Rocks and their microbes: a co-evolutionary partnership

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Miles beneath our feet, Earth’s rocky crust may seem a cold, dead place. On closer inspection it’s anything but.

Microbes have been making a home on and in rocks since…well, since the beginnings of life, some 3.5 billion years ago. The traditional view of rock-dwelling microbes is one of sparse, energy-starved survivalists eking out an existence on a hostile frontier. These critters are at the mercy of their environments, their numbers dictated by physical factors – things like temperature and pH.

Geobacter, a rock-dwelling bacteria coating iron oxide minerals. Credit: Wikipedia

Geobacter, a rock-dwelling bacteria coating iron oxide minerals. Credit: Eye of Science

A study published in Geomicrobiology Journal, led by graduate student Aaron Jones at the University of Texas at Austin, challenges the assumption that subsurface microbes are passive survivalists. In a series of lab experiments, Jones demonstrates that mineral-dwelling microbial communities are uniquely adapted to their environments on the scale of individual mineral grains. Moreover, his research suggests microbes and minerals share a partnership that stretches back through geologic time.

Jones was interested in how mineralogy- the composition of the minerals that form rocks- influences the abundance and diversity of microbes. In particular, he was interested in sulfur oxidizers- bacteria that strip electrons from sulfur to acquire energy. Sulfur oxidizers are common in other subsurface environments including deep ocean vents. Their metabolism generates strong acids that eat away at certain minerals, forming cracks, pores, even caves.

In the lab, Jones set up a series of “bioreactors”- tanks in which liquid of a specific chemical composition flows over thin sheets of rock. He used a range of rocks that differ in their mineralogy. These included several rocks composed of calcium carbonate (limestone, dolostone and calcite) and several made of silica-rich minerals (albite, microcline and quartz). Jones inoculated his bioreactors with samples of a sulfur-metabolizing biofilm collected from a Wyoming cave spring.

Example of a benchtop bioreactor. Bioreactors are commonly used in microbial biotechnology for cell culturing and fermentation. Credit: Wikimedia Commons

Example of a benchtop bioreactor. Bioreactors are commonly used in microbial biotechnology for cell culturing and fermentation. Credit: Wikimedia Commons

After three weeks of incubation, he measured bacterial growth, extracted DNA and used pyrosequencing to examine the composition and diversity of the microbial communities formed on different rock types.

Different rocks, different microbes

Growth patterns differed substantially across the carbonate and silicate rocks. On carbonates, bacteria grew in thick, filamentous biofilms. By contrast, growth was much sparser on silicates, with bacteria either accumulating in small clumps or spreading out individually.

Community composition also differed among different rocks. The bacterial phyla Alphaproteobacteria dominated on most carbonates. Sulfur oxidizers that thrive at near-neutral pH were common, representing 17-45 % of the community.

A different picture emerged on the silicate rocks. Members of the phyla Firmicutes and Actinobacteria- also known as Gram-positive bacteria– were found here. While community composition was relatively similar among the carbonates, communities that grew on quartz were very different than those found on albite or microcline. Jones observed fewer sulfur oxidizers on silicates, and those he found tended to be more acid-tolerant.

Composition of bacterial communities grown on different mineral surfaces. From Jones & Bennet 2014.

Composition of bacterial communities grown on different mineral surfaces. From Jones & Bennet 2014.

Perhaps the most obvious conclusion here is that microbes do not select rocks at random. Different growth patterns among rocks tell us this. Better microbial growth on carbonates probably reflects a higher density of essential nutrients, particularly phosphorus. Moreover, two of the silicate rocks, albite and microcline, contain high concentrations of aluminum, a toxic element that restricts bacterial growth.

More subtly, however, there appears to be “selective relationship” between mineralogy and microbial ecology. In other words, the microbes making a living on any given rock are uniquely adapted to taking advantage of, or protecting themselves from, the specific chemical properties of that rock.

This relationship bears itself out in many ways. For one Jones found more acid-tolerant microbial communities on silicate rocks. Remember, acid is a byproduct of sulfur metabolism. Carbonate rocks have a high “buffering capacity”, meaning their minerals react with and neutralize acid. By contrast, silicate rocks, especially quartz, are pretty unreactive. Rather than eating away at minerals, acid accumulates at silicate surfaces. The presence of acid-loving bacteria on quartz suggests a feedback loop between sulfur metabolism, mineralogy and microbial community structure.

The presence of Gram-positive bacteria on silicates also underscores the relationship between microbial community composition and the mineral environment. The cell walls of Gram-positives possess unique chemical properties that promote bonding with silicate surfaces. This microbe-mineral binding, known as adhesion, is a key element of survival for most rock-dwelling bacteria. By adhering to a solid surface, bacteria can mine nutrients and form protective biofilms.

Even microbes living on the undesirable aluminum-rich rocks may possess unique adaptations. While few bacteria grew on albite or microcline, these sparse populations were highly diverse. The unique challenges presented by a toxic environment may serve to maintain competition, preventing any single group of organisms from gaining dominance.

Our planet’s rocky subsurface is the largest microbial habitat, but we still know very little about its inhabitants. Most microbial ecologists prefer to study environments they can see, feel and intimately relate to. Jones’s research points to complex ecological interactions between microbes and minerals in the deep, dark depths of the world. Rocks shape microbial communities, but microbes may also shape rocks in profound ways. This study lends evidence to a deep synergy between the geological and biological evolution of our planet.

ResearchBlogging.org

Jones, A., & Bennett, P. (2014). Mineral Microniches Control the Diversity of Subsurface Microbial Populations Geomicrobiology Journal, 31 (3), 246-261 DOI: 10.1080/01490451.2013.809174

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Fossil viruses preserved in hot spring bacteria

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Viruses, such as the Avian flu virus depicted in this scanning electron microscope image, are neither alive nor quite dead, but may have a lot to tell us about the evolution of life on Earth. Credit: 3DScience.com

Viruses, such as the Avian flu virus depicted in this scanning electron microscope image, are neither alive nor quite dead, but may have a lot to tell us about the evolution of life on Earth. Credit: 3DScience.com

Fossilized microbes have provided scientists many clues about origins of life. By comparison, little attention is given to viruses in the fossil record. Although technically non-living, there is no question these tiny packets of protein-sheathed DNA have shaped the evolution of most life on earth, including humans. But can viral particles, a fractional size of even the smallest bacteria, actually become fossils? A study recently published in the journal Geobiology argues they can. Here, scientists present the first experimental evidence of silicification– the encasement in silicate minerals- of viruses living in hot spring biofilms. Moreover, these viruses can become fossils while still inside their host cells.

Before we get into viral fossilization, let’s back up for a minute and briefly review what a virus is. Viruses are simple beings that strip the notion of life down to a single, fundamental purpose: propagation of genetic code. While sharing this feature with all life on Earth, viruses are not considered living because they lack the means to carry out genetic reproduction on their own. Instead, viruses propagate by injecting their genetic material into a host cell. One inside a host, a virus uses one of two strategies, or “life cycles”, to reproduce. In the lysogenic cycle, viral DNA integrates with host DNA, lying low and allowing itself to be replicated along with the oblivious host cell. In our own chromosomes, many introns, or non-coding pieces of “junk DNA”, are thought to be ancient viral particles that “went to sleep” inside our cells and never woke up. In the more active (and deadly) lytic cycle, the virus co-opts the host cell’s replication machinery, creating a virus factory that eventually spews forth thousands of baby viruses which proceed to infect other cells.

Conceptual model of the two general modes of viral biology: lytic and lysogenic cycles. Credit: allbiologytutors.blogspot.com

Conceptual model of the two general modes of viral biology: lytic and lysogenic cycles. Credit: allbiologytutors.blogspot.com

Which of these cycles a virus chooses depends a lot on the external environment. If conditions are good in the host cell, it may be advantageous to hunker down and stay put. If, on the other hand, the host cell is already sick or dying, the virus may prefer to replicate as much as possible before abandoning ship.

Hot springs host communities of highly specialized extremeophiles. These bugs are not only hyperthermophilic, or super heat-tolerating, but often cope with extremely acid or alkali conditions. One way they do so is by modifying the pH within their cells, making it closer to neutral. Perhaps unsurprisingly, most hot spring viruses follow a lysogenic cycle, enjoying the less-extreme environment offered by their host.

It was in one such strange habitat, the Gumingquan hot spring in the Yunnan province of southern China, that Dr. Brian Jones and colleagues decided to investigate viral silicification. Why study fossilization in a geothermal hot spring? Geothermal waters, heated by subsurface magma from the Earth’s mantle, are often rich in silica and other minerals, such as calcium and iron, that promote fossilization. Moreover, thick, diverse biofilms hosting “virus-like” particles are known to inhabit the Gumingquan hot spring.

For their study, the researchers collected samples of these biofilms and brought them back to the lab. From biofilms containing many different Bacteria and Archaea, the scientists isolated Geobacillus lituanicus, a thermophilic, aerobic (oxygen-breathing) bacteria belonging to the phylum Firmicutes. They also isolated viral particles by liquifying samples of biofilm, separating out and removing all cells, and filtering the remaining liquid through a 0.22 µm filter- large enough to include viruses but small enough to remove most other microbial debris. Spherical, “virus-like” particles were further isolated and purified by infecting G.lituanicus cells with “viral extract”, and growing the infected cells on agar.

Once the researchers had purified both a host and virus-like particles, they set up a rather clever experiment to test for viral fossilization. They concocted a growth media for G. lituanicus containing sodium metasilicate, a silica-rich mineral. After adding G.lituanicus cells and virus-like particles to this media, they incubated the mixture for 22 days. At regular intervals, the scientists took samples and used transmission electron microscopy (TEM) to search for silicified viruses.

Using TEM, the scientists found numerous silica nanoparticles both inside and outside G. lituanicus cells. These circular particles consist of a core roughly 35-60 nanometers (nm) in diameter, surrounded by an outer shell approximately 30 nm thick. While these particles may just be small, silicified fragments of cellular detritus, there is good reason to think many of them are fossil viruses. For one, the fossil “cores” are similar in size and shape to the spherical virus-like particles used in the experiment. The silica nanoparticles had a “near normal”, or bell-curve, size distribution, another hallmark of biology. Finally, some of the silica nanoparticles had tails.

TEM images showing silica nanoparticles inside and outside cells. Note the light colored "virus-like cores" clearly visible in panels c and d, surrounded by a silicious shell. Credit: Peng et al. 2013

TEM images showing silica nanoparticles inside and outside cells. Note the light colored “virus-like cores” clearly visible in panels c and d, surrounded by a silicious shell. Credit: Peng et al. 2013

A few of you might be scratching your heads at this point, thinking something seems amiss. Remember, I told you most hot spring viruses are lysogenic (integrated into host DNA) most of the time. But in order for a virus to fossilize, it must have its own, independent structure. In other words, it must be lytic.

Does the discovery of fossil viruses within host cells negate the idea that hot spring viruses are typically dormant? Not necessarily. Actually, it may offer an explanation for when and why viral dormancy occurs. You see, all cells eventually die due to accumulated environmental stress. When a hot spring bacteria like G. lituanicus dies, its cell wall breaks open, causing hot, mineral-rich geothermal fluids to rush in. Suddenly, any viruses inside are exposed to all sorts of elements- heat, acid, etc.- they can’t necessarily tolerate. By precipitating a silica shell, these viruses may be able to survive long enough to find a new host.

Some of the proteins on a virus’s outer surface contain chemically-reactive molecules, such as carboxyl groups, that may aid in silica encrustation. The host cell itself may inadventently promote viral fossilization. Many alkaliphiles, like those in the alkaline Gumingquan waters, maintain their cellular pH at two or more units lower than the external environment. Geothermal waters that are undersaturated with silica at pH 12 may become supersaturated at pH 9 or 10, causing silicates to precipitate out as solids.

The strong evidence for viral silicification presented in this experiment suggests viruses may be preserved in the fossil record, particularly in rocks associated with alkaline geothermal systems. Some of the features of virus-like fossils noted in this study- distinct cores and shells, fossil “tails”, a concentration of fossil-like particles inside a host cell- may be useful for identifying the footprints of ancient viruses in the wild.

ResearchBlogging.org

Peng, X., Xu, H., Jones, B., Chen, S., & Zhou, H. (2013). Silicified virus-like nanoparticles in an extreme thermal environment: implications for the preservation of viruses in the geological record Geobiology DOI: 10.1111/gbi.12052

Spaceflight increases growth and affects community behavior of a bacterial pathogen

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Structured aggregates of bacteria known as biofilms are often able to survive under harsher environmental conditions than their free-living counterparts. One common problem resulting from biofilm hardiness is increased pathogenicity and drug-resistance. From common medical ailments such as urinary tract infections and cavities to life-threatening diseases such as cystic fibrosis and endocarditis, biofilms are often resistant to even the most potent antibiotics.

Electron micrograph image of a biofilm at 21, 850x magnification. Biofilms typically consist of structured communities of bacteria and an extracellular polysaccharide matrix that promotes surface adhesion.

Electron micrograph image of a biofilm at 21, 850x magnification. Biofilms typically consist of structured communities of bacteria and an extracellular polysaccharide matrix that promotes surface adhesion.

The hardiness and pathogenicity of biofilms poses additional risk for humans in one particularly extreme environment: space. On the Mir space station that operated in low Earth orbit until 2001, biofilms were abundant and tenacious, interfering with basic shipboard functions such as water purification. Furthermore, the little research to date suggests bacteria that grow in “microgravity”, the extremely low gravity conditions present in and on the surface of spacecrafts, can be more virulent than their Earth-bound counterparts. Unfortunately, human immune functions are also reduced in space. This unique situation creates a seemingly perfect storm of conditions under which, were a pathogenic species to proliferate, the results could be devastating.

The mir space station, owned and operated by the Soviet Union and then Russia, experienced problems with biofilms for the 15 years it was in orbit

The mir space station, owned and operated by the Soviet Union and then Russia, experienced problems with biofilms for the 15 years it was in orbit

NASA has recognized for years that a better understanding of how biofilms survive in microgravity is important for the success of any long-term manned space missions. But little is known about the development and physiology of biofilm mats in microgravity, making it hard to predict the likelihood of a pathogenic outbreak. To investigate this, scientists cultured Pseudomonas aeruginosa, a common model organism and a human pathogen, on two Space Shuttle Atlantis missions under microgravity conditions. They simultaneously ran ground control cultures at the Kennedy Space Center to compare biofilm growth rates, cell densities and architecture.

Conceptual model of the stages of P. aeruginosa biofilm development. Courtesy of MicrobeWiki

Conceptual model of the stages of P. aeruginosa biofilm development. Courtesy of MicrobeWiki

The results were startling. The number of viable cells, total biomass, and the thickness of biofilms were all significantly higher on spaceships than on the ground. This result was robust regardless of the carbon source or level of nutrients provided to the bacterial culture.

Depending on the organism and environmental conditions, biofilms can organize themselves in a variety of ways and produce characteristic structures. On Earth, P. aeruginosa biofilms are flat on static surfaces and mushroom-shaped under hydrodynamic conditions (i.e., with water flowing across them). Using confocal laser-scanning microscopy, the researchers compared the structures of P. aeruginosa biofilms grown in spaceflight and normal gravity. While under normal gravity P. aeruginosa formed flat biofilms, in space biofilms exhibited a novel “column and canopy” structure, with cells aggregating in tight-knit columns close to the adhesion surface, overlaid with a dense, canopy-like mat furthest from the surface.

Cellular flagella and pili important in maintaining biofilm structure and surface adhesion under normal gravity. A study recently published in Science even suggests that the appendages known as type IV pili may allow bacteria to stand upright and “walk” during the early stages of biofilm development. To test whether such cell structures are also important in space, the researchers created mutant strains that lacked the genetic capacity to produce flagella or pili. While mutants lacking pili were still able to produce column and canopy biofilms, mutants lacking flagella were not. Thus, flagellar motility appears to play a key role in the formation of unique column and canopy structures in space.

Artistic conception of a bacteria using type IV pili to walk during early biofilm development. Credit: UCLA

Artistic conception of a bacteria using type IV pili to walk during early biofilm development. Credit: UCLA

This study is the first carefully replicated scientific experiment demonstrating that spaceflight affects the community-level behavior of bacteria. The findings described here carry the profound implication that microbes are capable of adapting to non-terrestrial environments through altered behavioral patterns. More studies are clearly needed to understand the full range of behavioral adaptations exhibited by bacteria in microgravity, but it is clear any future long-term space missions will have to take these tiny organisms- and their potential human health risk- seriously.