Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Limitation of Microbial Processes at Saturation-Level Salinities in a Microbial Mat Covering a Coastal Salt Flat.

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ, at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi, photoheterotrophic Alphaproteobacteria, Bacteroidetes, and Archaea, the mat contained a distinct layer harboring filamentous Cyanobacteria, which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ. While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrations.

Microbial metal-sulfide oxidation in inactive hydrothermal vent chimneys suggested by metagenomic and metaproteomic analyses.

Metal-sulfides are wide-spread in marine benthic habitats. At deep-sea hydrothermal vents, they occur as massive sulfide chimneys formed by mineral precipitation upon mixing of reduced vent fluids with cold oxygenated sea water. Although microorganisms inhabiting actively venting chimneys and utilizing compounds supplied by the venting fluids are well studied, only little is known about microorganisms inhabiting inactive chimneys. In this study, we combined 16S rRNA gene-based community profiling of sulfide chimneys from the Manus Basin (SW Pacific) with radiometric dating, metagenome (n = 4) and metaproteome (n = 1) analyses. Our results shed light on potential lifestyles of yet poorly characterized bacterial clades colonizing inactive chimneys. These include sulfate-reducing Nitrospirae and sulfide-oxidizing Gammaproteobacteria dominating most of the inactive chimney communities. Our phylogenetic analysis attributed the gammaproteobacterial clades to the recently described Woeseiaceae family and the SSr-clade found in marine sediments around the world. Metaproteomic data identified these Gammaproteobacteria as autotrophic sulfide-oxidizers potentially facilitating metal-sulfide dissolution via extracellular electron transfer. Considering the wide distribution of these gammaproteobacterial clades in marine environments such as hydrothermal vents and sediments, microbially accelerated neutrophilic mineral oxidation might be a globally relevant process in benthic element cycling and a considerable energy source for carbon fixation in marine benthic habitats.

Heterotrophic Proteobacteria in the vicinity of diffuse hydrothermal venting.

Deep-sea hydrothermal vents are highly dynamic habitats characterized by steep temperature and chemical gradients. The oxidation of reduced compounds dissolved in the venting fluids fuels primary production providing the basis for extensive life. Until recently studies of microbial vent communities have focused primarily on chemolithoautotrophic organisms. In our study, we targeted the change of microbial community compositions along mixing gradients, focusing on distribution and capabilities of heterotrophic microorganisms. Samples were retrieved from different venting areas within the Menez Gwen hydrothermal field, taken along mixing gradients, including diffuse fluid discharge points, their immediate surroundings and the buoyant parts of hydrothermal plumes. High throughput 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization, and targeted metagenome analysis were combined with geochemical analyses. Close to diffuse venting orifices dominated by chemolithoautotrophic Epsilonproteobacteria, in areas where environmental conditions still supported chemolithoautotrophic processes, we detected microbial communities enriched for versatile heterotrophic Alpha- and Gammaproteobacteria. The potential for alkane degradation could be shown for several genera and yet uncultured clades. We propose that hotspots of chemolithoautotrophic life support a 'belt' of heterotrophic bacteria significantly different from the dominating oligotrophic microbiota of the deep sea.

Survival and rapid resuscitation permit limited productivity in desert microbial communities.

Microbial activity in drylands tends to be confined to rare and short periods of rain. Rapid growth should be key to the maintenance of ecosystem processes in such narrow activity windows, if desiccation and rehydration cause widespread cell death due to osmotic stress. Here, simulating rain with 2H2O followed by single-cell NanoSIMS, we show that biocrust microbial communities in the Negev Desert are characterized by limited productivity, with median replication times of 6 to 19 days and restricted number of days allowing growth. Genome-resolved metatranscriptomics reveals that nearly all microbial populations resuscitate within minutes after simulated rain, independent of taxonomy, and invest their activity into repair and energy generation. Together, our data reveal a community that makes optimal use of short activity phases by fast and universal resuscitation enabling the maintenance of key ecosystem functions. We conclude that desert biocrust communities are highly adapted to surviving rapid changes in soil moisture and solute concentrations, resulting in high persistence that balances limited productivity.

Both abundant and rare fungi colonizing Fagus sylvatica ectomycorrhizal root-tips shape associated bacterial communities.

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis.

Sugars dominate the seagrass rhizosphere.

Seagrasses are among the most efficient sinks of carbon dioxide on Earth. While carbon sequestration in terrestrial plants is linked to the microorganisms living in their soils, the interactions of seagrasses with their rhizospheres are poorly understood. Here, we show that the seagrass, Posidonia oceanica excretes sugars, mainly sucrose, into its rhizosphere. These sugars accumulate to µM concentrations-nearly 80 times higher than previously observed in marine environments. This finding is unexpected as sugars are readily consumed by microorganisms. Our experiments indicated that under low oxygen conditions, phenolic compounds from P. oceanica inhibited microbial consumption of sucrose. Analyses of the rhizosphere community revealed that many microbes had the genes for degrading sucrose but these were only expressed by a few taxa that also expressed genes for degrading phenolics. Given that we observed high sucrose concentrations underneath three other species of marine plants, we predict that the presence of plant-produced phenolics under low oxygen conditions allows the accumulation of labile molecules across aquatic rhizospheres.

Distribution of Mixotrophy and Desiccation Survival Mechanisms across Microbial Genomes in an Arid Biological Soil Crust Community.

Desert surface soils devoid of plant cover are populated by a variety of microorganisms, many with yet unresolved physiologies and lifestyles. Nevertheless, a common feature vital for these microorganisms inhabiting arid soils is their ability to survive long drought periods and reactivate rapidly in rare incidents of rain. Chemolithotrophic processes such as oxidation of atmospheric hydrogen and carbon monoxide are suggested to be a widespread energy source to support dormancy and resuscitation in desert soil microorganisms. Here, we assessed the distribution of chemolithotrophic, phototrophic, and desiccation-related metabolic potential among microbial populations in arid biological soil crusts (BSCs) from the Negev Desert, Israel, via population-resolved metagenomic analysis. While the potential to utilize light and atmospheric hydrogen as additional energy sources was widespread, carbon monoxide oxidation was less common than expected. The ability to utilize continuously available energy sources might decrease the dependency of mixotrophic populations on organic storage compounds and carbon provided by the BSC-founding cyanobacteria. Several populations from five different phyla besides the cyanobacteria encoded CO2 fixation potential, indicating further potential independence from photoautotrophs. However, we also found population genomes with a strictly heterotrophic genetic repertoire. The highly abundant Rubrobacteraceae (Actinobacteriota) genomes showed particular specialization for this extreme habitat, different from their closest cultured relatives. Besides the ability to use light and hydrogen as energy sources, they encoded extensive O2 stress protection and unique DNA repair potential. The uncovered differences in metabolic potential between individual, co-occurring microbial populations enable predictions of their ecological niches and generation of hypotheses on the dynamics and interactions among them.IMPORTANCE This study represents a comprehensive community-wide genome-centered metagenome analysis of biological soil crust (BSC) communities in arid environments, providing insights into the distribution of genes encoding different energy generation mechanisms, as well as survival strategies, among populations in an arid soil ecosystem. It reveals the metabolic potential of several uncultured and previously unsequenced microbial genera, families, and orders, as well as differences in the metabolic potential between the most abundant BSC populations and their cultured relatives, highlighting once more the danger of inferring function on the basis of taxonomy. Assigning functional potential to individual populations allows for the generation of hypotheses on trophic interactions and activity patterns in arid soil microbial communities and represents the basis for future resuscitation and activity studies of the system, e.g., involving metatranscriptomics.

Microaerobic Lifestyle at Nanomolar O2 Concentrations Mediated by Low-Affinity Terminal Oxidases in Abundant Soil Bacteria.

High-affinity terminal oxidases (TOs) are believed to permit microbial respiration at low oxygen (O2) levels. Genes encoding such oxidases are widespread, and their existence in microbial genomes is taken as an indicator for microaerobic respiration. We combined respiratory kinetics determined via highly sensitive optical trace O2 sensors, genomics, and transcriptomics to test the hypothesis that high-affinity TOs are a prerequisite to respire micro- and nanooxic concentrations of O2 in environmentally relevant model soil organisms: acidobacteria. Members of the Acidobacteria harbor branched respiratory chains terminating in low-affinity (caa3-type cytochrome c oxidases) as well as high-affinity (cbb3-type cytochrome c oxidases and/or bd-type quinol oxidases) TOs, potentially enabling them to cope with varying O2 concentrations. The measured apparent Km (Km(app)) values for O2 of selected strains ranged from 37 to 288 nmol O2 liter-1, comparable to values previously assigned to low-affinity TOs. Surprisingly, we could not detect the expression of the conventional high-affinity TO (cbb3 type) at micro- and nanomolar O2 concentrations but detected the expression of low-affinity TOs. To the best of our knowledge, this is the first observation of microaerobic respiration imparted by low-affinity TOs at O2 concentrations as low as 1 nM. This challenges the standing hypothesis that a microaerobic lifestyle is exclusively imparted by the presence of high-affinity TOs. As low-affinity TOs are more efficient at generating ATP than high-affinity TOs, their utilization could provide a great benefit, even at low-nanomolar O2 levels. Our findings highlight energy conservation strategies that could promote the success of Acidobacteria in soil but might also be important for as-yet-unrevealed microorganisms. IMPORTANCE Low-oxygen habitats are widely distributed on Earth, ranging from the human intestine to soils. Microorganisms are assumed to have the capacity to respire low O2 concentrations via high-affinity terminal oxidases. By utilizing strains of a ubiquitous and abundant group of soil bacteria, the Acidobacteria, and combining respiration kinetics, genomics, and transcriptomics, we provide evidence that these microorganisms use the energetically more efficient low-affinity terminal oxidases to respire low-nanomolar O2 concentrations. This questions the standing hypothesis that the ability to respire traces of O2 stems solely from the activity of high-affinity terminal oxidases. We propose that this energetically efficient strategy extends into other, so-far-unrevealed microbial clades. Our findings also demonstrate that physiological predictions regarding the utilization of different O2 concentrations based solely on the presence or absence of terminal oxidases in bacterial genomes can be misleading.

Acidobacteria are active and abundant members of diverse atmospheric H2-oxidizing communities detected in temperate soils.

Significant rates of atmospheric dihydrogen (H2) consumption have been observed in temperate soils due to the activity of high-affinity enzymes, such as the group 1h [NiFe]-hydrogenase. We designed broadly inclusive primers targeting the large subunit gene (hhyL) of group 1h [NiFe]-hydrogenases for long-read sequencing to explore its taxonomic distribution across soils. This approach revealed a diverse collection of microorganisms harboring hhyL, including previously unknown groups and taxonomically not assignable sequences. Acidobacterial group 1h [NiFe]-hydrogenase genes were abundant and expressed in temperate soils. To support the participation of acidobacteria in H2 consumption, we studied two representative mesophilic soil acidobacteria, which expressed group 1h [NiFe]-hydrogenases and consumed atmospheric H2 during carbon starvation. This is the first time mesophilic acidobacteria, which are abundant in ubiquitous temperate soils, have been shown to oxidize H2 down to below atmospheric concentrations. As this physiology allows bacteria to survive periods of carbon starvation, it could explain the success of soil acidobacteria. With our long-read sequencing approach of group 1h [NiFe]-hydrogenase genes, we show that the ability to oxidize atmospheric levels of H2 is more widely distributed among soil bacteria than previously recognized and could represent a common mechanism enabling bacteria to persist during periods of carbon deprivation.

Energetic Basis of Microbial Growth and Persistence in Desert Ecosystems.

Microbial life is surprisingly abundant and diverse in global desert ecosystems. In these environments, microorganisms endure a multitude of physicochemical stresses, including low water potential, carbon and nitrogen starvation, and extreme temperatures. In this review, we summarize our current understanding of the energetic mechanisms and trophic dynamics that underpin microbial function in desert ecosystems. Accumulating evidence suggests that dormancy is a common strategy that facilitates microbial survival in response to water and carbon limitation. Whereas photoautotrophs are restricted to specific niches in extreme deserts, metabolically versatile heterotrophs persist even in the hyper-arid topsoils of the Atacama Desert and Antarctica. At least three distinct strategies appear to allow such microorganisms to conserve energy in these oligotrophic environments: degradation of organic energy reserves, rhodopsin- and bacteriochlorophyll-dependent light harvesting, and oxidation of the atmospheric trace gases hydrogen and carbon monoxide. In turn, these principles are relevant for understanding the composition, functionality, and resilience of desert ecosystems, as well as predicting responses to the growing problem of desertification.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).

Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

Rapid microbial diversification of dissolved organic matter in oceanic surface waters leads to carbon sequestration.

The pool of dissolved organic matter (DOM) in the deep ocean represents one of the largest carbon sinks on the planet. In recent years, studies have shown that most of this pool is recalcitrant, because individual compounds are present at low concentrations and because certain compounds seem resistant to microbial degradation. The formation of the diverse and recalcitrant deep ocean DOM pool has been attributed to repeated and successive processing of DOM by microorganisms over time scales of weeks to years. Little is known however, about the transformation and cycling that labile DOM undergoes in the first hours upon its release from phytoplankton. Here we provide direct experimental evidence showing that within hours of labile DOM release, its breakdown and recombination with ambient DOM leads to the formation of a diverse array of new molecules in oligotrophic North Atlantic surface waters. Furthermore, our results reveal a preferential breakdown of N and P containing molecules versus those containing only carbon. Hence, we show the preferential breakdown and molecular diversification are the crucial first steps in the eventual formation of carbon rich DOM that is resistant to microbial remineralization.

Metaproteogenomic Profiling of Microbial Communities Colonizing Actively Venting Hydrothermal Chimneys.

At hydrothermal vent sites, chimneys consisting of sulfides, sulfates, and oxides are formed upon contact of reduced hydrothermal fluids with oxygenated seawater. The walls and surfaces of these chimneys are an important habitat for vent-associated microorganisms. We used community proteogenomics to investigate and compare the composition, metabolic potential and relative in situ protein abundance of microbial communities colonizing two actively venting hydrothermal chimneys from the Manus Basin back-arc spreading center (Papua New Guinea). We identified overlaps in the in situ functional profiles of both chimneys, despite differences in microbial community composition and venting regime. Carbon fixation on both chimneys seems to have been primarily mediated through the reverse tricarboxylic acid cycle and fueled by sulfur-oxidation, while the abundant metabolic potential for hydrogen oxidation and carbon fixation via the Calvin-Benson-Bassham cycle was hardly utilized. Notably, the highly diverse microbial community colonizing the analyzed black smoker chimney had a highly redundant metabolic potential. In contrast, the considerably less diverse community colonizing the diffusely venting chimney displayed a higher metabolic versatility. An increased diversity on the phylogenetic level is thus not directly linked to an increased metabolic diversity in microbial communities that colonize hydrothermal chimneys.

Niche partitioning of diverse sulfur-oxidizing bacteria at hydrothermal vents.

At deep-sea hydrothermal vents, primary production is carried out by chemolithoautotrophic microorganisms, with the oxidation of reduced sulfur compounds being a major driver for microbial carbon fixation. Dense and highly diverse assemblies of sulfur-oxidizing bacteria (SOB) are observed, yet the principles of niche differentiation between the different SOB across geochemical gradients remain poorly understood. In this study niche differentiation of the key SOB was addressed by extensive sampling of active sulfidic vents at six different hydrothermal venting sites in the Manus Basin, off Papua New Guinea. We subjected 33 diffuse fluid and water column samples and 23 samples from surfaces of chimneys, rocks and fauna to a combined analysis of 16S rRNA gene sequences, metagenomes and real-time in situ measured geochemical parameters. We found Sulfurovum Epsilonproteobacteria mainly attached to surfaces exposed to diffuse venting, while the SUP05-clade dominated the bacterioplankton in highly diluted mixtures of vent fluids and seawater. We propose that the high diversity within Sulfurimonas- and Sulfurovum-related Epsilonproteobacteria observed in this study derives from the high variation of environmental parameters such as oxygen and sulfide concentrations across small spatial and temporal scales.