons

Europa, Jupiter’s icy moon, has excited astrobiologists as a potential site for extraterrestrial life in our solar system. Credit: Wikimedia Commons

Lots of open questions remain regarding the ecology of Lake Vida’s ultrasmalls. Perhaps the biggest question is why exactly these microbes are so tiny. There are a number of possibilities to be explored. Smallness is a response to stressful environments across all domains of life. Hyperosmotic stress– the result of being bathed in a super salty liquid- may result in water loss and cell shrinkage. Or ultrasmalls may be expending so much energy dealing with the cold that they don’t have the extra resources required to grow bigger.

Answering these questions will help scientists understand how microbes may cope with extreme environments not only on Earth, but on icy extraterrestrial worlds as well.

ResearchBlogging.org

Kuhn, E., Ichimura, A., Peng, V., Fritsen, C., Trubl, G., Doran, P., & Murray, A. (2014). Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida, Antarctica Applied and Environmental Microbiology, 80 (12), 3687-3698 DOI: 10.1128/AEM.00276-14

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Resurrecting ancient microbes to understand evolution

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The ever-growing popularity of the zombie apocalypse is testament to our fascination with the idea of resurrection, or the reanimation of the dead. Credit: Robert D. Brown Inc.

The ever-growing popularity of the zombie apocalypse is testament to our fascination with the idea of resurrection, or the reanimation of the dead. Credit: Robert D. Brown Inc.

When you hear the word “resurrection”, what’s the first thing that comes to mind? Religious miracles? Zombie viruses? The end of the world?

Whatever your mental association, I’m willing to bet it’s not “an emerging scientific discipline.” Well, it just so happens a growing community of microbial ecologists are developing a new use for the traditionally mysticism-affiliated word. These scientists don’t just study modern microbes. Nor do they fashion themselves paleobiologists. No, these people actually straddle the boundary between life and death. They resurrect. And in doing so, they hope to gain insight into how evolution occurs: from single genes to entire communities.

I’ll admit Carl Zimmer beat me to this one , but the topic of resurrection ecology is too interesting and relevant for me to ignore. Zimmer’s article provides an excellent summary of many recent discoveries in the field- including the revival of a 1,500 year old Antarctic moss and the resurrection of a 30,000 year old virus frozen in Siberian permafrost. In previous posts, I’ve discussed both microbial resuscitation from ancient ice  as well as mechanisms some bacteria use to stay alive at subzero temperatures.

Today I’d like to dive a little deeper and really dissect what it is these resurrection ecologists are trying to do. Why resurrect an 8 million year old bacteria, or hatch 700 year old insect larvae? Other than the “cool” factor, is there any profound ecological or evolutionary insight to be gained from this research?

I’ll give you the punchline now: yes, there most certainly is. You see, resurrection of organisms from the past, a feat that was until quite recently considered impossible, offers scientists the opportunity to directly test hypotheses about why evolution occurs.

As an example, imagine a community of bacteria, happily swimming about in a shallow pool of water sitting on top of Siberian permafrost couple thousand years ago. One particularly cold winter, this water freezes and some of these bacteria get trapped in the ice. Perhaps it remains cold enough that the ice doesn’t melt again come spring. Over time, this ice, along with some hapless bacteria, get buried and compacted, frozen in space. But also frozen in time. While some of these guys may  have escaped cryosleep, continuing to grow, reproduce, and die, frozen bacteria don’t change. And because they remain static, evolution also freezes. No reproduction means no gene swapping, no adapting to new environmental pressures, no natural selection. Beyond their ice fortress, genetic cousins of the frozen bacteria are spreading their genes, mutating their DNA. Populations are growing, shrinking and migrating in response to their environment. They are evolving.

Fast forward to the present. Suppose you’re an ecologist studying the bacterial communities of a Siberian lake. You discover that a rare gene known as “cocA” is widespread. What sorts of selective pressures may have caused cocA to become so common? By collecting samples from the surrounding permafrost, you find traces of bacteria frozen in the ice. You extract and sequence their DNA, and determine, to your delight, these frozen organisms are a close genetic match to some of your lake bacteria. Similar, but different enough to infer that some evolution has occurred. Moreover, in all of your frozen samples, a different flavor of the cocA gene- let’s call it “colA” – is dominant.

Here’s where the resurrection part comes in. You’d really like to know how your bacterial communities shifted from colA-dominated to cocA-dominated. To do so, you try growing some of your frozen bacteria in the lab. You give them heat, carbon, and nutrients. Soon, you’ve got fossil bacteria happily multiplying on your lab bench. You have resurrected life.

 More importantly, you’ve got yourself a situation most paleontologists would kill for. You now have living replica of ancient organisms, and their  modern descendants. You set out to conduct a series of lab experiments in which you vary different environmental conditions- pH, nutrient concentrations, temperature- and monitor how your fossil bacteria change. Specifically, you track whether and how your gene of interest (colA) changes over time. What you are really doing is experimentally simulating evolution on an ancient organism. Ultimately, you might be able to determine what sorts of environmental changes must have occurred to lead to the modern, cocA-dominated descendants.

The concept of resurrection ecology is a powerful one. In no other subfield of biology can scientists directly study the ecology or evolution of an organism from a different time. Normally, scientists have to rely on fossils and other “proxies” to make inferences about past species, their ecology, and ultimately, the environment. Imagine what we might learn if we could resurrect a family of dinosaurs and tinker in the lab until we figured out what sorts of environmental conditions lead to the evolution of feathers.

Obviously this is a preposterous idea. Safety considerations aside (we all know how Jurassic Park turned out), dinosaurs reproduce too slowly to observe evolutionary change on useful timescales. Moreover their ranges are too large, and their species interactions too complex, to accurately simulate their habitats.

Microbes are a different story. They require only a petri dish to grow. They reproduce in minutes to hours. Changes in the genetic make-up of microbial communities can be observed within days or weeks. Antibiotic and pesticide resistance are powerful testimonial to the swift pace of microbial evolution.

Resurrection ecology thus represents a compelling new tool for understanding how genes, populations and communities evolve. But its significance may not be limited to the past. By informing population models with “biological archives”, scientists may be able to forecast adaption to future environmental changes. In other words, by helping scientists develop a deeper understanding of how evolution works, resurrection ecology may inform our expectations for the future, and allow us to prepare for it.

*******

A quick update: National Geographic’s cover story last week was on reviving extinct species! Check it out here.

ResearchBlogging.org

Orsini, L., Schwenk, K., De Meester, L., Colbourne, J., Pfrender, M., & Weider, L. (2013). The evolutionary time machine: using dormant propagules to forecast how populations can adapt to changing environments Trends in Ecology & Evolution, 28 (5), 274-282 DOI: 10.1016/j.tree.2013.01.009

Arctic bacteria gene swap to detoxify mercury

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Mercury, also known as quicksilver, is unusual among metals for being liquid or gaseous at room temperature. This also makes it extremely dangerous for most living organisms. Credit: iStock

Mercury, also known as quicksilver, is unusual among metals for being liquid or gaseous at room temperature. This also makes it extremely dangerous for most living organisms. Credit: iStock

On the list of substances you really don’t want to mess with, elemental mercury is pretty high up. Inhalation or absorption of the volatile metal can quickly lead to irreversible poisoning. Unfortunately, Hg is also a hallmark of industrial society. Fossil fuel combustion releases small amounts of gaseous, elemental mercury, or Hg0,into the atmosphere. Problem is, it usually doesn’t stay that way for long. Free radicals, such as hydroxyl, strip electrons off elemental mercury, converting the metal into its oxidized form, Hg2+

With two electrons missing, mercury falls out of the atmosphere, entering soils and waterways where it can travel readily into plants, animals and people, hungry to restore its charge balance. It turns out oxidized mercury has a high affinity for sulfur. Sulfur, while not abundant in our bodies like carbon or hydrogen, plays an essential role in protein structure. It forms bridges and cross-links, folding our proteins into the shapes needed to carry out their functions. Once in your bloodstream, mercury can scavenge the sulfur out of your proteins, causing them to unravel.

While most higher life forms have no defenses against the devastating effects of mercury poisoning, bacteria have evolved mechanisms to fight the deadly metal. Mercury-resistant bacteria carry a collection of Hg-detoxifying genes, including the enzyme mercuric reductase, which converts Hg2+ back into gaseous H0. How abundant are these guys, and how did mercury resistance evolve? These questions are important for understanding the natural detoxification of mercury-contaminated environments.

In a study recently published in the journal FEMS Microbiology Ecology, Dr. Annette Møller and colleagues seek answers from an unlikely place: the ice sheets and cold, briny waters of the high arctic. Why study mercury detoxifiers in the Arctic? Mercury deposition at the north pole has been increasing for decades due to mercury-enriched air currents making their way poleward from industrializing Asia. Roughly 300 tons of mercury are now deposited across the Arctic every year.

Industrial activity has greatly increased the amount of mobile mercury cycling through the biosphere.

Industrial activity has greatly increased the amount of mobile mercury cycling through the biosphere.

For their study, Møller and colleagues collected snow, fresh water and arctic sea-ice brine from Station Nord in Northeastern Greenland. To isolate mercury resisters, they grew bacteria in media containing low concentrations of mercuric chloride. Once grown, the researchers extracted DNA from their cultures. From these DNA extracts, they determined the identity of mercury detoxifiers by sequencing the 16S ribosomal RNA gene (a marker used to identify different bacterial taxa) . They also sequenced merA, the gene encoding mercuric reductase. By examining the similarity between merA genes from different bacteria, scientists can study the evolutionary history of mercury detoxification and understand how it is transferred through the environment.

In total, the researchers were able to isolate 72 different mercury-resistant bacteria, roughly 10% of the total culturable bacteria. To their surprise, the scientists only found the merA gene in 32% of their mercury-resistant isolates. This may suggest the presence of novel merA genes that are very different from the ones we already know. It could also mean arctic bacteria have different, entirely novel mechanisms of detoxifying mercury.

What of the merA genes the scientists found? merA, it turns out, is diverse, both in its genetic code and taxonomic distribution. Many distantly related bacteria posses the ability to detoxify mercury. Upon further investigation, the scientists discovered that two-thirds of merA carriers also contain plasmids– tiny circular bits of “mobile” DNA that are physically separate from the bacterium’s main chromosome. Plasmids are important agents of horizontal gene transfer, the release of tiny bits of DNA into the environment, where they can be taken up and used by others. The diverse distribution of merA, coupled with the presence of merA plasmids in some bacteria, suggests mercury-resistance may jump through microbial communities, bypassing reproduction and species boundaries.

Plasmids, small DNA molecules physically separate from a bacteria's main chromosome, can be used to transfer DNA between different microorganisms. Credit: Wkipedia commons

Plasmids, small DNA molecules physically separate from a bacteria’s main chromosome, can be used to transfer DNA between different microorganisms. Credit: Wikipedia commons

Thus, arctic microbial communities may adapt to increased mercury concentrations by swapping detoxification genes: much in the same way a piece of code can be shared to patch computer software. This is promising news for folks interested in bioremediation. By releasing mercury detoxifiers in contaminated sites, we may be able to spread the “genetic knowledge”, powering up local microbial communities to combat mercury.

ResearchBlogging.org

Møller, A., Barkay, T., Hansen, M., Norman, A., Hansen, L., Sørensen, S., Boyd, E., & Kroer, N. (2014). Mercuric reductase genes and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine
FEMS Microbiology Ecology, 87 (1), 52-63 DOI: 10.1111/1574-6941.12189

Frozen bacteria repair their DNA at -15ºC

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Bacteria encased in ice can be resuscitated after thousands, perhaps even millions of years. How these hardy bugs manage to survive deep freeze is something of a mystery.  If nothing else, the low levels of radiation hitting Earth’s surface should cause any ice-bound bacterium’s DNA to break apart over time, eventually leading to irreparable damage. Some scientists think bacteria survive cryosleep by encasing their DNA in protective shells known as spores and entering a state of dormancy. Following spore formation, a bacterium can withstand harsh environmental conditions, including desiccation, strong acids, heat and UV radiation.

Spores don't actually have eyes like they do in the popular video game, but they can resist drought, fire, ice, acid, and even antibiotics.

Spores don’t actually have eyes like they do in the popular video game, but they can resist drought, fire, ice, acid, and even antibiotics.

But other researchers think we aren’t giving enough credit to the ice dwellers. Recent studies have shown that some psycrhophiles– technical-speak for cold-loving bacteria – are able to maintain basic metabolic functions at subzero temperatures. Could psychrophiles trapped in ice be repairing their DNA faster than the UV radiation bombarding our planet pulls it apart? Microbiologist Markus Dieser at Lousiana State University was interested in finding out. In a study published  in the journal Applied and Environmental Microbiology, Dieser and colleagues show for the first time that one bacteria- Psychrobacter arcticus– can repair it’s DNA at temperatures as low as -15ºC, or 5ºF. Moreover, it can do so 100,000 times faster than damage occurs.

P. articus is an innocuous little bacteria that is famous for one thing: it really likes the cold. It can grow and metabolize at -10 ºC, making it one of the most psychrophilic organisms on Earth. To investigate P. articus’’s ability to repair DNA in deep freeze, Dieser and colleagues isolated viable P. articus cells from Siberian permafrost that has been frozen for 20 to 30 thousand years. In the lab, the researchers dosed their cell cultures with a large pulse of ionizing radiation- roughly equal to what P. articus might experience over 225 thousand years of field exposure. By using such an intense burst of radiation, the team hoped to induce many “double-strand breaks”, or breaks that cause small DNA fragments to separate off from P. articus’s main chromosome.They incubated the irradiated cultures at -15ºC and monitored their survival over the course of 505 days.

Rather astoundingly, the scientists found no significant difference between the survival rates of irradiated and non-irradiated bacteria over the year and a half long study. While this finding alone suggests P. articus can repair its DNA at subzero temperatures, Dieser and colleagues wanted direct evidence.  They used pulse-field electrophoresis, a technique which separates DNA fragments by size, to determine how may DNA double-strand breaks occurred after radiation exposure, and whether the DNA fragments reassembled themselves over time. Like Humpty Dumpty rebuilding himself, the scientists could literally watch P. articus reassemble its genome. On average, P. articus was able to patch thirteen double-strand DNA breaks over the course of the study-  quite close to the roughly sixteen breaks inducted by radiation.

DNA ligase repairing a DNA molecule that has suffered a double-strand break. Credit: Wikipedia

DNA ligase repairing a DNA molecule that has suffered a double-strand break. Credit: Wikipedia

Not only can P. articus repair its DNA at subzero temperatures, it can do so really fast. Using annual radiation exposure data collected in the field, Dieser estimates that P. articus can repair double-strand breaks 100,000 times faster than they occur. The discovery has important implications for the survival of life in extreme environments, including cold extraterrestrial environments. For instance on the surface of Mars, where radiation levels are ~400 times greater than the Siberian permafrost, P. articus can still patch DNA breaks 280 times faster than they would accrue. As scientists continue exploring the “cold limit” to essential cellular functions such as DNA repair, they will continue to refine, and perhaps expand, our understanding of the fundamental boundaries for life.

ResearchBlogging.org

Markus Dieser, John R. Battista, & Brent C. Christner (2013). DNA Double-Strand Break Repair at −15°C Applied and Environmental Microbiology DOI: 10.1128/AEM.02845-13

Cryogenics, gene popsicles and the oldest life on Earth

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While the notion of “cryogenic freezing”, or putting a person into a state of frozen suspension, has been a common theme in science fiction for decades (think the Alien movies, Star Wars, Sleeper, Vanilla Sky) bacteria have probably been doing their own version of cryogenic sleep for billions of years.

Researchers studying ice cores from the Dry Valleys of Antarctica have found viable, frozen bacteria that are thousands to millions of years old. The ice in this region of the Dry Valleys ranges from modern to about ten million years old, making it some of the oldest known ice on earth. By analyzing the ice crystal structure and isotopic data, these researchers determined their ice samples had likely been permanently frozen (i.e., no thawing/refreezing), implying that the bacteria encased within the ice have been trapped since its formation.

Resuscitation

The scientists incubated meltwater from ice core samples at temperatures just above freezing for up to 300 days, adding supplemental nutrients to encourage bacterial growth. The samples they incubated represented a broad range of timescales, with ages ranging from 10,000 years to 8 million years. Astoundingly, bacterial growth was observed in all samples, though growth rates declined with sample age: bacteria that had been encased in ice for shorter periods of time grew much more rapidly than bacteria frozen for millions of years.

Caveats to cryogenic

The study concluded that even bacteria cannot maintain cryogenic preservation forever. In addition to slower growth rates for older bacteria, the study found an exponential decline in the size of the community DNA pool over time, suggesting the DNA is slowly degrading, even in a deep freeze. Very slowly. The estimated half-life for the reduction in DNA pool size (i.e., the amount of time it takes to reduce the amount of DNA in a sample by 50%) was 1.1 million years. (I think I just heard the microbial ecologists breathe a collective sigh of relief.) So, it may be perfectly reasonable to find frozen bacteria that are hundreds of thousands of years, even a couple million years old, that can still be resuscitated.

{An aside: why does DNA degrade, even in a deep freeze? The jury is still out, though one suspect in the present study is cosmic radiation (high-energy particles that bombard the Earth from space). Antarctica receives the highest levels of incoming cosmic radiation on the planet.}

Gene popsicles in a melting world

Bacteria encased in ice for thousands to millions of years are literally a gene bank. Collectively, the community DNA frozen in ice can be thought of as a “gene popsicle” that provides a snapshot into the past and another clue scientists can use to piece together ancient Earth environments. Moreover, it is well known that bacteria are capable of transferring genes amongst each other in a process known as lateral gene transfer. Could the periodic melting of ice sheets, due to shifts from glacial to interglacial periods, result in an influx of ancient genes into modern bacterial communities? Could genetic information perhaps be preserved for hundreds of millions, or even billions of years, through freezing, melting, and re-uptake of ancient genes by living bacteria?

And finally, the million dollar question: what are the implications for of melting gene popsicles on present-day Earth? As glaciers and ice sheets across the world continue to melt due to climate change, will hordes of ancient bacteria start to “wake up”? Could they plague the world with ancient diseases that no modern humans have resistance to? (Hmm…sounds like a good idea for a science fiction story 🙂 The answer to the former question is, probably yes, the latter, probably not. But time, and a lot more research on the microbial ecology of melting ice sheets is needed to answer these questions.

Journal reference: Proceedings of the National Academy of Sciences (DOI: 10.1073/pnas.0702196104)