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Background: Sulfate-reducing bacteria (SRB) are frequently encountered in anoxic-to-oxic transition zones, where they are transiently exposed to microoxic or even oxic conditions on a regular basis. This can be marine tidal sediments, microbial mats, and freshwater wetlands like peatlands. In the latter, a cryptic but highly active sulfur cycle supports their anaerobic activity. Here, we aimed for a better understanding of how SRB responds to periodically fluctuating redox regimes., Results: To mimic these fluctuating redox conditions, a bioreactor was inoculated with peat soil supporting cryptic sulfur cycling and consecutively exposed to oxic (one week) and anoxic (four weeks) phases over a period of > 200 days. SRB affiliated to the genus Desulfosporosinus (Bacillota) and the families Syntrophobacteraceae, Desulfomonilaceae, Desulfocapsaceae, and Desulfovibrionaceae (Desulfobacterota) successively established growing populations (up to 2.9% relative abundance) despite weekly periods of oxygen exposures at 133 µM (50% air saturation). Adaptation mechanisms were analyzed by genome-centric metatranscriptomics. Despite a global drop in gene expression during oxic phases, the perpetuation of gene expression for energy metabolism was observed for all SRBs. The transcriptional response pattern for oxygen resistance was differentiated across individual SRBs, indicating different adaptation strategies. Most SRB transcribed differing sets of genes for oxygen consumption, reactive oxygen species detoxification, and repair of oxidized proteins as a response to the periodical redox switch from anoxic to oxic conditions. Noteworthy, a Desulfosporosinus, a Desulfovibrionaceaea, and a Desulfocapsaceaea representative maintained high transcript levels of genes encoding oxygen defense proteins even under anoxic conditions, while representing dominant SRB populations after half a year of bioreactor operation., Conclusions: In situ-relevant peatland SRB established large populations despite periodic one-week oxygen levels that are one order of magnitude higher than known to be tolerated by pure cultures of SRB. The observed decrease in gene expression regulation may be key to withstand periodically occurring changes in redox regimes in these otherwise strictly anaerobic microorganisms. Our study provides important insights into the stress response of SRB that drives sulfur cycling at oxic-anoxic interphases. Video Abstract., (© 2024. The Author(s).)

Blue holes are vertical water-filled openings in carbonate rock that exhibit complex morphology, ecology, and water chemistry. In this study, macroscopic microbial mat structures found in complete anoxic conditions in the Faanu Mudugau Blue Hole (Maldives) were studied by metagenomic methods. Such communities have likely been evolutionary isolated from the surrounding marine environment for more than 10,000 years since the Blue Hole formation during the last Ice Age. A total of 48 high-quality metagenome-assembled genomes (MAGs) were recovered, predominantly composed of the phyla Chloroflexota, Proteobacteria and Desulfobacterota. None of these MAGs have been classified to species level (<95% ANI), suggesting the discovery of several new microbial taxa. In particular, MAGs belonging to novel bacterial genera within the order Dehalococcoidales accounted for 20% of the macroscopic mat community. Genome-resolved metabolic analysis of this dominant microbial fraction revealed a mixotrophic lifestyle based on energy conservation via fermentation, hydrogen metabolism and anaerobic CO 2 fixation through the Wood-Ljungdahl pathway. Interestingly, these bacteria showed a high proportion of ancestral genes in their genomes providing intriguing perspectives on mechanisms driving microbial evolution in this peculiar environment. Overall, our results provide new knowledge for understanding microbial life under extreme conditions in blue hole environments., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Background: Cable bacteria are filamentous members of the Desulfobulbaceae family that are capable of performing centimetre‑scale electron transport in marine and freshwater sediments. This long‑distance electron transport is mediated by a network of parallel conductive fibres embedded in the cell envelope. This fibre network efficiently transports electrical currents along the entire length of the centimetre‑long filament. Recent analyses show that these fibres consist of metalloproteins that harbour a novel nickel‑containing cofactor, which indicates that cable bacteria have evolved a unique form of biological electron transport. This nickel‑dependent conduction mechanism suggests that cable bacteria are strongly dependent on nickel as a biosynthetic resource. Here, we performed a comprehensive comparative genomic analysis of the genes linked to nickel homeostasis. We compared the genome‑encoded adaptation to nickel of cable bacteria to related members of the Desulfobulbaceae family and other members of the Desulfobulbales order., Results: Presently, four closed genomes are available for the monophyletic cable bacteria clade that consists of the genera Candidatus Electrothrix and Candidatus Electronema. To increase the phylogenomic coverage, we additionally generated two closed genomes of cable bacteria: Candidatus Electrothrix gigas strain HY10‑6 and Candidatus Electrothrix antwerpensis strain GW3‑4, which are the first closed genomes of their respective species. Nickel homeostasis genes were identified in a database of 38 cable bacteria genomes (including 6 closed genomes). Gene prevalence was compared to 19 genomes of related strains, residing within the Desulfobulbales order but outside of the cable bacteria clade, revealing several genome‑encoded adaptations to nickel homeostasis in cable bacteria. Phylogenetic analysis indicates that nickel importers, nickel‑binding enzymes and nickel chaperones of cable bacteria are affiliated to organisms outside the Desulfobulbaceae family, with several proteins showing affiliation to organisms outside of the Desulfobacterota phylum. Conspicuously, cable bacteria encode a unique periplasmic nickel export protein RcnA, which possesses a putative cytoplasmic histidine‑rich loop that has been largely expanded compared to RcnA homologs in other organisms., Conclusion: Cable bacteria genomes show a clear genetic adaptation for nickel utilization when compared to closely related genera. This fully aligns with the nickel‑dependent conduction mechanism that is uniquely found in cable bacteria., (© 2024. The Author(s).)

Background: Biological nitrogen fixation is a fundamental process sustaining all life on earth. While distribution and diversity of N 2 -fixing soil microbes have been investigated by numerous PCR amplicon sequencing of nitrogenase genes, their comprehensive understanding has been hindered by lack of de facto standard protocols for amplicon surveys and possible PCR biases. Here, by fully leveraging the planetary collections of soil shotgun metagenomes along with recently expanded culture collections, we evaluated the global distribution and diversity of terrestrial diazotrophic microbiome., Results: After the extensive analysis of 1,451 soil metagenomic samples, we revealed that the Anaeromyxobacteraceae and Geobacteraceae within Deltaproteobacteria are ubiquitous groups of diazotrophic microbiome in the soils with different geographic origins and land usage types, with particular predominance in anaerobic soils (paddy soils and sediments)., Conclusion: Our results indicate that Deltaproteobacteria is a core bacterial taxon in the potential soil nitrogen fixation population, especially in anaerobic environments, which encourages a careful consideration on deltaproteobacterial diazotrophs in understanding terrestrial nitrogen cycling. Video Abstract., (© 2024. The Author(s).)

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene ( bcrABCD ). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ . For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans , and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments., Competing Interests: The authors declare no conflict of interest.

Syntrophus aciditrophicus strain SB is a model syntroph that degrades benzoate and alicyclic acids. The structure of a putative 3-hydroxypimelyl-CoA dehydrogenase from S. aciditrophicus strain SB (SaHcd1) was resolved at 1.78 Å resolution. SaHcd1 contains sequence motifs and structural features that belong to the short-chain dehydrogenase/reductase (SDR) family of NADPH-dependent oxidoreductases. SaHcd1 is proposed to concomitantly reduce NAD + or NADP + to NADH or NADPH, respectively, while converting 3-hydroxypimelyl-CoA to 3-oxopimeyl-CoA. Further enzymatic studies are needed to confirm the function of SaHcd1.

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO 2 , formate, and H 2 that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially N ε -acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, N ε -acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Sulfate-reducing microorganisms (SRM) in subsurface sediments live under constant substrate and energy limitation, yet little is known about how they adapt to this mode of life. We combined controlled chemostat cultivation and transcriptomics to examine how the marine sulfate reducer, Desulfobacterium autotrophicum, copes with substrate (sulfate or lactate) limitation. The half-saturation uptake constant (K m ) for lactate was 1.2 µM, which is the first value reported for a marine SRM, while the K m for sulfate was 3 µM. The measured residual lactate concentration in our experiments matched values observed in situ in marine sediments, supporting a key role of SRM in the control of lactate concentrations. Lactate limitation resulted in complete lactate oxidation via the Wood-Ljungdahl pathway and differential overexpression of genes involved in uptake and metabolism of amino acids as an alternative carbon source. D. autotrophicum switched to incomplete lactate oxidation, rerouting carbon metabolism in response to sulfate limitation. The estimated free energy was significantly lower during sulfate limitation (-28 to -33 kJ mol -1 sulfate), suggesting that the observed metabolic switch is under thermodynamic control. Furthermore, we detected the upregulation of putative sulfate transporters involved in either high or low affinity uptake in response to low or high sulfate concentration., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Electroactive bacteria are living catalysts, mediating energy-generating reactions at anodes or energy storage reactions at cathodes via extracellular electron transfer (EET). The Cathode-ANode (CANode) biofilm community was recently shown to facilitate both reactions; however, the identities of the primary constituents and underlying molecular mechanisms remain unknown. Here, we used metagenomics and metatranscriptomics to characterize the CANode biofilm. We show that a previously uncharacterized member of the family Desulfobulbaceae, Desulfobulbaceae -2, which had <1% relative abundance, had the highest relative gene expression and accounted for over 60% of all differentially expressed genes. At the anode potential, differential expression of genes for a conserved flavin oxidoreductase (Flx) and heterodisulfide reductase (Hdr) known to be involved in ethanol oxidation suggests a source of electrons for the energy-generating reaction. Genes for sulfate and carbon dioxide reduction pathways were expressed by Desulfobulbaceae -2 at both potentials and are the proposed energy storage reactions. Reduction reactions may be mediated by direct electron uptake from the electrode or from hydrogen generated at the cathode potential. The Desulfobulbaceae -2 genome is predicted to encode at least 85 multiheme (≥3 hemes) c -type cytochromes, some with as many as 26 heme-binding domains, that could facilitate reversible electron transfer with the electrode. Gene expression in other CANode biofilm species was also affected by the electrode potential, although to a lesser extent, and we cannot rule out their contribution to observed current. Results provide evidence of gene expression linked to energy storage and energy-generating reactions and will enable development of the CANode biofilm as a microbially driven rechargeable battery. IMPORTANCE Microbial electrochemical technologies (METs) rely on electroactive bacteria to catalyze energy-generating and energy storage reactions at electrodes. Known electroactive bacteria are not equally capable of both reactions, and METs are typically configured to be unidirectional. Here, we report on genomic and transcriptomic characterization of a recently described microbial electrode community called the Cathode-ANode (CANode). The CANode community is able to generate or store electrical current based on the electrode potential. During periods where energy is not needed, electrons generated from a renewable source, such as solar power, could be converted into energy storage compounds to later be reversibly oxidized by the same microbial catalyst. Thus, the CANode system can be thought of as a living "rechargeable battery." Results show that a single organism may be responsible for both reactions demonstrating a new paradigm for electroactive bacteria.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H 2 , CO 2 , or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO 2 to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO 2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO 2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms., Competing Interests: The authors declare no competing interest.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911 T ) and Pelobacter propionicus (DSM 2379 T ), Ac. wieringae (DSM 1911 T ) and Anaerotignum neopropionicum (DSM 3847 T ), Ac. wieringae strain JM and P. propionicus (DSM 2379 T ), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847 T ). Propionate was produced by all the cocultures, with the highest titer (∼24 mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum , which also produced isovalerate (∼4 mM), butyrate (∼1 mM), and isobutyrate (0.3 mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior. IMPORTANCE Syngas fermentation has great potential for the sustainable production of chemicals from wastes (via prior gasification) and flue gases containing CO/CO 2 . Research efforts need to be directed toward expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study provides the basis for a microbial process to produce propionate from CO using synthetic cocultures composed of acetogenic and propionigenic bacteria and elucidates the metabolic pathways involved. Furthermore, based on proteomics results, we hypothesize that the two bacterial species engage in an interaction that results in amino acid exchange, which subsequently promotes isovalerate and isobutyrate production. These findings provide a new understanding of gas fermentation and a coculturing strategy for expanding the product spectrum of microbial conversion of CO/CO 2 .

Background: Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA., Results: Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B 12 -dependent mutase, and a NAD + -dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B 12 -dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B 12 -dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone., Conclusions: According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70‍ ‍d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3‍ ‍mg L -1 d -1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.

Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure-function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca 2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca 2+ ; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca 2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a "locally closed" conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca 2+ modulation and periplasmic NTDs., Competing Interests: The authors declare no competing interest.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM., Competing Interests: The authors have declared that no competing interests exist.

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with 13 C (bicarbonate and propionate) and 15 N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the "community service" performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H 2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1 T and D. butyratoxydans MSL71 T , of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

Organohalide respiration is an important process in the global halogen cycle and for bioremediation. In this study, we compared the global transcriptomic and proteomic analyses of Desulfoluna spongiiphila strain AA1, an organohalide-respiring member of the Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as an electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster ( rdh16 ), which was significantly upregulated with the addition of 2,6-dibromophenol. The corresponding protein, reductive dehalogenase RdhA16032, was detected in the proteome under treatment with 2,6-dibromophenol but not with sulfate only. There was no significant difference in corrinoid biosynthesis gene expression levels between the two treatments, indicating that the production of corrinoid in D. spongiiphila is constitutive or not specific for organohalide versus sulfate respiration. Electron-transporting proteins or mediators unique for reductive dehalogenation were not revealed in our analysis, and we hypothesize that reductive dehalogenation may share an electron-transporting system with sulfate reduction. The metabolism of D. spongiiphila , predicted from transcriptomic and proteomic results, demonstrates high metabolic versatility and provides insights into the survival strategies of a marine sponge symbiont in an environment rich in organohalide compounds and other secondary metabolites. IMPORTANCE Respiratory reductive dehalogenation is an important process in the overall cycling of both anthropogenic and natural organohalide compounds. Marine sponges produce a vast array of bioactive compounds as secondary metabolites, including diverse halogenated compounds that may enrich for dehalogenating bacteria. Desulfoluna spongiiphila strain AA1 was originally enriched and isolated from the marine sponge Aplysina aerophoba and can grow with both brominated compounds and sulfate as electron acceptors for respiration. An understanding of the overall gene expression and the protein production profile in response to organohalides is needed to identify the full complement of genes or enzymes involved in organohalide respiration. Elucidating the metabolic capacity of this sponge-associated bacterium lays the foundation for understanding how dehalogenating bacteria may control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle., (Copyright © 2020 American Society for Microbiology.)

Cable bacteria of the family Desulfobulbaceae couple spatially separated sulfur oxidation and oxygen or nitrate reduction by long-distance electron transfer, which can constitute the dominant sulfur oxidation process in shallow sediments. However, it remains unknown how cells in the anoxic part of the centimeter-long filaments conserve energy. We found 16S rRNA gene sequences similar to groundwater cable bacteria in a 1-methylnaphthalene-degrading culture (1MN). Cultivation with elemental sulfur and thiosulfate with ferrihydrite or nitrate as electron acceptors resulted in a first cable bacteria enrichment culture dominated >90% by 16S rRNA sequences belonging to the Desulfobulbaceae. Desulfobulbaceae-specific fluorescence in situ hybridization (FISH) unveiled single cells and filaments of up to several hundred micrometers length to belong to the same species. The Desulfobulbaceae filaments also showed the distinctive cable bacteria morphology with their continuous ridge pattern as revealed by atomic force microscopy. The cable bacteria grew with nitrate as electron acceptor and elemental sulfur and thiosulfate as electron donor, but also by sulfur disproportionation when Fe(Cl) 2 or Fe(OH) 3 were present as sulfide scavengers. Metabolic reconstruction based on the first nearly complete genome of groundwater cable bacteria revealed the potential for sulfur disproportionation and a chemo-litho-autotrophic metabolism. The presence of different types of hydrogenases in the genome suggests that they can utilize hydrogen as alternative electron donor. Our results imply that cable bacteria not only use sulfide oxidation coupled to oxygen or nitrate reduction by LDET for energy conservation, but sulfur disproportionation might constitute the energy metabolism for cells in large parts of the cable bacterial filaments.

Redox-active iron minerals can act as energy sources or electron-transferring mediators in microbial syntrophic associations, being important means of interspecies metabolic cooperation in sedimentary environments. Alkaline conditions alter the thermodynamic stability of iron minerals, influencing their availability for interspecies syntrophic interactions. We have modeled anaerobic alkaliphilic microbial associations in ethanol-oxidizing co-culture of an obligate syntroph Candidatus "Contubernalis alkalaceticum" and a facultative lithotroph Geoalkalibacter ferrihydriticus, which is capable of dissimilatory Fe(III) reduction and homoacetogenic oxidation of Fe(II) with CO 2 . The co-cultures were cultivated with thermodynamically metastable ferric-containing ferrihydrite, or ferrous-containing siderite, or without minerals. Mössbauer spectral analysis revealed the transformation of both minerals to the stable magnetite. In the presence of ferrihydrite, G. ferrihydriticus firstly reduced Fe(III) with ethanol and then switched to syntrophic homoacetogenesis, providing the growth of obligate syntroph on ethanol. The ability of G. ferrihydriticus to accept hydrogen from its syntrophic partner and produce extra acetate from carbonate during ethanol oxidation was confirmed by co-culture growth without minerals. In the presence of siderite, G. ferrihydriticus performed homoacetogenesis using two electron donors simultaneously- siderite and hydrogen. Pieces of evidence for direct and indirect hydrogen-mediated electron exchange between partner organisms were obtained. Relative abundancies of partner organisms and the rate of acetate production by their co-cultures were strongly determined by thermodynamic benefits, which G. ferrihydriticus got from redox transformations of iron minerals. Even the minor growth of G. ferrihydriticus sustained the growth of the syntroph. Accordingly, microbe-to-mineral interactions could represent underestimated drivers of syntrophic interactions in alkaline sedimentary environments.

( R )-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and β-oxidation involving a specific ( R )-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens , revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for β-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all β-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria ) or by functional specialization (e.g., clade T for anaerobic toluene degradation). IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other β-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria., (Copyright © 2019 American Society for Microbiology.)

Iron sheet piles are widely used in flood protection, dike construction, and river bank reinforcement. Their corrosion leads to gradual deterioration and often makes replacement necessary. Natural deposit layers on these sheet piles can prevent degradation and significantly increase their life span. However, little is known about the mechanisms of natural protective layer formation. Here, we studied the microbially diverse populations of corrosion-protective deposit layers on iron sheet piles at the Gouderak pumping station in Zuid-Holland, the Netherlands. Deposit layers, surrounding sediment and top sediment samples were analyzed for soil physicochemical parameters, microbially diverse populations, and metabolic potential. Methanogens appeared to be enriched 18-fold in the deposit layers. After sequencing, metagenome assembly and binning, we obtained four nearly complete draft genomes of microorganisms ( Methanobacteriales , two Coriobacteriales , and Syntrophobacterales ) that were highly enriched in the deposit layers, strongly indicating a potential role in corrosion protection. Coriobacteriales and Syntrophobacterales could be part of a microbial food web degrading organic matter to supply methanogenic substrates. Methane-producing Methanobacteriales could metabolize iron, which may initially lead to mild corrosion but potentially stimulates the formation of a carbonate-rich protective deposit layer in the long term. In addition, Methanobacteriales and Coriobacteriales have the potential to interact with metal surfaces via direct interspecies or extracellular electron transfer. In conclusion, our study provides valuable insights into microbial populations involved in iron corrosion protection and potentially enables the development of novel strategies for in situ screening of iron sheet piles in order to reduce risks and develop more sustainable replacement practices. IMPORTANCE Iron sheet piles are widely used to reinforce dikes and river banks. Damage due to iron corrosion poses a significant safety risk and has significant economic impact. Different groups of microorganisms are known to either stimulate or inhibit the corrosion process. Recently, natural corrosion-protective deposit layers were found on sheet piles. Analyses of the microbial composition indicated a potential role for methane-producing archaea. However, the full metabolic potential of the microbial communities within these protective layers has not been determined. The significance of this work lies in the reconstruction of the microbial food web of natural corrosion-protective layers isolated from noncorroding metal sheet piles. With this work, we provide insights into the microbiological mechanisms that potentially promote corrosion protection in freshwater ecosystems. Our findings could support the development of screening protocols to assess the integrity of iron sheet piles to decide whether replacement is required., (Copyright © 2019 American Society for Microbiology.)

Oxygen minimum zones (OMZs) are marine regions where O 2 is undetectable at intermediate depths. Within OMZs, the oxygen-depleted zone (ODZ) induces anaerobic microbial processes that lead to fixed nitrogen loss via denitrification and anammox. Surprisingly, nitrite oxidation is also detected in ODZs, although all known marine nitrite oxidizers (mainly Nitrospina) are aerobes. We used metagenomic binning to construct metagenome-assembled genomes (MAGs) of nitrite oxidizers from OMZs. These MAGs represent two novel Nitrospina-like species, both of which differed from all known Nitrospina species, including cultured species and published MAGs. Relative abundances of different Nitrospina genotypes in OMZ and non-OMZ seawaters were estimated by mapping metagenomic reads to newly constructed MAGs and published high-quality genomes of members from the Nitrospinae phylum. The two novel species were present in all major OMZs and were more abundant inside ODZs, which is consistent with the detection of higher nitrite oxidation rates in ODZs than in oxic seawaters and suggests novel adaptations to anoxic environments. The detection of a large number of unclassified nitrite oxidoreductase genes in the dataset implies that the phylogenetic diversity of nitrite oxidizers is greater than previously thought.

Community compositional changes and the corrosion of carbon steel in the presence of different electron donor and acceptor combinations were examined with a methanogenic consortium enriched for its ability to mineralize paraffins. Despite cultivation in the absence of sulfate, metagenomic analysis revealed the persistence of several sulfate-reducing bacterial taxa. Upon sulfate amendment, the consortium was able to couple C28H58 biodegradation with sulfate reduction. Comparative analysis suggested that Desulforhabdus and/or Desulfovibrio likely supplanted methanogens as syntrophic partners needed for C28H58 mineralization. Further enrichment in the absence of a paraffin revealed that the consortium could also utilize carbon steel as a source of electrons. The severity of both general and localized corrosion increased in the presence of sulfate, regardless of the electron donor utilized. With carbon steel as an electron donor, Desulfobulbus dominated in the consortium and electrons from iron accounted for ∼92% of that required for sulfate reduction. An isolated Desulfovibrio spp. was able to extract electrons from iron and accelerate corrosion. Thus, hydrogenotrophic partner microorganisms required for syntrophic paraffin metabolism can be readily substituted depending on the availability of an external electron acceptor and a single paraffin-degrading consortium harbored microbes capable of both chemical and electrical microbially influenced iron corrosion., (© FEMS 2019.)

Methanogenic degradation of n -alkanes is prevalent in n -alkane-impacted anoxic oil reservoirs and oil-polluted sites. However, little is known about the initial activation mechanism of the substrate, especially n -alkanes with a chain length above C 16 Here, a methanogenic C 16 to C 20 n -alkane-degrading enrichment culture was established from production water of a low-temperature oil reservoir. At the end of the incubation (364 days), C 16 to C 20 (1-methylalkyl)succinates were detected in the n -alkane-amended enrichment culture, suggesting that fumarate addition had occurred in the degradation process. This evidence is supported further by the positive amplification of the assA gene encoding the alpha subunit of alkylsuccinate synthase. A phylogenetic analysis shows these assA amplicons to be affiliated with Smithella and Desulfatibacillum clades. Together with the high abundance of these clades in the bacterial community, these two species are postulated to be the key players in the degradation of C 16 to C 20 n -alkanes in the present study. Our results provide evidence that long n -alkanes are activated via a fumarate addition mechanism under methanogenic conditions. IMPORTANCE Methanogenic hydrocarbon degradation is the major process for oil degradation in subsurface oil reservoirs and is blamed for the formation of heavy oil and oil sands. Addition of n -alkanes to fumarate yielding alkyl-substituted succinates is a well-characterized anaerobic activation mechanism for hydrocarbons and is the most common activation mechanism in the anaerobic biodegradation of n -alkanes with chain lengths less than C 16 However, the activation mechanism involved in the methanogenic biodegradation of n -alkanes longer than C 16 is still uncertain. In this study, we analyzed a methanogenic enrichment culture amended with a mixture of C 16 to C 20 n -alkanes. These n -alkanes can be activated via fumarate addition by mixed cultures containing Smithella and Desulfatibacillum species under methanogenic conditions. These observations provide a fundamental understanding of long- n -alkane metabolism under methanogenic conditions and have important applications for the remediation of oil-contaminated sites and for energy recovery from oil reservoirs., (Copyright © 2019 American Society for Microbiology.)

A sulfide-oxidizing microorganism, Desulfurivibrio alkaliphilus (DA), generates a consistent enrichment of sulfur-34 ( 34 S ) in the produced sulfate of +12.5 per mil or greater. This observation challenges the general consensus that the microbial oxidation of sulfide does not result in large 34 S enrichments and suggests that sedimentary sulfides and sulfates may be influenced by metabolic activity associated with sulfide oxidation. Since the DA-type sulfide oxidation pathway is ubiquitous in sediments, in the modern environment, and throughout Earth history, the enrichments and depletions in 34 S in sediments may be the combined result of three microbial metabolisms: microbial sulfate reduction, the disproportionation of external sulfur intermediates, and microbial sulfide oxidation.

The inoculum source plays a crucial role in the anaerobic treatment of wastewaters. Lipids are present in various wastewaters and have a high methanogenic potential, but their hydrolysis results in the production of long chain fatty acids (LCFAs) that are inhibitory to anaerobic microorganisms. Screening of inoculum for the anaerobic treatment of LCFA-containing wastewaters has been performed at mesophilic and thermophilic conditions. However, an evaluation of inocula for producing methane from LCFA-containing wastewater has not yet been conducted at low temperatures and needs to be undertaken. In this study, three inocula (one granular sludge and two municipal digester sludges) were assessed for methane production from LCFA-containing synthetic dairy wastewater (SDW) at low temperatures (10 and 20°C). A methane yield (based on mL-CH 4 /g-COD added ) of 86-65% with acetate and 45-20% with SDW was achieved within 10 days using unacclimated granular sludge, whereas the municipal digester sludges produced methane only at 20°C but not at 10°C even after 200 days of incubation. The acetotrophic activity in the inoculum was found to be crucial for methane production from LCFA at low temperatures, highlighting the role of Methanosaeta (acetoclastic archaea) at low temperatures. The presence of bacterial taxa from the family Syntrophaceae ( Syntrophus and uncultured taxa) in the inoculum was found to be important for methane production from SDW at 10°C. This study suggests the evaluation of acetotrophic activity and the initial microbial community characteristics by high-throughput amplicon sequencing for selecting the inoculum for producing methane at low temperatures (up to 10°C) from lipid-containing wastewaters.

Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isophthalate (3-carboxybenzoate). It is a xenobiotic compound which has accumulated in the environment for more than 50 years due to its global industrial usage and can cause negative effects on the environment. Isophthalate degradation by the strictly anaerobic S. aromaticivorans was investigated to advance our understanding of the degradation of xenobiotics introduced into nature, and to identify enzymes that might have ecological significance for bioremediation. Differential proteome analysis of isophthalate- vs benzoate-grown cells revealed over 400 differentially expressed proteins of which only four were unique to isophthalate-grown cells. The isophthalate-induced proteins include a phenylacetate:CoA ligase, a UbiD-like decarboxylase, a UbiX-like flavin prenyltransferase, and a hypothetical protein. These proteins are encoded by genes forming a single gene cluster that putatively codes for anaerobic conversion of isophthalate to benzoyl-CoA. Subsequently, benzoyl-CoA is metabolized by the enzymes of the anaerobic benzoate degradation pathway that were identified in the proteomic analysis. In vitro enzyme assays with cell-free extracts of isophthalate-grown cells indicated that isophthalate is activated to isophthalyl-CoA by an ATP-dependent isophthalate:CoA ligase (IPCL), and subsequently decarboxylated to benzoyl-CoA by a UbiD family isophthalyl-CoA decarboxylase (IPCD) that requires a prenylated flavin mononucleotide (prFMN) cofactor supplied by UbiX to effect decarboxylation. Phylogenetic analysis revealed that IPCD is a novel member of the functionally diverse UbiD family (de)carboxylases. Homologs of the IPCD encoding genes are found in several other bacteria, such as aromatic compound-degrading denitrifiers, marine sulfate-reducers, and methanogenic communities in a terephthalate-degrading reactor. These results suggest that metabolic strategies adapted for degradation of isophthalate and other phthalate are conserved between microorganisms that are involved in the anaerobic degradation of environmentally relevant aromatic compounds.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps 1-3 , and through ethane-dependent sulfate reduction in slurries 4-7 . Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown 8 . Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum' 9 . Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO 2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C 2 H 6 ) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (C n H 2n+2 ) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps 10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13 C 2 -choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13 C 2 -choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment.

The formation of the potent neurotoxic methylmercury (MeHg) is a microbially mediated process that has raised much concern because MeHg poses threats to wildlife and human health. Since boreal forest soils can be a source of MeHg in aquatic networks, it is crucial to understand the biogeochemical processes involved in the formation of this pollutant. High-throughput sequencing of 16S rRNA and the mercury methyltransferase, hgcA, combined with geochemical characterisation of soils, were used to determine the microbial populations contributing to MeHg formation in forest soils across Sweden. The hgcA sequences obtained were distributed among diverse clades, including Proteobacteria, Firmicutes, and Methanomicrobia, with Deltaproteobacteria, particularly Geobacteraceae, dominating the libraries across all soils examined. Our results also suggest that MeHg formation is also linked to the composition of non-mercury methylating bacterial communities, likely providing growth substrate (e.g. acetate) for the hgcA-carrying microorganisms responsible for the actual methylation process. While previous research focused on mercury methylating microbial communities of wetlands, this study provides some first insights into the diversity of mercury methylating microorganisms in boreal forest soils.

A highly convergent access to the late-stage biosynthetic intermediates projerangolid and jerangolid E is presented, and its utility is demonstrated by the synthesis of novel non-natural jerangolid derivatives. The key steps are fragment couplings by Julia-Kocienski olefination and olefin cross metathesis, as well as a stereoselective tetrahydropyran formation by intramolecular oxa-Michael addition. Bioconversion experiments with the tailoring O-methyltransferase JerF confirmed its proposed biosynthetic role and revealed relaxed substrate specificity of this enzyme as well as tolerance to organic cosolvents.

The energetic situation of terminal fermentations in methanogenesis was analyzed by pool size determinations in sediment cores taken in the oligotrophic Lake Constance, Germany. Distribution profiles of fermentation intermediates and products were measured at three different water depths (2, 10, and 80 m). Methane concentrations were constant below 10 cm of sediment depth. Within the methanogenic zone, concentrations of formate, acetate, propionate, and butyrate varied between 1 and 40 μM, and hydrogen was between 0.5 and 5 Pa. From the distribution profiles of the fermentation intermediates, Gibbs free energy changes for their interconversion were calculated. Pool sizes of formate and hydrogen were energetically nearly equivalent, with -5 ± 5 kJ per mol difference of free energy change (ΔG) for a hypothetical conversion of formate to hydrogen plus CO 2 The ΔG values for conversion of fatty acids to methanogenic substrates and their further conversion to methane and CO 2 were calculated with hydrogen and with formate as intermediates. Syntrophic propionate oxidation reached energetic equilibrium with formate as the sole electron carrier but was sufficiently exergonic if at least some of the electrons were transferred via hydrogen. The energetic consequences of formate versus hydrogen transfer in secondary and methanogenic fermentations indicate that both carrier systems are probably used simultaneously to optimize the energy yields for the partners involved. IMPORTANCE In the terminal steps of methane formation in freshwater lake sediments, fermenting bacteria cooperate syntrophically with methanogens and homoacetogens at minimum energy increments via interspecies electron transfer. The energy yields of the partner organisms in these cooperations have so far been calculated based mainly on in situ hydrogen partial pressures. In the present study, we also analyzed pools of formate as an alternative electron carrier in sediment cores of an oligotrophic lake. The formate and hydrogen pools appeared to be energetically nearly equivalent and are likely to be used simultaneously for interspecies electron transfer. Calculations of reaction energies of the partners involved suggest that propionate degradation may also proceed through the Smithella pathway, which converts propionate via butyrate and acetate to three acetate residues, thus circumventing one energetically difficult fatty acid oxidation step., (Copyright © 2018 American Society for Microbiology.)

Background: The sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet., Results: The pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsβ (phenylphosphate synthase beta subunit), ppcβ (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg 2+ , Mn 2+ , K + as co-factors., Conclusions: The genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H 2 ) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H 2 derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.

Exposure to dietary sources of methylmercury (MeHg) is the focus of public health concerns with environmental mercury (Hg) contamination. MeHg is formed in anoxic environments by anaerobic microorganisms. This process has been studied mostly with single-species culture incubations, although the relevance of such studies to Hg(II)-methylation in situ is limited because microbial activities in the environment are critically modulated by interactions among microbial functional groups. Here we describe experiments in which Hg(II)-methylation was examined within the context of various microbial syntrophies. We show enhanced Hg(II)-methylation under conditions that established syntrophy by interspecies hydrogen and acetate transfer. Relative to activity of monocultures, interactions of Hg(II) methylating sulfate-reducing bacteria with a methanogen stimulated potential Hg(II)-methylation rates 2-fold to 9-fold, and with Syntrophobacter sp. 1.7-fold to 1.8-fold; those of a Hg(II) methylating Syntrophobacter sp. with a methanogen increased Hg(II)-methylation 2-fold. Under sulfate-depleted conditions, higher Hg(II)-methylation rates in the syntrophic incubations corresponded to higher free energy yields (ΔG°') than in the monocultures. Based on energetic considerations, we therefore propose that syntrophic microbial interactions are likely a major source of MeHg in sulfate- and iron-limited anoxic environments while in sulfate-replete environments, MeHg formation via sulfate reduction dominates.

Sulfate reducing bacteria (SRB) was used to stabilize cadmium (Cd) in sediments spiked with Cd. The study found that the Cd in sediments (≤600 mg kg -1 ) was successfully stabilized after 166 d SRB bio-treatment. This was verified by directly and indirectly examining Cd speciation in sediments, mobilization index, and Cd content in interstitial water. After 166 d bio-treatment, compared with control groups, Cd concentrations in interstitial water of Cd-spiked sediments were reduced by 77.6-96.4%. The bioavailable fractions of Cd (e.g., exchangeable and carbonate bound phases) were reduced, while more stable fractions of Cd (e.g., Fe-Mn oxide, organic bound, and residual phases) were increased. However, Cd mobilization in sediment was observed during the first part of bio-treatment (32 d), leading to an increase of Cd concentrations in the overlying water. Bacterial community composition (e.g., richness, diversity, and typical SRB) played an important role in Cd mobilization, dissolution, and stabilization. Bacterial community richness and diversity, including the typical SRB (e.g., Desulfobacteraceae and Desulfobulbaceae), were enhanced. However, bacterial communities were also influenced by Cd content and its speciations (especially the exchangeable and carbonate bound phases) in sediments, as well as total organic carbon in overlying water., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phylogenetically diverse environmental ANME archaea and sulfate-reducing bacteria cooperatively catalyze the anaerobic oxidation of methane oxidation (AOM) in multicelled consortia within methane seep environments. To better understand these cells and their symbiotic associations, we applied a suite of electron microscopy approaches, including correlative fluorescence in situ hybridization-electron microscopy (FISH-EM), transmission electron microscopy (TEM), and serial block face scanning electron microscopy (SBEM) three-dimensional (3D) reconstructions. FISH-EM of methane seep-derived consortia revealed phylogenetic variability in terms of cell morphology, ultrastructure, and storage granules. Representatives of the ANME-2b clade, but not other ANME-2 groups, contained polyphosphate-like granules, while some bacteria associated with ANME-2a/2c contained two distinct phases of iron mineral chains resembling magnetosomes. 3D segmentation of two ANME-2 consortium types revealed cellular volumes of ANME and their symbiotic partners that were larger than previous estimates based on light microscopy. Polyphosphate-like granule-containing ANME (tentatively termed ANME-2b) were larger than both ANME with no granules and partner bacteria. This cell type was observed with up to 4 granules per cell, and the volume of the cell was larger in proportion to the number of granules inside it, but the percentage of the cell occupied by these granules did not vary with granule number. These results illuminate distinctions between ANME-2 archaeal lineages and partnering bacterial populations that are apparently unified in their ability to perform anaerobic methane oxidation. IMPORTANCE Methane oxidation in anaerobic environments can be accomplished by a number of archaeal groups, some of which live in syntrophic relationships with bacteria in structured consortia. Little is known of the distinguishing characteristics of these groups. Here, we applied imaging approaches to better understand the properties of these cells. We found unexpected morphological, structural, and volume variability of these uncultured groups by correlating fluorescence labeling of cells with electron microscopy observables., (Copyright © 2018 American Society for Microbiology.)

Electron transfer is central to cellular life, from photosynthesis to respiration. In the case of anaerobic respiration, some microbes have extracellular appendages that can be utilised to transport electrons over great distances. Two model organisms heavily studied in this arena are Shewanella oneidensis and Geobacter sulfurreducens. There is some debate over how, in particular, the Geobacter sulfurreducens nanowires (formed from pilin nanofilaments) are capable of achieving the impressive feats of natural conductivity that they display. In this article, we outline the mechanisms of electron transfer through delocalised electron transport, quantum tunnelling, and hopping as they pertain to biomaterials. These are described along with existing examples of the different types of conductivity observed in natural systems such as DNA and proteins in order to provide context for understanding the complexities involved in studying the electron transport properties of these unique nanowires. We then introduce some synthetic analogues, made using peptides, which may assist in resolving this debate. Microbial nanowires and the synthetic analogues thereof are of particular interest, not just for biogeochemistry, but also for the exciting potential bioelectronic and clinical applications as covered in the final section of the review., Statement of Significance: Some microbes have extracellular appendages that transport electrons over vast distances in order to respire, such as the dissimilatory metal-reducing bacteria Geobacter sulfurreducens. There is significant debate over how G. sulfurreducens nanowires are capable of achieving the impressive feats of natural conductivity that they display: This mechanism is a fundamental scientific challenge, with important environmental and technological implications. Through outlining the techniques and outcomes of investigations into the mechanisms of such protein-based nanofibrils, we provide a platform for the general study of the electronic properties of biomaterials. The implications are broad-reaching, with fundamental investigations into electron transfer processes in natural and biomimetic materials underway. From these studies, applications in the medical, energy, and IT industries can be developed utilising bioelectronics., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO 3 2- ) is oxidized to phosphate (PO 4 3- ), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO 2 reduction. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax ( Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria , which has no known cultured isolates. Genes for phosphite oxidation and for CO 2 reduction to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO 2 reduction to formate, which is then assimilated into biomass via the reductive glycine pathway., Competing Interests: The authors declare no conflict of interest.

The possibility that bacteria other than Geobacter species might contain genes for electrically conductive pili (e-pili) was investigated by heterologously expressing pilin genes of interest in Geobacter sulfurreducens. Strains of G. sulfurreducens producing high current densities, which are only possible with e-pili, were obtained with pilin genes from Flexistipes sinusarabici, Calditerrivibrio nitroreducens and Desulfurivibrio alkaliphilus. The conductance of pili from these strains was comparable to native G. sulfurreducens e-pili. The e-pili derived from C. nitroreducens, and D. alkaliphilus pilin genes are the first examples of relatively long (>100 amino acids) pilin monomers assembling into e-pili. The pilin gene from Candidatus Desulfofervidus auxilii did not yield e-pili, suggesting that the hypothesis that this sulfate reducer wires itself with e-pili to methane-oxidizing archaea to enable anaerobic methane oxidation should be reevaluated. A high density of aromatic amino acids and a lack of substantial aromatic-free gaps along the length of long pilins may be important characteristics leading to e-pili. This study demonstrates a simple method to screen pilin genes from difficult-to-culture microorganisms for their potential to yield e-pili; reveals new sources for biologically based electronic materials; and suggests that a wide phylogenetic diversity of microorganisms may use e-pili for extracellular electron exchange.

Paraffinic n -alkanes (>C 17 ) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n -octacosane (C 28 H 58 ). Compared with that in controls, the consumption of n -octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C 28 H 58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C 28 H 58 -degrading cultures compared with that in controls, suggesting n -octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C 28 H 58 -degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery. IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (17 ) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C 17 ) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkane n -octacosane can be biodegraded under methanogenic conditions by a presumed Smithella phylotype. Compared with that of controls, we show an increased abundance and expression of the assA gene, which is known to be important for anaerobic n -alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only in n -octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments., (Copyright © 2017 American Society for Microbiology.)

Oil and gas percolate profusely through the sediments of the Gulf of Mexico, leading to numerous seeps at the seafloor, where complex microbial, and sometimes animal communities flourish. Sediments from three areas (two cold seeps with contrasting hydrocarbon composition and a site outside any area of active seepage) of the Gulf of Mexico were investigated and compared. Consistent with the existence of a seep microbiome, a distinct microbial community was observed in seep areas compared to sediment from outside areas of active seepage. The microbial community from sediments without any influence from hydrocarbon seepage was characterized by Planctomycetes and the metabolic potential was consistent with detrital marine snow degradation. By contrast, in seep samples with methane as the principal hydrocarbon, methane oxidation by abundant members of ANME-1 was likely the predominant process. Seep samples characterized by fluids containing both methane and complex hydrocarbons, were characterized by abundant Chloroflexi (Anaerolinaceae) and deltaproteobacterial lineages and exhibited potential for complex hydrocarbon degradation. These different metabolic capacities suggested that microorganisms in cold seeps can potentially rely on other processes beyond methane oxidation and that the hydrocarbon composition of the seep fluids may be a critical factor structuring the seafloor microbial community composition and function.

This study evaluates the transcriptionally active, dissimilatory sulfur- and arsenic-cycling components of the microbial community in alkaline, hypersaline Mono Lake, CA, USA. We sampled five depths spanning the redox gradient (10, 15, 18, 25 and 31 m) during maximum thermal stratification. We used custom databases to identify transcripts of genes encoding complex iron-sulfur molybdoenzyme (CISM) proteins, with a focus on arsenic (arrA, aioA and arxA) and sulfur cycling (dsrA, aprA and soxB), and assigned them to taxonomic bins. We also report on the distribution of transcripts related to the ars arsenic detoxification pathway. Transcripts from detoxification pathways were not abundant in oxic surface waters (10 m). Arsenic cycling in the suboxic and microaerophilic zones of the water column (15 and 18 m) was dominated by arsenite-oxidizing members of the Gammaproteobacteria most closely affiliated with Thioalkalivibrio and Halomonas, transcribing arxA. We observed a transition to arsenate-reducing bacteria belonging to the Deltaproteobacteria and Firmicutes transcribing arsenate reductase (arrA) in anoxic bottom waters of the lake (25 and 31 m). Sulfur cycling at 15 and 18 m was dominated by Gammaproteobacteria (Thioalkalivibrio and Thioalkalimicrobium) oxidizing reduced S species, with a transition to sulfate-reducing Deltaproteobacteria at 25 and 31 m. Genes related to arsenic and sulfur oxidation from Thioalkalivibrio were more highly transcribed at 15 m relative to other depths. Our data highlight the importance of Thioalkalivibrio to arsenic and sulfur biogeochemistry in Mono Lake and identify new taxa that appear capable of transforming arsenic.

Background: Deep-sea hydrothermal vents are hotspots for productivity and biodiversity. Thermal pyrolysis and circulation produce fluids rich in hydrocarbons and reduced compounds that stimulate microbial activity in surrounding sediments. Several studies have characterized the diversity of Guaymas Basin (Gulf of California) sediment-inhabiting microorganisms; however, many of the identified taxa lack cultures or genomic representations. Here, we resolved the metabolic potential and community-level interactions of these diverse communities by reconstructing and analyzing microbial genomes from metagenomic sequencing data., Results: We reconstructed 115 microbial metagenome-assembled genomes comprising 27 distinct archaeal and bacterial phyla. The archaea included members of the DPANN and TACK superphyla, Bathyarchaeota, novel Methanosarcinales (GoM-Arc1), and anaerobic methane-oxidizing lineages (ANME-1). Among the bacterial phyla, members of the Bacteroidetes, Chloroflexi, and Deltaproteobacteria were metabolically versatile and harbored potential pathways for hydrocarbon and lipid degradation and a variety of respiratory processes. Genes encoding enzymes that activate anaerobic hydrocarbons for degradation were detected in Bacteroidetes, Chloroflexi, Latescibacteria, and KSB1 phyla, while the reconstructed genomes for most candidate bacteria phyla (Aminicenantes, Atribacteria, Omnitrophica, and Stahlbacteria) indicated a fermentative metabolism. Newly obtained GoM-Arc1 archaeal genomes encoded novel pathways for short-chain hydrocarbon oxidation by alkyl-coenzyme M formation. We propose metabolic linkages among different functional groups, such as fermentative community members sharing substrate-level interdependencies with sulfur- and nitrogen-cycling microbes., Conclusions: Overall, inferring the physiologies of archaea and bacteria from metagenome-assembled genomes in hydrothermal deep-sea sediments has revealed potential mechanisms of carbon cycling in deep-sea sediments. Our results further suggest a network of biogeochemical interdependencies in organic matter utilization, hydrocarbon degradation, and respiratory sulfur cycling among deep-sea-inhabiting microbial communities.

Acetylene (C 2 H 2 ) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C 2 H 2 block assays of N 2 O reductase, and Pelobacter acetylenicus was shown to grow on C 2 H 2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N 2 ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N 2 ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N 2 ase. Diazotrophic growth was observed under N 2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N 2 ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days. IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants., (Copyright © 2017 American Society for Microbiology.)

This study demonstrates that the deltaproteobacterium Desulfurivibrio alkaliphilus can grow chemolithotrophically by coupling sulfide oxidation to the dissimilatory reduction of nitrate and nitrite to ammonium. Key genes of known sulfide oxidation pathways are absent from the genome of D. alkaliphilus Instead, the genome contains all of the genes necessary for sulfate reduction, including a gene for a reductive-type dissimilatory bisulfite reductase (DSR). Despite this, growth by sulfate reduction was not observed. Transcriptomic analysis revealed a very high expression level of sulfate-reduction genes during growth by sulfide oxidation, while inhibition experiments with molybdate pointed to elemental sulfur/polysulfides as intermediates. Consequently, we propose that D. alkaliphilus initially oxidizes sulfide to elemental sulfur, which is then either disproportionated, or oxidized by a reversal of the sulfate reduction pathway. This is the first study providing evidence that a reductive-type DSR is involved in a sulfide oxidation pathway. Transcriptome sequencing further suggests that nitrate reduction to ammonium is performed by a novel type of periplasmic nitrate reductase and an unusual membrane-anchored nitrite reductase. IMPORTANCE Sulfide oxidation and sulfate reduction, the two major branches of the sulfur cycle, are usually ascribed to distinct sets of microbes with distinct diagnostic genes. Here we show a more complex picture, as D. alkaliphilus , with the genomic setup of a sulfate reducer, grows by sulfide oxidation. The high expression of genes typically involved in the sulfate reduction pathway suggests that these genes, including the reductive-type dissimilatory bisulfite reductases, are also involved in as-yet-unresolved sulfide oxidation pathways. Finally, D. alkaliphilus is closely related to cable bacteria, which grow by electrogenic sulfide oxidation. Since there are no pure cultures of cable bacteria, D. alkaliphilus may represent an exciting model organism in which to study the physiology of this process., (Copyright © 2017 Thorup et al.)