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Time-Sharing Between Biofilms

Electrical and chemical communication among microbes has been an active area of research for a while, but this new study looks at how B. subtilis communicates not just among individuals, but between distinct biofilms. They demonstrate that resource limitation prompts an anti-phase growth pattern synchronization - the suggestion being that time with full resource availability is better than the same amount of time with half of that resource. That seems logical enough, but why the growth phase periods are the same between the two cases (suggesting that fewer nutrients overall are being consumed), and why anti-phase relationships aren't also favored under nutrient-replete conditions, is unclear. Perhaps most surprisingly, the researchers show that under limitation, the average growth rate is actually higher in two anti-phase biofilms than one big biofilm! Lots of outstanding questions, but this is a great place to start with community-level biochemical communication.

Link [here], abstract below:

Bacteria within communities can interact to organize their behavior. It remains unclear whether such interactions extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to anti-phase oscillations. This results in time-sharing behavior where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

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New Methyl-Reducing Methanogenic Pathway

When methylotrophic methanogens run their metabolism, they typically dismutate the methyl group - they process some of the CH3 groups to CO2 (oxidation) and some to CH4 (reduction). But the genomic reconstruction and physiological examination of two newly discovered organisms from hypersaline lakes in Siberia suggest that something different is happening: only the reductive branch is operational. And yet, the microbes still possess the genes for the oxidative pathway - they just don't seem to be active. The study has interesting implications for the evolution of methane metabolism, exposing a lineage of organisms that may be phasing methanogenesis out of its metabolic repertoire. The authors propose a new methanogenic class, the "Methanonatronarchaeia". Full text here, abstract below:

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage ‘Methanonatronarchaeia’ that is most closely related to the class Halobacteria. Similar to the Halobacteria, ‘Methanonatronarchaeia’ are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that ‘Methanonatronarchaeia’ employ the ‘salt-in’ osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C1-methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO2 as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate ‘Methanonatronarchaeia’ from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.

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Can Mineralogy be a Biosignature in SLiMEs?

Subsurface lithoautotrophic microbial ecosystems lead to one of geobiology's better acronyms - SLiMEs - but how can we evaluate the viability of such systems? In basaltic aquifers, iron oxidation can generate molecular hydrogen, which is a juicy electron donor for many organisms. In the presence of carbon dioxide, methanogenesis is particularly favorable. A recent paper from Lisa Mayhew, Graham Lau, and Alexis Templeton looks at how a methanogenic culture can take advantage of this hydrogen to generate energy (but not necessarily proliferate) and potentially alter the mineralogy. The results show that mineral phases are altered - to a Fe-bearing pyroxene - but not always in a repeatable or predictable way. Life does seem to influence the geochemistry and the mineralogy, but mineral-based biosignatures aren't likely to be too conclusive in this system.

Abstract below, full paper [here]:

Microbial CO2-driven methanogenesis, the biologically mediated conversion of hydrogen and carbon dioxide to methane, is potentially one of the earliest metabolic strategies to appear on Earth and likely originated in ecosystems sustained by water/rock interaction. The potential for methanogens to influence secondary mineralization pathways during water–rock reactions is not well known. Yet geochemical signatures of the presence and activity of life in rock-hosted environments are of great interest in the search for life and signs of life preserved on Earth and other planets. We designed a laboratory experiment to evaluate microbial growth and secondary mineralization pathways in the presence and absence of microbial methanogenesis. Methanothermobacter thermoflexus, a moderately thermophilic methanogen was supported by the continuous production of H2 from water–basalt–Fe0 reactions at 55 °C for over one year. As H2 was converted to CH4 in culture experiments, the H2 concentration was maintained at 0.1–0.5 μM, orders of magnitude lower than the concentration in comparable abiotic experiments. The pH in active cultures was lower than in abiotic experiments. The concentration of CO2(g), and thus CO2(aq), was higher in the active cultures vs. abiotic and inactive culture experiments. These differences in chemistry between the two systems led to the prediction, from geochemical equilibrium models, of distinct Fe-bearing secondary phases stable in the active cultures (carbonate) vs. abiotic and inactive culture experiments (phyllosilicate). The application of synchrotron X-ray absorption spectroscopic techniques enabled the detection of rare and microscale Fe-bearing solid-phase reaction products. A phyllosilicate dominated the Fe-bearing secondary mineralogy of the abiotic and inactive culture experiments. In contrast, the phyllosilicate was not formed in the active cultures. However, a carbonate phase was not robustly detected. Surprisingly, an Fe-bearing pyroxene was detected in some cultures, and formed through an unknown pathway. Thus distinct secondary mineralization occurred in the presence vs. absence of microbial methanogenesis, whether it was directly or indirectly caused by in-situ biological activity.

 

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One Step Closer to Engineered Methanotrophic Microbes

An important priority for many metabolic engineers is the production of a fast growing, easily controllable microbe that can turn methane - a strong greenhouse gas - into biofuel. A multi-team initiative funded by the Department of Energy's Advanced Research Projects Agency (of which I am a part) is pursuing this goal, and a recent paper has made a pretty substantial step forward. They've identified the genes needed to produce F430, a critical nickel-containing cofactor for the Mcr (methyl coenzyme M reductase) enzyme. F430 is where the catalytic magic of methane oxidation and production happens. If these five genes can be expressed along with the Mcr subunits and any accessory proteins needed to activate the enzyme itself, then methane's carbon - long regarded as a tough nut to crack - could become available for E. coli's central carbon metabolism.

Abstract below, full paper at Science is [here].

Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

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Linking Function with Identity, at Scale, with epicPCR

Now this is pretty cool. Figuring out which microbes are performing a certain type of metabolism is an age old challenge. The main competing technologies represent a trade-off between scale (e.g., PCR targeting specific genes or a metagenome that looks at a system's entire genetic complement) and specificity (e.g., single cell genomes). With the former, you can sample the enormous diversity that characterizes most microbial systems; with the latter, you can connect identity to a range of functions by seeing which genes are linked with a given 16S rRNA gene.

A new approach called Emulsion, Paired Isolation and Concatenation PCR (or, clearly, epicPCR) tries to bridge the gap. The method isolates individual cells and performs a snazzy fusion PCR to link the 16S rRNA gene with a functional gene of choice. In a survey of sulfate reducer diversity, two million fused dsrB-16S rRNA fusions were observed, exposing novel diversity at high-throughput. 

Full paper [here], abstract below:

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

 

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A Glass-Half-Full Perspective for World Oceans Day

I'm a bit late on this, but World Oceans Day occurred last month, setting off celebrations and reflections around the world. This [Nature editorial] focuses in large part on one of the most charismatic of marine habitats - coral reefs. While news of pervasive coral bleaching has been coming fast and furious recently, a new paper by Joshua Cinner et al. compiled data from more than 2,500 reefs around the world to evaluate conditions that lead to dramatically improved reef prospects...and worse than average outcomes. Among the findings:

We surveyed local experts about social, institutional, and environmental conditions at these sites to reveal that bright spots are characterized by strong sociocultural institutions such as customary taboos and marine tenure, high levels of local engagement in management, high dependence on marine resources, and beneficial environmental conditions such as deep-water refuges. Alternatively, dark spots are characterized by intensive capture and storage technology and a recent history of environmental shocks.

The full paper, optimistically titled "Bright Spots Among the World's Coral Reefs," can be found [here].

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Methane Hydrates Seemingly Safe from the Next Century of Global Warming

One of the more apocalyptic scenarios surrounding anthropogenic climate change is the destabilization of the planet's vast methane hydrate reserves. Under this nightmare contingency, warming oceans cause the gas hydrate stability zone to migrate downward, leaving substantial stores of the ice-like substance in a metastable state. As they dissolve, methane is released into the water column and ultimately the atmosphere, where it wreaks havoc as a strong greenhouse gas. In a recent paper, Kretschmer et al. use ocean circulation simulations and other models to show how bottom water temperatures may change over the next century, producing a fascinating map of anticipated thermal changes. Shallow shelf regions are expected to experience the largest changes, up to a 3 degree C increase. Overall, a minimal global influence on methane hydrates is anticipated - roughly 0.03% of seafloor stocks. Abstract below, full paper [here].

Large amounts of methane hydrate locked up within marine sediments are vulnerable to climate change. Changes in bottom water temperatures may lead to their destabilization and the release of methane into the water column or even the atmosphere. In a multimodel approach, the possible impact of destabilizing methane hydrates onto global climate within the next century is evaluated. The focus is set on changing bottom water temperatures to infer the response of the global methane hydrate inventory to future climate change. Present and future bottom water temperatures are evaluated by the combined use of hindcast high-resolution ocean circulation simulations and climate modeling for the next century. The changing global hydrate inventory is computed using the parameterized transfer function recently proposed by Wallmann et al. (2012). We find that the present-day world's total marine methane hydrate inventory is estimated to be 1146 Gt of methane carbon. Within the next 100 years this global inventory may be reduced by ∼0.03% (releasing ∼473 Mt methane from the seafloor). Compared to the present-day annual emissions of anthropogenic methane, the amount of methane released from melting hydrates by 2100 is small and will not have a major impact on the global climate. On a regional scale, ocean bottom warming over the next 100 years will result in a relatively large decrease in the methane hydrate deposits, with the Arctic and Blake Ridge region, offshore South Carolina, being most affected.

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Seafloor Hydrocarbon Seeps Influence Sea-surface Microbiology

Hydrocarbon seeps are typically viewed as seafloor phenomena: with vast quantities of juicy organic molecules streaming out of the crust, sediment-hosted microbes are the primary recipients of the energetic windfall. But at particularly high-flow seeps, hydrocarbons can emerge into the bottom water, and, running the gauntlet of benthic aerobic heterotrophs, even reach the water's surface layers. A new study from Nigel D'souza and colleagues demonstrates that these seafloor features actually result in measurable increases in surface-water microbial activity. By quantifying chlorophyll concentrations, the team shows that seafloor venting of many kinds may have a larger effect on the surface world than previously believed.

Abstract below, full paper [here].

Natural hydrocarbon seeps occur on the sea floor along continental margins, and account for up to 47% of the oil released into the oceans1. Hydrocarbon seeps are known to support local benthic productivity2, but little is known about their impact on photosynthetic organisms in the overlying water column. Here we present observations with high temporal and spatial resolution of chlorophyll concentrations in the northern Gulf of Mexico using in situ and shipboard flow-through fluorescence measurements from May to July 2012, as well as an analysis of ocean-colour satellite images from 1997 to 2007. All three methods reveal elevated chlorophyll concentrations in waters influenced by natural hydrocarbon seeps. Temperature and nutrient profiles above seep sites suggest that nutrient-rich water upwells from depth, which may facilitate phytoplankton growth and thus support the higher chlorophyll concentrations observed. Because upwelling occurs at natural seep locations around the world1, 2, 3, we conclude that offshore hydrocarbon seeps, and perhaps other types of deep ocean vents and seeps at depths exceeding 1,000 m, may influence biogeochemistry and productivity of the overlying water column.

 

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The Missing Carbonates of Mars

Billions of years ago, liquid water flowed across the surface of Mars, as indicated by the pervasive fluvial features that cover the planet's surface to this day. To stably maintain this phase of H2O, the martian atmosphere would have needed to be much, much thicker that it is today. As the atmosphere is ~95% carbon dioxide, there are billions of tons of carbon that are unaccounted for. Atmospheric physics and chemistry suggest that sequestration of CO2 into carbonate rocks would be the most likely scenario, yet a corresponding quantity of carbonate-bearing rocks has not been identified by satellite-based analyses. Except for a chunk of such rock found at Nili Fossae. In a new analysis of that outcrop and estimates of carbon quantities, a new study from Christopher Edwards and Bethany Ehlmann at Caltech lays out three possibilities: 1) the atmosphere may have been thick in the past, but it had a lower proportion of CO2 than it does today; 2) much of the carbon-bearing atmosphere was lost to space; or 3) large carbonate rock repositories - about 34 Nili Fossae equivalents - remain buried, yet to be discovered...

Full paper here, abstract below:

On Earth, carbon sequestration in geologic units plays an important role in the carbon cycle, scrubbing CO2 from the atmosphere for long-term storage. While carbonate is identified in low abundances within the dust and soils of Mars, at <1 wt% in select meteorites, and in limited outcrops, no massive carbonate rock reservoir on Mars has been identified to date. Here, we investigate the largest exposed carbonate-bearing rock unit, the Nili Fossae plains, combining spectral, thermophysical, and morphological analyses to evaluate the timing and carbon sequestration potential of rocks on Mars. We find that the olivine-enriched (∼20%–25%) basalts have been altered, by low-temperature in situ carbonation processes, to at most ∼20% Fe-Mg carbonate, thus limiting carbon sequestration in the Nili Fossae region to ∼0.25–12 mbar of CO2 during the late Noachian–early Hesperian, before or concurrent with valley network formation. While this is large compared to modern-day CO2 reservoirs, the lack of additional, comparably sized post–late Noachian carbonate-bearing deposits on Mars indicates ineffective carbon sequestration in rock units over the past ∼3.7 b.y. This implies a thin atmosphere (≲500 mbar) during valley network formation, extensive post-Noachian atmospheric loss to space, or diffuse, deep sequestration by a yet-to-be understood process. In stark contrast to Earth’s biologically mediated crust:atmosphere carbon reservoir ratio of ∼104–105, Mars’ ratio is a mere ∼10–103, even if buried pre-Noachian crust holds multiple bars.

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Could Fungi Survive on Mars?

It seems, based on some experimental work done on the International Space Station and published recently in the journal Astrobiology, that the answer is yes. Fungi are eukaryotes and posses cell walls made partially of chitin, a polymer of acetylated glucose that also comprises arthropod exoskeletons. In this experiment, endolithic Antarctic fungi and dried colonies were subjected to 18 months of simulated martian conditions; upon return and rehydration, the scientists evaluated whether the cells were viable - could they grow once more now that conditions were more luxurious? About 10% of the cells proved viable, compared to roughly 50% of control cells that were kept in a dark Earth-based laboratory. Intact DNA was recovered from about 75% of endolithic fungi, but just 10-15% of fully exposed organisms. While the work shows that martian conditions are tolerable to some organisms and confirms the importance of radiation shielding in the form of rock-hosted habitats, it doesn't necessarily offer fundamentally new hope of encountering life on Mars. For one thing, martian soil chemistry is likely more treacherous than Antarctic sandstone, given photochemical reactions and free radical production. And metabolic activity - a prerequisite for doubling and a vibrant, sustainable biosphere, was not assessed. Cell and DNA survivability studies under martian conditions would perhaps be more useful in determining the likelihood of remnant biochemicals. Abstract for the paper below:

Dehydrated Antarctic cryptoendolithic communities and colonies of the rock inhabitant black fungi Cryomyces antarcticus (CCFEE 515) and Cryomyces minteri (CCFEE 5187) were exposed as part of the Lichens and Fungi Experiment (LIFE) for 18 months in the European Space Agency's EXPOSE-E facility to simulated martian conditions aboard the International Space Station (ISS). Upon sample retrieval, survival was proved by testing colony-forming ability, and viability of cells (as integrity of cell membrane) was determined by the propidium monoazide (PMA) assay coupled with quantitative PCR tests. Although less than 10% of the samples exposed to simulated martian conditions were able to proliferate and form colonies, the PMA assay indicated that more than 60% of the cells and rock communities had remained intact after the “Mars exposure.” Furthermore, a high stability of the DNA in the cells was demonstrated. The results contribute to assessing the stability of resistant microorganisms and biosignatures on the surface of Mars, data that are valuable information for further search-for-life experiments on Mars.

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A Deep Dive Into Methane Seep Carbonates

The carbonate rocks that comprise the dominant structural features at methane seeps (see Research page) contain active methanotrophic organisms and a range of different organisms. But the degree of community dynamism and its ability to adapt to changing geochemical and energetic conditions was entirely unknown...until now. Through an ambitious set of in situ seafloor transplantation experiments, DNA sequencing, and ecological statistics, David Case led a team (of which I was a part) that simulated the "activation" and "deactivation" of methane seeps. In general, Case shows that community composition doesn't change too drastically with seep dormancy, but that certain microbial constituents are primed and ready to pounce when methane supply "reappears".

The full open-access paper can be found [here], and the abstract is below:

Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages.

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Protein-Protein Interactions by the Thousands

Detecting protein-protein interactions is usually a low-throughput endeavor: a specific bait protein or peptide is affixed to a column, and unsuspecting prey passers-by get involved. Subsequent elutions leave only the interacting complex, which can be sequenced by LC-MS/MS. Because of the time and financial expenses, many weaker interactions are not pursued, accentuating the false narrative that such interactions are not important. But now, through a Herculean effort of cloning and mass spectrometry, a group led by Anthony Hyman and Matthias Mann conducted nearly 4,000 MS runs to detect over 28,000 interactions. The most revealing trend indicated that weak interactions dominate, both quantitatively and in terms of network topology determination. Full paper here, abstract below:

The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

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