Microbial highlights from ESA 2014

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The Ecological Society of America (ESA) annual conference, once dominated by discussions of lions, tigers and bears (or so I’ll pretend), has become a convergence point for microbial ecologists. From amphibian skin microbiomes to biological soil crusts and more, the diversity of microbial talks this year was astounding.

As the conference winds down, I wanted to share just a few of the coolest microbial findings that stuck with me from the last couple days at ESA. What was your favorite microbial talk of the week? Post about it in the comments, or better yet, tweet about it! And don’t forget to tag me (@themadstone) as well as the official ESA microbial ecology twitter (@ESAmicrobe), if you do 😉

Can dormancy explain microbial biogeography?

Dormancy is a condition that afflicts the best of us.

Dormancy is a condition that affects the best of us.

Dr. Jay Lennon gave a great talk on dormancy; a subject that’s particularly fascinating to me in light of the vast (and probably rather dormant) microbial populations living in deep, subterranean environments.

Dormancy, otherwise known as metabolic inactivity, is an important strategy for surviving stressful environments. Conditions such as extreme drought, cold, or lack of food can cause microbes to enter a dormant state. Dormant microbes are fascinating not only for their ability to survive extreme conditions, but to do so for thousands to millions of years.

Lennon’s research group is particularly interested in applying principles of biogeography- how diversity scales across space- to microbial ecology. But understanding spatial patterns in microbial diversity is tricky because:  1) microorganisms are enormously diverse compared with macroorganisms and 2) unlike, say, mountain lions, which may be very genetically similar across kilometer scales but diverse over thousands of kilometers, genetically distinct populations of microbes can exist at the millimeter scale. Scientists find that microbial communities in soil samples taken 1 centimeter apart from each other can be up to 90% genetically distinct.

Here’s where dormancy comes in. Lennon’s group can separate the active from dormant members of microbial communities, by looking at the number of RNA transcript copies for a ubiquitous bacterial gene. RNA transcripts, the intermediate product between a DNA “blueprint” and an active protein, are well correlated with metabolic activity. Lennon finds differences in how diversity scales with space when examining the dormant versus active portions of a microbial community. Simply put, dormant microbial communities are more homogenous across space than their active counterparts. Furthermore, active microbial communities tend to become dominated by select taxa, probably those with some fitness advantage in the local environment. Dormant communities are less likely to be dominated by a few individual taxa, but rather represent a well-distributed “seed-bank” of genetic diversity.

The difference in how dormant versus active microbial communities scale with space may seem like a nuance, but this information could go a long way toward helping ecologists understand the distribution of microbial diversity across our planet. Lennon’s results suggest dormant microbes may represent up to 40% of a community. That’s no small portion of the total genetic diversity. Furthermore, dormant microbes, as a potentially long-term seed-bank, may help replenish the diversity of a community following extinction events.

Pine needle bacteria that soak up nitrogen

Scots Pine, Poland. Pine needles may host communities of nitrogen fixing bacteria that aid in survival in low-nitrogen environments. Credit: Encyclopedia of Life

Scots Pine, Poland. Pine needles may host communities of nitrogen fixing bacteria that aid in survival in low-nitrogen environments. Credit: Encyclopedia of Life

Did you know that nearly all leaves on Earth host their own microbiomes? Neither did I! But perhaps it’s not too surprising, given we’re now studying the microbiomes of cell phones and sewers. Anyway, it’s pretty cool. It’s extra cool in alpine ecosystems, where scientists are finding that these leaf-dwellers may play an important role in making nitrogen available to plants.

Dr. Carolin Frank’s lab studies nitrogen cycling in high, cold alpine ecosystems dominated by coniferous trees. Alpine ecosystems have long puzzled ecologists, because their soils and vegetation often contain more nitrogen than can be easily explained. Dr. Frank and colleagues hypothesized the “missing nitrogen” might come from nitrogen fixers- bacteria that carry a special enzyme which allows them to convert atmospheric N2 gas to ammonia- found in an unlikely place.

The team went out to a high-altitude forest in Colorado, collecting pine needles and bringing them back to the lab. In the lab, they placed the needles in sealed jars and added a chemical called acetylene. Acetylene, a simple carbon compound, can be chemically converted by nitrogenase, the same enzyme used to fix dinitrogen gas. This is a quick and cheap way of assessing whether a sample contains the nitrogenase enzyme, and hence nitrogen-fixing bacteria.

After finding that the pine needles did indeed host nitrogen fixers, the group used genetic techniques to look more closely at who exactly is there. The most prominent bacteria in all pine needle samples was genetically similar to Gluconacetobacter -a nitrogen fixer found in sugarcane.

So, nitrogen fixers are living in pine needles and helping their host trees take up nitrogen. If replicated in other coniferous forests, these findings could transform our understanding of alpine and boreal ecosystems, helping explain how coniferous trees persist on very nitrogen-limited soils.

Ocean acidification and marine calcifiers: it’s more complicated than you thought.

Coccolithophore, a single celled marine phytoplankton that produces calcium carbonate scales. Credit: Encyclopedia of Life

Coccolithophore, a single celled marine phytoplankton that produces calcium carbonate scales. Credit: Encyclopedia of Life

Or at least, it’s much more complicated than an ignorant terrestrial scientist like myself thought. See, my understanding of ocean acidification went something like this: more CO2 in the atmosphere means more CO2 dissolving in the surface ocean. When this CO2 dissolves, it reacts with water to produce acid, lowering the pH of the ocean. This is bad news for organisms that build exoskeletons from calcium carbonate (CaCO3), because the protons that create acidity get in the way of calcium carbonate formation. Sound good?

Well, like most natural phenomena when examined closely, the answer is quite a bit more complicated. Dr. Deborah Iglesias-Rodruigez enlightened me today as to some of that complexity. Iglesias-Rodruigez studies Coccolithophores, a common group of marine phytoplankton (photosynthetic organisms) that precipitate a beautiful, calcium carbonate exoskeleton. But in order for these little guys to create their exoskeletons, they must first take up some form of carbon into their cells.

It turns out Coccolithophores have evolved to take up the predominant form of carbon found in seawater: bicarbonate, or HCO3. And it also turns out this is the particular form of carbon that becomes more abundant during ocean acidification. So- does that mean acidification is good for calcium carbonate formation, after all?

Well, not quite. Although Coccolithophores seem to take up more bicarbonate under more acidic conditions, this isn’t exactly a good thing. Iglesias-Rodruigez finds that, in acidic conditions, Coccolithophores can start over-calcifying: precipitating more calcium carbonate than they need to, which is energetically wasteful. Moreover, once that calcium carbonate is precipitated, it gets slowly eaten away by the more-acidic seawater.

There was more detail to be found in this presentation, including some interesting stuff about differences in microbial cell volume mediating ocean acidification effects, but I’ll leave that for another day. One ocean chemistry lesson today was enough for my brain to juice on.

 

Could magnetic bacteria be the next generation of microbots?

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The cutting edge of robotics may not be a smarter Siri or a less-creepy humanoid Japanese robot. It might be a swarm of bacteria, compelled to do our bidding through a remotely controlled magnetic field.

Some of the biggest technological advances of the past two decades have involved scaling things down. The development and continual improvement of microprocessors has revolutionized home computing. And the emerging field of nanomedicine promises to transform biomedical science in many ways: from targeted drug delivery to tissue repair at the cellular level.

However, working at the microscale (thousandths of a millimeter) or nanoscale (millionths of a millimeter) poses major challenges. To create a circuit out of microscopic components, we need very small tools that can do the work for us. We need microscopic robots, or microbots.

Artist's depiction of a nanobot performing cell surgery. Credit:

Artist’s depiction of a nanobot performing cell surgery. Source: yalescientific.org 

But building useful microbots is no easy task. Power supply has always been one of the toughest challenges. Some bots contain a very lightweight battery. Others possess a coin cell that scavenges vibrational or light energy from its surroundings. Telling our microbots where to go is another hurdle. Microbots typically don’t work alone- we usually need a swarm of them to perform a task. And we’d like to be able to direct that swarm with the utmost precision.

Some scientists think the solution to our microbot woes can be found in nature. Bacteria are essentially organic “machines” that use light or chemical energy to move about and do work. They fit the size criteria, and they’re everywhere. Rather than re-invent the wheel, what if we could train bacteria to do work for us?

To answer this question, microroboticists are now looking to a group of bacteria that possesses an astounding property: magnetism.

So-called magnetotactic bacteria are promising because their motion can be guided by an externally applied magnetic field. Given the right magnetic field, we might be able to coordinate the motion of thousands to millions of magnetotactic bacteria at once.

Source: iGEM2009

Source: iGEM2009

Magnetotactic bacteria are a genetically diverse group of organisms thought to have evolved during the early Proterozoic, some 2.5 billion years ago, when rising atmospheric oxygen concentrations were reducing the amount of dissolved iron in Earth’s oceans. It’s believed this caused some bacteria to start stockpiling iron. Eventually, these iron stores were adapted to form magnetosomes, crystalline structures found in the cell membranes of modern magnetotactic bacteria.  Magnetosomes align in a chain, allowing the bacterium to orient itself like a compass needle to the local magnetic field.

The most obvious advantage to a magnetotactic microbot is that its motion can be guided by a user-generated magnetic field. But bacteria are also advantageous because they possess flagella. These rotating, tail-like structures act as a propeller, allowing bacteria to swim about quickly and change direction with ease. And the energy required to turn a flagellum is generated by the bacterium’s own metabolism. Power source, check. Motor, check.

Helicobacter pylori electron micrograph, showing multiple flagella on the cell surface Electron micrograph of H. pylori possessing multiple flagella (negative staining). Credit: Wikipedia

Helicobacter pylori electron micrograph, showing multiple flagella on the cell surface Electron micrograph of H. pylori possessing multiple flagella (negative staining). Credit: Wikipedia

So, what’s it going to take for magnetotactic bacteria to become our microbots of choice? One challenge scientists are now focusing on is 3-D aggregation. Using a single electromagnet, it’s simple business to line a bunch of magnetic bacteria up in a 2-D sheet. Forming a 3-D swarm is more challenging. But if doable, 3-D configurations would have major advantages. A 3-D swarm would be much easier to guide through the intricacies of the human vascular system. It would also be able to build 3-D objects more efficiently.

In a paper recently published in the Journal of International Robotics Research, scientists used Magnetococcus marinus, a spherical, magnetotactic bacterium possessing 2 bundles of flagellar “propellors”, to explore the possibility of creating 3-D bacterial clusters with magnets.

Using remotely powered pairs of electromagnetic coils, the researchers applied three different time-varying magnetic field sequences to a liquid culture of M. marinus. They were able to generate both 2-D and 3-D bacterial configurations. Then they managed to steer these swarms through a complex network of glass tubes intended to mimic capillaries.

These basic steps are laying the foundation of being able to one-day guide armies of magnetotactic bacteria to carry out many tasks. In the future, swarms of magnetic “biobots” might be used for targeted cancer treatments, transporting microscale objects, assembling microcircuits, and even microscale magnetic resonance imaging.

ResearchBlogging.org

de Lanauze, D., Felfoul, O., Turcot, J., Mohammadi, M., & Martel, S. (2013). Three-dimensional remote aggregation and steering of magnetotactic bacteria microrobots for drug delivery applications The International Journal of Robotics Research, 33 (3), 359-374 DOI: 10.1177/0278364913500543

From farm to table: insects as a conduit for antibiotic resistant bacteria

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The love affair between industrial agriculture and the antibiotic industry has come into an uncomfortable spotlight of late. In 2011, 7.7 million pounds of antibiotics were sold to treat sick people in the United States. This compares with a whopping 29.9 million pounds of antibiotics fed to cattle, pigs and poultry.1 Regular antibiotics doses keep perpetually overcrowded animals from falling ill and dying en masse, but antibiotics are also widely used to hasten growth, shortening an animal’s time to slaughter and increasing profit.

Concentrated animal feeding operations, or CAFOs, have come to dominate the meat industry over the past fifty years. Swine operations such as the one depicted here represent an enormous source of environmental pollution and are a breeding ground for antibiotic resistant bacteria. Credit: Wikimedia commons

Concentrated animal feeding operations, or CAFOs, have come to dominate the meat industry over the past fifty years. Swine operations such as the one depicted here represent an enormous source of environmental pollution and are a breeding ground for antibiotic resistant bacteria. Credit: Wikimedia commons

What’s the consequence of all this unfettered antibiotic use? Multi-drug resistant strains, or “superbugs” are on the rise. Our ability to keep pace with resistance by producing new antibiotics is diminishing. It’s even been suggested that we’re now entering a post-antibiotic era.

In 2010, representatives of the FDA, U.S. Department of Agriculture and Center for Disease Control and Prevention testified before Congress that a definitive link exists between the overuse of antibiotics in animal agriculture and antibiotic resistant diseases in humans.

Credit: pewhealth.org

Credit: pewhealth.org

But in spite of mounting evidence, the meat industry has largely succeeded in lobbying against any antibiotic restrictions. A major thrust of the industry’s argument is the lack of direct evidence linking antibiotic resistant bacteria bred on animal farms to human disease.

Now, proponents of antibiotic regulation may have some powerful new evidence to fuel their case. Microbial ecologist Ludek Zuerkand colleagues at Kansas State University are finding that insects- particularly houseflies and cockroaches- may represent the missing link between animal farms and human population centers.

Their review paper on insects and antibiotic resistance is currently in press in the journal Applied and Environmental Microbiology.

Zurek’s research team focuses on Enterococci, a group of bacteria responsible for illnesses ranging from urinary-tract infections to meningitis. Enterococci are also rather infamous for developing multi-drug antibiotic resistance. In one study, researchers measured the abundance of Enterococci in two swine production facilities in Kansas and North Carolina. The scientists examined houseflies, roaches and pig feces collected at both sites, finding Enterococci in 89% of all samples. Multi-drug resistant strains were found everywhere. Moreover, the drug-resistant strains found in flies and roaches were genetically identical to the strains found in swine feces, indicating insects acquired their pathogens from pigs.

In another study, the researchers screened houseflies collected from five fast food restaurants in a town in northeastern Kansas. Ninety seven percent of flies harbored Enterococci. The most abundant strain, Enterococcus faecalis, showed resistance to broad-spectrum antibiotics including tetracycline, erythromycin, ciprofloxacin and kanamycin. The scientists also identified transposons– snippets of DNA bacteria can swap during conjugation, their version of sex- that are associated with antibiotic resistant traits.

Ready-to-eat food from the same restaurants was also contaminated with antibiotic-resistant bacteria. Contamination was higher in summer than winter, corresponding with increased numbers of houseflies in restaurants.

From these investigations, the researchers concluded that “food served in restaurants is commonly contaminated with antibiotic-resistant Enterococci and that houseflies may play a role in this contamination.”

The common housefly may be more than just a nuisance: new research highlights this insect's important role in spreading antibiotic resistant bacteria.

The common housefly may be more than just a nuisance: new research highlights this insect’s important role in spreading antibiotic resistant bacteria. Credit: Wikimedia commons

Not wishing to lose points for a lack of thoroughness, the scientists decided to test directly whether insects from animal farms can contaminate food. In another study, they collected flies from a cattle feedlot and brought them back to the lab. Within thirty minutes, the flies deposited roughly 1,000 antibiotic-resistant Enterococci on a hapless beef patty. This experiment was carried out using as few as five flies.

Houseflies give bacteria more than just a free ride from farm to food. They may also serve as an incubator. Several studies have shown that pathogenic strains of E.coli proliferate in the gut of common houseflies and can be transferred during feeding.

Using a fluorescent protein to tag and track bacteria, Zurek’s research team found Enteroccoccus density peaks in the fly’s crop, or foregut, roughly 48 hours after ingestion. Significantly, houseflies regurgitate the contents of their crop while feeding. In doing so, they can disseminate bacteria into their food and water.  Zurek suggests houseflies serve as a “bioenhanced vector for bacteria” because of their dual role as incubator and locomotion.

The work of Zurek and his fellow scientists has profound public health implications.  Through many lines of evidence, this body of research demonstrates a direct link between the antibiotic resistant bacteria on factory farms and antibiotic resistant bacteria in our food.

Of course, none of this is terribly surprising, is it? We’ve known since biblical times that flies are harbingers of disease. Included in the ten Biblical Plagues in the Book of Exodus is the Plague of Flies, which “came [as a] grievous swarm of flies into the house of Pharaoh, and into his servants’ houses, and into all the land of Egypt: the land was corrupted by reason of the swarm of flies.”

 However, when it comes to an issue as personal (and political) as food, we sometimes tend to forget unpleasant truths. In his book in Eating Animals, an acclaimed work of investigative journalism on the modern meat industry, Jonathan Safran Foer writes, “Food choices are determined by many factors, but reason (even consciousness) is generally not high on the list.” As hard scientific evidence accumulates on the link between antibiotic resistance on animal farms and public health, one can only hope growing consumer consciousness will force the meat industry to take a hard look at its practices.

1. Pew Campaign on Human Health and Industrial Farming

ResearchBlogging.org

Zurek, L., & Ghosh, A. (2014). Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits Applied and Environmental Microbiology, 80 (12), 3562-3567 DOI: 10.1128/AEM.00600-14

 

 

Microbe, enzyme or mineral? A riddle in the soil.

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Soil is the most microbially diverse habitat on Earth, and contains twice as much carbon as living plants and the atmosphere combined. Source: National Geographic

Soil is the most microbially diverse habitat on Earth, and contains twice as much carbon as living plants and the atmosphere combined. Source: National Geographic

When you look at soil, you probably see dirt. When I look at soil, I see billions of microorganisms, crawling atop one another, consuming the dead in a feasting frenzy that stops for nothing save a deep freeze. I see microbes and their enzymes, the digestive juices that break down, transform and release all the energy tied up in our planet’s terrestrial ecosystems.

Through their remarkable ability to decompose nearly anything that comes their way, soil microbes collectively represent a planetary recycling factory, one that takes carbon- the structural unit behind all living matter- from dead organic matter back to the atmosphere as carbon dioxide (CO2). Soils “exhale” CO2 for the same reason you and I exhale CO2. We’ve eaten something, broken down the carbon bonds that hold it together, and extracted all the energy that we could. CO2 is the generic waste product of cellular metabolism, the last bit of carbon that our own metabolic inefficiency precludes us from using. Ecologists have coined the term soil respiration to describe the collective exhale of microbial carbon decomposition.

Breathing matters, but in soil, it’s complicated

This earthly exhale is an event of profound significance to the biosphere. It dictates how much carbon remains in soil, and how much returns to the atmosphere, where acts as a heat-trapping greenhouse gas that helps regulate Earth’s climate. Biogeochemists- scientists who study the planetary cycling of elements, like carbon- are working to understand how microbes contribute to the global exhale of Earth’s soils. What makes them breathe slower? Faster? How does this influence the distribution of carbon across a forest? Across the planet? Over a year? A century? Understanding what makes soil microbes breathe will allow scientists to make better predictions about our planet’s future. How will human disturbances, like climate change, urbanization, or fertilization affect this important carbon pathway?

In order to predict why soils exhale, we need to first understand who or what’s breaking down carbon. And it turns out, when you really get down into the dirt, things get a bit more complicated. You see, microbes don’t actually accomplish all that carbon digestion by themselves. Enzymes- proteins that act as catalysts by facilitating specific reactions- mediate nearly every step in soil carbon decomposition. Some of these enzymes reside inside living microbes, but many are released outside the cell, into the environment. These “extracellular enzymes”, or exoenzymes, act as independent entities from their microbial producers. This means exoenzymes can-and often do- persist in soil long after their microbial parent dies. By chemically associating with soil minerals, exoenzymes can form stable complexes that resist drought, pH changes, even attack and degradation by other enzymes.

And finally, to make matters even more complicated, some carbon decomposition occurs by entirely abiotic (non-living) processes. Certain soil minerals, such as iron oxides, strip electrons form organic matter in a process known as oxidation. Oxidation is important for the breakdown of large, complex molecules such as lignin, the primary building block of wood.

Scanning electron microscope image of iron oxidizing bacteria Acidovorax sp. BoFeN1, encrusted in iron minerals. Microbes, enzymes and minerals are often intimately associated in soils, making it difficult to separate out their contributions to biochemical reactions. Source: Eye of Science, Reutlingen

Scanning electron microscope image of iron oxidizing bacteria Acidovorax sp. BoFeN1, encrusted in iron minerals. Microbes, enzymes and minerals are often intimately associated in soils, making it difficult to separate out their contributions to biochemical reactions. Source: Eye of Science, Reutlingen

Exoenzymes and soil microbes represent two different pieces of the decomposition puzzle. Abiotic carbon oxidation represents a third. Since these different aspects of decomposition may respond differently to environmental change, we’d like a way of separately measuring their contributions to the soil carbon cycle. But separating exoenzymes, microbes and minerals, all of which not only coexist within nanometers of each other, but are often chemically bonded, is no easy task. Soil microbial ecologists have long recognized, sometimes acknowledged, but largely ignored our inability to separate the living from the non-living in soil carbon decomposition.

Separating the living and the dead

Finally, we may have a roadmap for solving a problem that has eluded microbial ecologists for years. Last month, a group of researchers led by Dr. Joshua Schimel at the University of California, Santa Barbara, published the first study that rigorously attempts to separate cellular carbon metabolism from exoenzyme activity in soils. The study is currently in press in the journal Soil Biology & Biochemistry.

To separate cellular from extracellular metabolism in soil, the researchers recognized the need for a method that could disable living cells while leaving exoenzymes intact. This is no trivial problem. Heat can be used to sterilize soil, but high heat can also disrupt exoenzyme activity and accelerate the break-down of other organic compounds. Toxic chemicals like sodium azide (NaN3) can inhibit most microbial activity, but a small fraction of microbes develop resistance.

Two potential methods the researchers decided to test are chloroform fumigation and gamma irradiation. Chloroform, a nonpolar and hydrophobic (water-avoiding) molecule, is chemically inert but nonetheless deadly. Chloroform buries itself in the lipid bilayer of cell membranes, causing membranes to become disorganized, and eventually rupture. While some exoenzymes are clearly not affected by chloroform, others may be. Gamma radiation is a powerful, ionizing radiation that punctures cell membranes and causes them to burst. Gamma radiation is frequently used to sterilize food and medical equipment. Very few studies have measured the effect of gamma radiation on exoenzymes. The scientists compared the efficacy of these to techniques to autoclaving, a more traditional sterilization method in which a sample is subjected to very high heat.

Having chosen several sterilization methods, the researchers set up an experiment to test how cellular and extracellular soil carbon metabolism would respond. They collected soils from a California grassland, and sterilized samples using each method. From each sterilized soil, they attempted to culture viable cells. They also added a vital stain (one that differentiates living and dead cells), and used fluorescent microscopy to evaluate the number of living and dead cells.

To evaluate extracellular metabolism, they measured the activity of eight exoenzymes produced by a wide range of soil microorganisms. The eight chosen enzymes degrade common components of soil organic matter (lignin, cellulose, chitin and proteins). They included six hydrolases, enzymes that break specific chemical bonds, and two oxidases, which indiscriminately strip electrons from organic molecules. Hydrolases like beta-glucosidase are important for degrading molecules with predictable, repeating structures, such as cellulose. Oxidases play a central role in breaking down larger, more complex structures, similarly to abiotic oxidation.

To evaluate cellular metabolism, the researchers used a technique called “substrate induced respiration” (SIR). SIR involves adding a small molecule (typically an amino acid or glucose) that cells can metabolize completely, and measuring CO2 loss. The CO2 generated from an SIR experiment indicates the metabolic potential of live cells in a sample.

They found chloroform and gamma irradiation both led to a 1,000 fold reduction in the number of living cells. Autoclaving was a more powerful sterilization technique; reducing the number of live cells approximately 100,000 fold. Most exoenzyme activities were only modestly reduced by chloroform or gamma irradiation. However, sterilization did reduce the activity of two enzymes, alpha-glucosidase and beta- xylosidase by approximately 75%. It’s possible that consistent reductions in activity across all enzymes may have had a proportionally greater effect on these two, which had the lowest activity in non-sterilized soils.

From these initial findings, what can we conclude? Both chloroform and gamma radiation effectively sterilize soils (if we can accept the fact that a few hardy spores might resist these treatments). Both treatments also keep exoenzyme activity intact (keeping in mid low-activity soils will probably experience proportionally larger reductions). A few caveats here and there, but by and large both techniques appear promising.

But what about cellular metabolism? Did sterilization successfully eliminate this, as well? Your gut reaction might be that this is a silly question. If we’re killing all living cells, we must also be killing cellular activity, right? That’s at least what the researchers initially thought.

The SIR results tell a different story. The scientists added a number of different simple carbon substrates, all of which can are broken down during basic cellular metabolic processes like glycolysis and the citric acid cycle, or TCA cycle.Following sterilization, respiration from glucose and amino acid substrates was halted. However, respiration from TCA-cycle substrates, including pyruvate, citrate and ketoglutarate, was reduced but not eliminated. How could dead cells continue respiring? The authors speculate that certain dehydrogenases, the enzymes that break down TCA cycle-substrates, are able continue functioning outside the controlled environment of the cell. This finding was completely unexpected. It calls into question many assumptions about one of the most fundamental energy-harvesting processes in cellular biology, while further blurring the line between microbial metabolism and environmental metabolism.

Another surprise came from the oxidase enzymes. While all hydrolytic enzymes were killed by high heat in the autoclaving treatment, oxidative reactions persisted. The authors speculate the oxidative activity they measured in autoclaved soil is the result of abiotic processes, supporting the notion that a large amount of oxidative decomposition in soils can be caused by mineral catalysis.

Taken as a whole, this study finds both chloroform fumigation and gamma irradiation to be promising methods for separating cellular and extracellular metabolism. Some refinements are certainly needed- finding a way to separate living from non-living oxidation, for instance. But both methods already promise to unravel new mechanisms for microbial carbon metabolism, as highlighted by the finding that some TCA- cycle enzymes can persist after cell death. Now scientists can start using these sterilization treatments to measure the lifetime of exoenzymes in the environment. Right now we can only guess at how long exoenzymes stick around once they’re produced. Understanding the long-term persistence of these enzymes in the environment will help us to better gauge the capacity of soils- even in the absence of living cells- to continue digesting the planet’s carbon.

ResearchBlogging.org

Blankinship, J., Becerra, C., Schaeffer, S., & Schimel, J. (2014). Separating cellular metabolism from exoenzyme activity in soil organic matter decomposition Soil Biology and Biochemistry, 71, 68-75 DOI: 10.1016/j.soilbio.2014.01.010

When infection is a good thing: sulfur-eating bacteria enlist viruses to help acquire energy

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“Black smokers”, deep sea vents where hot, sulfur-rich fluids bubble up from beneath the ocean floor, are considered hotspots of microbial activity. They may even be where life originated. Credit: Wikimedia Commons

Life is no cake walk at the ocean floor, where carbon is scarce and light nonexistent.  At least near deep ocean vents, mineral-rich water bubbles up from magma beneath the crust, providing both heat and a source of energy. In these alien environments, lithotrophs– bacteria that eat minerals instead of organic carbon- have staked out a niche by evolving some creative metabolic strategies.

But minerals are a poor source of energy compared to organic matter. Lithotrophs are slow-growing critters, easily outcompeted when carbon is abundant. They need all the help they can get. And it turns out, they might very well get help… from an unlikely source. A study published last week in Science Express reports how viruses may be helping deep marine bacteria eat sulfur.

Viruses are everywhere- in soils, skies, oceans, plants and animals, even deep beneath the ocean floor.  In spite of their ubiquity, it’s not well-known how viruses influence marine bacteria and the nutrient cycles they drive.

To study the impact of viruses on sulfur-eating bacteria, a team of scientists led by Dr. Karthik Anatharaman at the University of Michigan collected samples from five different hydrothermal vents : four in the Western Pacific Ocean and one in the Gulf of California. They used shotgun metagenomic sequencing to look at the genomes of the bacteria and viruses present in these environments.

The researchers found five different virus “types” that carried two genes involved in sulfur metabolism. Known as rdsrA and  rdsrC, these genes encode different pieces of dissimilatory sulfite reductase, an enzyme that breaks down elemental sulfur.

How and why did viruses come to acquire these genes? Not technically considered alive, viruses don’t really need enzymes because they don’t perform metabolism on their own. A virus’s sole purpose is to infect a host with its genetic code so that it can turn the host cell into a virus factory. But during viral replication, pieces of  DNA can be accidentally snipped out of the host and integrated into the viral genome. The scientists found bacteria in the same samples carrying both the rdsrA and rdsrC genes. It’s likely that accidental incorporation of host DNA is how viruses ended up with sulfur genes in the first place.

While acquiring sulfur genes may have been a complete accident, viruses appear to have been actively maintaining those genes for a long time.

When the scientists compared rdsrA and rdsrC from viral and bacterial genomes, they found something interesting: completely different DNA sequences surrounding the genes. If viruses had acquired sulfur genes during recent replication errors, some of the nearby DNA would probably have come along for the ride as well. That completely different DNA sequences surround the sulfur metabolism genes in bacteria and viruses suggests viruses have been passing along rdsr genes, generation to generation, for a very long time. In other words, sulfur metabolism genes are being maintained  in viruses by natural selection.

Why would deep sea viruses bother to maintain sulfur metabolism genes they don’t use?  It turns out these viruses may actually have a use for sulfur genes, albeit an indirect one: helping out their hosts. By carrying genes that bacteria can make use of, viruses may assist their hosts in acquiring energy. The ability of sulfur-eating bacteria to acquire energy is ultimately limited by how quickly they can decode their sulfur genes to build enzymes that can do the work. But if a bacteria were to contain viruses with extra copies of the genes it needs, that could help speed the process along. In essence, when a sulfur virus infects a sulfur bacteria, it donates genes that its host can use.

What’s a virus to gain from all of this? Remember, a virus’s sole purpose is replication. Staying in your host’s good graces has its perks. An “infected” bacteria that can acquire energy faster than its neighbors may be able to grow and reproduce faster- leading to more infected offspring, and more viruses. By supplementing host metabolism, viruses may help ensure their continued survival.

This study sheds light on a potentially widespread, mutually beneficial ecological interaction between bacteria and viruses. Lithotrophs in the deep ocean play an important role in the global sulfur cycle and have done so for billions of years. By giving an adaptive advantage to sulfur-eaters and helping them survive, it’s possible viruses have played an equally important role in the geochemical evolution of our planet.

ResearchBlogging.org

Anantharaman, K., Duhaime, M., Breier, J., Wendt, K., Toner, B., & Dick, G. (2014). Sulfur Oxidation Genes in Diverse Deep-Sea Viruses Science DOI: 10.1126/science.1252229

Who cheats and who eats? An evolutionary conundrum.

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Say what you will about our other vices, human beings did not invent cheating. Microbes have been doing it for billions of years. You see, for microbes, cheating can sometimes be an evolutionary advantage. And this can cause it to get out of hand really quickly.

Bacteria “cheat” by stealing each other’s lunch. They do it everywhere, all the time, and unwittingly. To understand why, we first need to consider how bacteria feed themselves.

Has it ever occurred to you what a convenience it is that most things we like eat are comparable to us in size? Probably not. But think about it for a second. Sure, some foods are quite a bit smaller than us (think nuts, berries and grains), while cows, bison and mastodons are certainly larger. But, in general, most things we eat are close enough to us in size that we can see, touch and handle them.

If this seems like a trivial observation, you may be surprised to learn it’s not always the case. Most of the things a bacteria would like to eat are thousands to millions of times too large for it to ingest. But bacteria have devised a clever way around this situation. They secrete enzymes, specialized proteins, out of their cells and into their environments. These so-called exoenzymes swim around chiseling bite sized fragments off anything they can: leaf scraps, insect remains, animal carcasses. Those fragments, sometimes a single molecule in size, can then be ingested by microbes and converted into energy.

Many bacteria rely entirely on exoenzymes for their food. However, enzymes are costly to build. All of the carbon and nutrients that go into making an enzyme could be spent elsewhere- on growth, reproduction, or cellular repair. Therefore, exoenzyme production is strictly regulated according to a simple rule anyone who has ever taken an introductory econ class will be familiar with: marginal revenues must exceed marginal costs. In other words, if you don’t get much benefit from that enzyme you just built, don’t build another one.

Here’s where cheating comes in. Exoenzymes are costly for the individual to produce, but they increase resource availability for the community. In econ-speak, exoenzymes are a public good. Once released into the “wild”, an enzyme is free to diffuse away from its producer. It can get stuck to a mineral, become inactivated by freezing or desiccation, or be eaten by another enzyme.  Given the risk associated with enzyme production, cheating can become an attractive alternative. Why produce enzymes yourself if you can enjoy free lunch thanks to your neighbors’ enzymes?

This type of microbial freeloading can become a major problem. The more cheaters are present in a population, the less return a producer will get on its’ exoenzymes. Too many cheaters, and suddenly it’s not worth anyone’s while to produce enzymes at all. Paradoxically, this leads to a situation where everyone’s starving, because no one can spare the resources needed to get food.

From a microbial perspective an exoenzyme is a tool for acquiring food. But on a global scale, exoenzymes serve a higher purpose. They are the engine that digests the dead, recycling carbon and nutrients back the living. They are the reason the surface of our planet is not piled high with the dead bodies of every living organism that ever was. Clearly, then, microbes have found ways of coping with the potentially debilitating effect of cheaters.

How, then, do microbial communities keep the cheaters in check? A group of microbial ecologists led by Dr. Steve Allison at the University of California, Irvine, devised a simple experiment using Pseudomonas fluorescens, a common soil bacteria, to investigate how and when cheating occurs. The study was published last week in the journal Frontiers in Microbiology.

The scientists first obtained two different strains of P. fluorescens. One strain had the genetic capacity to produce protease– a protein-decomposing enzyme. The other strain, a cheater, lacked the ability to produce the protease enzyme. The researchers created mixed cultures of these two strains and monitored the abundance of cheaters, producers, and protease over time.

When cheaters and producers were grown in 50/50 mixed cultures, protease activity declined to near zero. Rampant cheating apparently deterred everyone from making protease. More surprisingly, in cultures that contained only protease producers, enzyme activity still declined to near zero. Why? In an environment full of enzyme producers, cheaters have a selective advantage because they can get a free lunch. It’s possible that a genetic mutation resulting in an inability to produce protease- a cheater mutation- swept through the population.

So, given that cheaters cheat, and producers become cheaters who then cheat, how is it that microbial decomposition hasn’t ground to a halt? The answer, it turns out, may lie in the structure of the microbial environment. The experiments I just described were performed in liquid cultures. In liquid, enzymes move about freely via diffusion. The result is a cheater’s paradise, an environment where resources are abundant everywhere. At least for a short while.

But what about in a more structured environment, like soil? In soil, bacteria and enzymes adhere to solid particles. Depending on moisture levels, diffusion in soil can be rapid or very slow. If a producer’s exoenzymes stay close to home, they are more likely to provide their maker with benefits. In this case, the producers may actually have incentive to continue making enzymes. To test this idea, the researchers repeated their experiment, only this time, they grew P. fluorescens on solid agar in petri dishes. The results were quite different. Cheaters did not sweep the population. Instead, populations remained a patchwork of producers and cheaters.

In spite of its simplicity, this experiment has a profound implication for our understanding of microbial ecology. It demonstrates that environments with greater spatial structure favor a diversity of life strategies. In other words, increased environmental heterogeneity facilitates coexistence.

This finding is not unique to microbial ecology- we see a similar principle playing out across much larger scales. The introduction of invasive plants and animals to new ecosystems represents a breakdown of spatial boundaries; this results in the mixing of once-separate communities. We need only go to a kudzu-ridden forest in the Southern US, or read scifi-like stories of the cane toad devastation in Australia, to see for ourselves the link between spatial isolation and diversity. When populations are separate, they experience unique environmental challenges, leading to diverse adaptations and evolution. When systems become too mixed, diversity can lose out to a lower common denominator: who can hoard the most resources, grow and reproduce the fastest.

Ultimately, as systems become too well-mixed, too homogenous, they grow vulnerable to collapse. Remember, if everyone’s a cheater, no one eats.

ResearchBlogging.org
Allison, S., Lu, L., Kent, A., & Martiny, A. (2014). Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates Frontiers in Microbiology, 5 DOI: 10.3389/fmicb.2014.00169

Amazon Mycorenewal Project launches campaign to clean up world’s largest oil spill with microbes

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Deep in the heart of the Ecuadorian Amazon lies one of the worst environmental disasters in human history. Over the past several decades, oil companies have discharged more than 18 billion gallons of petroleum contaminated wastewater into the Sucumbíos region in northeastern Ecuador. The contamination, which spans a geographic region roughly the size of Rhode Island, is described by the media as “Amazonian Chernobyl.”

Oil drilling in Lago Agrio, one of the many oil-contaminated sites in Sucombios, Ecuador

Oil drilling in Lago Agrio, one of the many oil-contaminated sites in Sucombios, Ecuador

Three years ago, an Ecuadorian court ruled that Chevron should pay $18 billion US dollars of damages for the pollution in Sucumbíos. However, the oil giant has so far refused to pay remediation costs, and a bitter legal battle continues to rage.

Now a grassroots effort known as the Amazon Mycorenewal Project (AMP) hopes to take remediation into their own hands- and to the people of Sucumbíos. The scientists, international volunteers and Ecuadorians driving the AMP believe the key to remediation lies in microorganisms thriving in petroleum-contaminated soils. Today, the AMP launched an indiegogo campaign to raise money for research that will determine whether naturally occurring bacteria, fungi and plants can be used to degrade the toxic petro-waste that has plagued the region.

Bioremediation, or using microbes to clean up our environmental messes, is not a new concept. Many microorganisms have been deployed across the world to degrade a range of environmental pollutants, including PCBs, gasoline, radioactive waste and mercury. Several years ago, a group of undergraduate researchers from Yale University visited the Amazon and discovered fungi that eat polyurethane plastic, a synthetic, petroleum-derived material. The enzymes microbes use to degrade synthetics are probably used in nature to decompose lignocellulose, the stuff that makes wood “woody”.

What makes the AMP unique is their ecological approach to bioremediation. By culturing bacteria and fungi that already thrive in petroleum-contaminated environments, the AMP hopes to develop communities of bioremediators that are “naturally suited” to their habitat. Preliminary studies conducted over the last several years show that local fungi, grown in the lab on petroleum-enriched substrates, develop increased resistance to petroleum toxicity.

Cultures of "petrophiles", wild fungi isolated from petroleum contaminated soils in the Ecuadorian Amazon

Cultures of “petrophiles”, wild fungi isolated from petroleum contaminated soils in the Ecuadorian Amazon

The other unique aspect of AMP’s approach is the organization’s integration with local communities. Mia Maltz, a fungal ecologist and PhD student at the University of California, Irvine, has been working with the AMP since 2007. Over the last seven years Mia and other members of the AMP have taught mushroom cultivation and mycoremediation (bioremediation using fungi) techniques to locals. By designing and teaching low-cost bioremediation strategies, AMP is empowering locals to do bioremediation on their own.

 “A lot of what we’re doing is very low-tech, easy for local people to do” says Mia. “That’s really the goal here. We want things to be inexpensive and cost-effective. The science here is very simple-  encouraging the natural process of lignocellulose decomposition. We want to show people the simplicity of what’s happening, help them internalize it, so they can tap into a process that’s a huge part of the web of life.”

This summer, a team of scientists and volunteers will work in Sucumbíos to identify and cultivate microorganisms capable of degrading different hydrocarbons present in petroleum. In collaboration with scientists in the United States, the AMP hopes to use metagenomic analyses to profile entire microbial communities and document key microbial taxa and genes involved in petroleum degradation.

AMP volunteer searching for petro-tolerant fungi in contaminated sites

AMP volunteer searching for petro-tolerant fungi in contaminated sites

Ultimately, AMP aims to develop inexpensive biofiltration systems. Biofiltration is the process of filtering contaminated water through living organisms (microbes and plants) which detoxify and remove pollutants. The AMP envisions a biofiltration system consisting of several chambers containing different assemblages of petro-degrading bacteria and fungi. After microbial filtration, the wastewater will be fed to plants that can tolerate high concentrations of heavy metals and other residual contaminants.

AMP volunteers are currently developing prototype biofiltration systems. “We want these to be modular and flexible” says Mia. “We plan on licensing everything in the creative commons to make our technology accessible to local communities around the world.”

The Amazon Mycorenewal Project brings together a broad range of expertise to tackle this enormous pollution challenge. Co-founder Bob Rawson is president of two bioremediation companies (International Wastewater Solutions and the Pseudonym Corporation) and has over 36 years of experience cleaning up contaminated soils. Joanna Zlotnik, program director for AMP, has worked as an environmental geologist for ten years, conducting site assessments and doing remediation work. Collaborator Tradd Cotter is the founder and director of Mushroom Mountain LLC. Tradd brings over 22 years of experience in commercial and experimental mushroom cultivation and mycoremediation research.

Check out the Amazon Mycorenewal Project indiegogo campaign and make a contribution here.