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Unraveling the geochemical and microbial controls on methylmercury (MeHg) dynamics in mangrove sediments is important, as MeHg can potentially pose risks to marine biota and people that rely on these ecosystems. While the important role of sulfate-reducing bacteria in MeHg formation has been examined in this ecologically important habitat, the contribution of non-Hg methylating communities on MeHg production remains particularly unclear. Here, we collected sediment samples from 13 mangrove forests in south China and examined the geochemical parameters and microbial communities related to the Hg methylation. MeHg concentrations were significantly correlated to the OM-related parameters such as organic carbon content, total nitrogen, and dissolved organic carbon concentrations, suggesting the importance of OM in the MeHg production. Sulfate-reducing bacteria were the major Hg-methylators in mangrove sediments. Desulfobacteraceae and Desulfobulbaceae dominated the Hg-methylating microbes. Classification random forest analysis detected strong co-occurrence between Hg methylators and putative non-Hg methylators, thus suggesting that both types of microorganisms contribute to the MeHg dynamics in the sediments. Our study provides an overview of MeHg contamination in south China and advances our understanding of Hg methylation in mangrove ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.)

Various environmental factors, including H 2 availability, metabolic tradeoffs, optimal growth temperature, stochasticity, and hydrology, were examined to determine if they affect microbial competition between three autotrophic thermophiles. The thiosulfate reducer Desulfurobacterium thermolithotrophum ( T opt 72°C) was grown in mono- and coculture separately with the methanogens Methanocaldococcus jannaschii ( T opt 82°C) at 72°C and Methanothermococcus thermolithotrophicu s ( T opt 65°C) at 65°C at high and low H 2 concentrations. Both methanogens showed a metabolic tradeoff shifting from high growth rate-low cell yield at high H 2 concentrations to low growth rate-high cell yield at low H 2 concentrations and when grown in coculture with the thiosulfate reducer. In 1:1 initial ratios, D. thermolithotrophum outcompeted both methanogens at high and low H 2 , no H 2 S was detected on low H 2 , and it grew with only CO 2 as the electron acceptor indicating a similar metabolic tradeoff with low H 2 . When the initial methanogen-to-thiosulfate reducer ratio varied from 1:1 to 10 4 :1 with high H 2 , D. thermolithotrophum always outcompeted M. jannaschii at 72°C. However, M. thermolithotrophicus outcompeted D. thermolithotrophum at 65°C when the ratio was 10 3 :1. A reactive transport model that mixed pure hydrothermal fluid with cold seawater showed that hyperthermophilic methanogens dominated in systems where the residence time of the mixed fluid above 72°C was sufficiently high. With shorter residence times, thermophilic thiosulfate reducers dominated. If residence times increased with decreasing fluid temperature along the flow path, then thermophilic methanogens could dominate. Thermophilic methanogen dominance spread to previously thiosulfate-reducer-dominated conditions if the initial ratio of thermophilic methanogen-to-thiosulfate reducer increased., Importance: The deep subsurface is the largest reservoir of microbial biomass on Earth and serves as an analog for life on the early Earth and extraterrestrial environments. Methanogenesis and sulfur reduction are among the more common chemolithoautotrophic metabolisms found in hot anoxic hydrothermal vent environments. Competition between H2-oxidizing sulfur reducers and methanogens is primarily driven by the thermodynamic favorability of redox reactions with the former outcompeting methanogens. This study demonstrated that competition between the hydrothermal vent chemolithoautotrophs Methanocaldococcus jannaschii , Methanothermococcus thermolithotrophicus , and Desulfurobacterium thermolithotrophum is also influenced by other overlapping factors such as staggered optimal growth temperatures, stochasticity, and hydrology. By modeling all aspects of microbial competition coupled with field data, a better understanding is gained on how methanogens can outcompete thiosulfate reducers in hot anoxic environments and how the deep subsurface contributes to biogeochemical cycling., Competing Interests: The authors declare no conflict of interest.

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).)

Sulfate reduction is an essential metabolism that maintains biogeochemical cycles in marine and terrestrial ecosystems. Sulfate reducers are exclusively prokaryotic, phylogenetically diverse, and may have evolved early in Earth's history. However, their origin is elusive and unequivocal fossils are lacking. Here we report a new microfossil, Qingjiangonema cambria, from ∼518-million-year-old black shales that yield the Qingjiang biota. Qingjiangonema is a long filamentous form comprising hundreds of cells filled by equimorphic and equidimensional pyrite microcrystals with a light sulfur isotope composition. Multiple lines of evidence indicate Qingjiangonema was a sulfate-reducing bacterium that exhibits similar patterns of cell organization to filamentous forms within the phylum Desulfobacterota, including the sulfate-reducing Desulfonema and sulfide-oxidizing cable bacteria. Phylogenomic analyses confirm separate, independent origins of multicellularity in Desulfonema and in cable bacteria. Molecular clock analyses infer that the Desulfobacterota, which encompass a majority of sulfate-reducing taxa, diverged ∼2.41 billion years ago during the Paleoproterozoic Great Oxygenation Event, while cable bacteria diverged ∼0.56 billion years ago during or immediately after the Neoproterozoic Oxygenation Event. Taken together, we interpret Qingjiangonema as a multicellular sulfate-reducing microfossil and propose that cable bacteria evolved from a multicellular filamentous sulfate-reducing ancestor. We infer that the diversification of the Desulfobacterota and the origin of cable bacteria may have been responses to oxygenation events in Earth's history., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

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.

Fats, oil and grease, and their hydrolyzed counterparts-long chain fatty acids (LCFA) make up a large fraction of numerous wastewaters and are challenging to degrade anaerobically, more so, in low temperature anaerobic digestion (LtAD) systems. Herein, we perform a comparative analysis of publicly available Illumina 16S rRNA datasets generated from LCFA-degrading anaerobic microbiomes at low temperatures (10 and 20 °C) to comprehend the factors affecting microbial community dynamics. The various factors considered were the inoculum, substrate and operational characteristics, the reactor operation mode and reactor configuration, and the type of nucleic acid sequenced. We found that LCFA-degrading anaerobic microbiomes were differentiated primarily by inoculum characteristics (inoculum source and morphology) in comparison to the other factors tested. Inoculum characteristics prominently shaped the species richness, species evenness and beta-diversity patterns in the microbiomes even after long term operation of continuous reactors up to 150 days, implying the choice of inoculum needs careful consideration. The generalised additive models represented through beta diversity contour plots revealed that psychrophilic bacteria RBG-13-54-9 from family Anaerolineae, and taxa WCHB1-41 and Williamwhitmania were highly abundant in LCFA-fed microbial niches, suggesting their role in anaerobic treatment of LCFAs at low temperatures of 10-20 °C. Overall, we showed that the following bacterial genera: uncultured Propionibacteriaceae, Longilinea, Christensenellaceae R7 group, Lactivibrio, candidatus Caldatribacterium, Aminicenantales, Syntrophus, Syntrophomonas, Smithella, RBG-13-54-9, WCHB1-41, Trichococcus, Proteiniclasticum, SBR1031, Lutibacter and Lentimicrobium have prominent roles in LtAD of LCFA-rich wastewaters at 10-20 °C. This study provides molecular insights of anaerobic LCFA degradation under low temperatures from collated datasets and will aid in improving LtAD systems for treating LCFA-rich wastewaters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2 T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2 T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.)

Peatlands are responsible for over half of wetland methane emissions, yet major uncertainties remain regarding carbon flow, especially when increased availability of electron acceptors stimulates competing physiologies. We used microcosm incubations to study the effects of sulfate on microorganisms in two temperate peatlands, one bog and one fen. Three different electron donor treatments were used (13C-acetate, 13C-formate and a mixture of 12C short-chain fatty acids) to elucidate the responses of sulfate-reducing bacteria (SRB) and methanogens to sulfate stimulation. Methane production was measured and metagenomic sequencing was performed, with only the heavy DNA fraction sequenced from treatments receiving 13C electron donors. Our data demonstrate stimulation of dissimilatory sulfate reduction in both sites, with contrasting community responses. In McLean Bog (MB), hydrogenotrophic Deltaproteobacteria and acetotrophic Peptococcaceae lineages of SRB were stimulated, as were lineages with unclassified dissimilatory sulfite reductases. In Michigan Hollow Fen (MHF), there was little stimulation of Peptococcaceae populations, and a small stimulation of Deltaproteobacteria SRB populations only in the presence of formate as electron donor. Sulfate stimulated an increase in relative abundance of reads for both oxidative and reductive sulfite reductases, suggesting stimulation of an internal sulfur cycle. Together, these data indicate a stimulation of SRB activity in response to sulfate in both sites, with a stronger growth response in MB than MHF. This study provides valuable insights into microbial community responses to sulfate in temperate peatlands and is an important first step to understanding how SRB and methanogens compete to regulate carbon flow in these systems., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Magnetite-binding proteins are in high demand for the functionalization of magnetic nanoparticles. Binding analysis of six previously uncharacterized proteins from the magnetotactic Deltaproteobacterium Desulfamplus magnetovallimortis BW-1 identified two new magnetite-binding proteins (Mad10, Mad11). These proteins can be utilized as affinity tags for the immobilization of recombinant fusion proteins to magnetite.

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.

Soil biogeochemical processes are not only gauged by the dominant taxa in the microbiome but also depend on the critical functions of its 'rare biosphere' members. Here, we evaluated the biogeographical pattern of 'rare biosphere' propionate-oxidizing syntrophs in 113 paddy soil samples collected across China. The relative abundance, activity and growth potential of propionate-oxidizing syntrophs were analysed to provide a panoramic view of syntroph biogeographical distribution at the continental scale. The relative abundances of four syntroph genera, Syntrophobacter, Pelotomaculum, Smithella and Syntrophomonas were significantly greater at the warm low latitudes than at the cool high latitudes. Correspondingly, propionate degradation was faster in the low latitude soils compared with the high latitude soils. The low rate of propionate degradation in the high latitude soils resulted in a greater increase of the total syntroph relative abundance, probably due to their initial low relative abundances and the longer incubation time for propionate consumption. The mean annual temperature (MAT) is the most important factor shaping the biogeographical pattern of propionate-oxidizing syntrophs, with the next factor being the soil's total sulfur content (TS). We suggest that the effect of MAT is related to the thermodynamic conditions, in which the endergonic constraint of propionate oxidation is leveraged with the increase of MAT. The TS effect is likely due to the ability of some propionate syntrophs to facultatively perform sulfate respiration., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

Magnetotactic microorganisms can be found as unicellular prokaryotes, as cocci, vibrions, spirilla and rods, and as multicellular organisms. Multicellular magnetotactic prokaryotes are magnetotactic microorganisms composed by several magnetotactic bacteria organized almost in a spherical helix, and one of the most studied is Candidatus Magnetoglobus multicellularis. Several studies have shown that Ca. M. multicellularis displays forms of behavior not well explained by magnetotaxis. One of these is escape motility, also known as "ping-pong" motion. Studies done in the past associated the "ping-pong" motion to some magnetoreceptive behavior, but those studies were never replicated. In the present manuscript a characterization of escape motility trajectories of Ca. M. multicellularis was done for several magnetic fields, considering that this microorganism swims in cylindrical helical trajectories. It was observed that the escape motility can be separated into three phases: (I) when the microorganism jumps from the drop border, (II) where the microorganism moves almost perpendicular to the magnetic field and (III) when the microorganism returns to the drop border. The total time of the whole escape motility, the time spent in phase II and the displacement distance in phase I decreases when the magnetic field increases. Our results show that the escape motility has several characteristics that depend on the magnetic field and cannot be understood by magnetotaxis, with a magnetoreceptive mechanism being the best explanation.

Wetlands are common sites of active Hg methylation by anaerobic microbes; however, the amount of methylmercury produced varies greatly, as Hg methylation is dependent upon both the availability of Hg and the composition and activity of the microbial community involved. In this study, we identified the major microbial guilds responsible for Hg methylation along a trophic gradient composed of two sites and three different types of wetlands: a bog-fen peatland gradient and a black alder swamp, serving as net sources and a sink for methylmercury respectively. Iron-reducing bacteria in the Geobacteraceae were important Hg methylators across all wetlands and seasons examined, as evidenced by abundant 16S rRNA and hgcA transcripts clustering with this family. Molybdate inhibited Hg methylation more efficiently in the peatlands than in the swamp, suggesting an increasing role of sulfate-reducing bacteria and/or related syntrophs in the methylation of Hg with decreasing trophic status. Sulfate addition failed to increase Hg methylation rates in the peatlands, suggesting that SRBs/syntrophs were instead likely metabolizing alternative substrates such as syntrophic fermentation of organic compounds with methanogens. These results highlight the interconnectivity of anaerobic metabolism and importance of community dynamics on the methylation of Hg in wetlands with different trophic status., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The aromatic hydrocarbon naphthalene, which occurs in coal and oil, can be degraded by aerobic or anaerobic microorganisms. A wide-spread electron acceptor for the latter is sulfate. Evidence for in situ naphthalene degradation stems in particular from the detection of 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate in oil field samples. Because such intermediates are usually not detected in laboratory cultures with high sulfate concentrations, one may suppose that conditions in reservoirs, such as sulfate limitation, trigger metabolite release. Indeed, if naphthalene-grown cells of marine sulfate-reducing Deltaproteobacteria (strains NaphS2, NaphS3 and NaphS6) were transferred to sulfate-free medium, they released 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate while still consuming naphthalene. With 2-naphthoate as initial substrate, cells produced [5,6,7,8]-tetrahydro-2-naphthoate and the hydrocarbon, naphthalene, indicating reversibility of the initial naphthalene-metabolizing reaction. The reactions in the absence of sulfate were not coupled to observable growth. Excretion of naphthalene-derived metabolites was also achieved in sulfate-rich medium upon addition of the protonophore carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone or the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. In conclusion, obstruction of electron flow and energy gain by sulfate limitation offers an explanation for the occurrence of naphthalene-derived metabolites in oil reservoirs, and provides a simple experimental tool for gaining insights into the anaerobic naphthalene oxidation pathway from an energetic perspective., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Strain 28bB2T T is a sulfate-reducing bacterium isolated in a previous study, obtained from a p-xylene-degrading enrichment culture. Physiological, phylogenetic and genomic characterizations of strain 28bB2T T were performed to establish the taxonomic status of the strain. Cells of strain 28bB2T T were short oval-shaped (0.8-1.2×1.2-2.7μm), motile, and Gram-negative. For growth, the optimum pH was pH 6.5-7.0 and the optimum temperature was 28-32°C. Strain 28bB2T T oxidized toluene but could not utilize p-xylene. Sulfate and thiosulfate were used as electron acceptors. The G+C content of the genomic DNA was 53.8mol%. The genome consisted of an approximately 8.3 Mb of chromosome and two extrachromosomal elements. On the basis of 16S rRNA gene analysis, strain 28bB2T T was revealed to belong to the genus Desulfosarcina, with high sequence identities to Desulfosarcina ovata oXyS1 T (99.5%) and Desulfosarcina cetonica DSM 7267 T (98.7%). Results of Average Nucleotide Identity (ANI) calculation and digital DNA-DNA hybridization (dDDH) analysis showed that the strain 28bB2T T should be classified as a subspecies under D. ovata. Based on physiological and phylogenetic data, strain 28bB2T T (=NBRC 106234 =DSM 23484) is proposed as the type strain of a novel species in genus Desulfosarcina, Desulfosarcina ovata subsp. sediminis subsp. nov., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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.

Methylmercury is a neurotoxin that bioaccumulates from seawater to high concentrations in marine fish, putting human and ecosystem health at risk. High methylmercury levels have been found in the oxic subsurface waters of all oceans, but only anaerobic microorganisms have been shown to efficiently produce methylmercury in anoxic environments. The microaerophilic nitrite-oxidizing bacteria Nitrospina have previously been suggested as possible mercury methylating bacteria in Antarctic sea ice. However, the microorganisms responsible for processing inorganic mercury into methylmercury in oxic seawater remain unknown. Here, we show metagenomic and metatranscriptomic evidence that the genetic potential for microbial methylmercury production is widespread in oxic seawater. We find high abundance and expression of the key mercury methylating genes hgcAB across all ocean basins, corresponding to the taxonomic relatives of known mercury methylating bacteria from Deltaproteobacteria, Firmicutes and Chloroflexi. Our results identify Nitrospina as the predominant and widespread microorganism carrying and actively expressing hgcAB. The highest hgcAB abundance and expression occurs in the oxic subsurface waters of the global ocean where the highest MeHg concentrations are typically observed., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Anaerobic methanotrophic archaea (ANME) consume methane in marine sediments, limiting its release to the water column, but their responses to changes in methane and sulfate supplies remain poorly constrained. To address how methane exposure may affect microbial communities and methane- and sulfur-cycling gene abundances in Arctic marine sediments, we collected sediments from offshore Svalbard that represent geochemical horizons where anaerobic methanotrophy is expected to be active, previously active, and long-inactive based on reaction-transport biogeochemical modelling of porewater sulfate profiles. Sediment slurries were incubated at in situ temperature and pressure with different added methane concentrations. Sediments from an active area of seepage began to reduce sulfate in a methane-dependent manner within months, preceding increased relative abundances of anaerobic methanotrophs ANME-1 within communities. In previously active and long-inactive sediments, sulfur-cycling Deltaproteobacteria became more dominant after 30 days, though these communities showed no evidence of methanotrophy after nearly 8 months of enrichment. Overall, enrichment conditions, but not methane, broadly altered microbial community structure across different enrichment times and sediment types. These results suggest that active ANME populations may require years to develop, and consequently microbial community composition may affect methanotrophic responses to potential large-scale seafloor methane releases in ways that provide insight for future modelling studies., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Temperature influences microbiological growth and catabolic rates. Between 15 and 35 °C the growth rate and cell specific sulfate reduction rate of the sulfate reducing bacterium Desulfococcus multivorans increased with temperature. Sulfur isotope fractionation during sulfate reduction decreased with increasing temperature from 27.2 ‰ at 15 °C to 18.8 ‰ at 35 °C which is consistent with a decreasing reversibility of the metabolic pathway as the catabolic rate increases. Oxygen isotope fractionation, in contrast, decreased between 15 and 25 °C and then increased again between 25 and 35 °C, suggesting increasing reversibility in the first steps of the sulfate reducing pathway at higher temperatures. This points to a decoupling in the reversibility of sulfate reduction between the steps from the uptake of sulfate into the cell to the formation of sulfite, relative to the whole pathway from sulfate to sulfide. This observation is consistent with observations of increasing sulfur isotope fractionation when sulfate reducing bacteria are living near their upper temperature limit. The oxygen isotope decoupling may be a first signal of changing physiology as the bacteria cope with higher temperatures., (© FEMS 2020.)

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.

Protein-surface interactions play a pivotal role in processes as diverse as biomineralization, biofouling, and the cellular response to medical implants. In biomineralization processes, biomacromolecules control mineral deposition and architecture via complex and often unknown mechanisms. For studying these mechanisms, the formation of magnetite nanoparticles in magnetotactic bacteria has become an excellent model system. Most interestingly, nanoparticle morphologies have been discovered that defy crystallographic rules (e.g., in the species Desulfamplus magnetovallimortis strain BW-1). In certain conditions, this strain mineralizes bullet-shaped magnetite nanoparticles, which exhibit defined (111) crystal faces and are elongated along the [100] direction. We hypothesize that surface-specific protein interactions break the nanoparticle symmetry, inhibiting the growth of certain crystal faces and thereby favoring the growth of others. Screening the genome of BW-1, we identified Mad10 ( Ma gnetosome- a ssociated d eep-branching) as a potential magnetite-binding protein. Using atomic force microscope (AFM)-based single-molecule force spectroscopy, we show that a Mad10-derived peptide, which represents the most conserved region of Mad10, binds strongly to (100)- and (111)-oriented single-crystalline magnetite thin films. The peptide-magnetite interaction is thus material- but not crystal-face-specific. It is characterized by broad rupture force distributions that do not depend on the retraction speed of the AFM cantilever. To account for these experimental findings, we introduce a three-state model that incorporates fast rebinding. The model suggests that the peptide-surface interaction is strong in the absence of load, which is a direct result of this fast rebinding process. Overall, our study sheds light on the kinetic nature of peptide-surface interactions and introduces a new magnetite-binding peptide with potential use as a functional coating for magnetite nanoparticles in biotechnological and biomedical applications.

( 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.)

Around the world, several dozen deep sedimentary aquifers are being used for storage of natural gas. Ad hoc studies of the microbial ecology of some of them have suggested that sulfate reducing and methanogenic microorganisms play a key role in how these aquifers' communities function. Here, we investigate the influence of gas storage on these two metabolic groups by using high-throughput sequencing and show the importance of sulfate-reducing Desulfotomaculum and a new monophyletic methanogenic group. Aquifer microbial diversity was significantly related to the geological level. The distance to the stored natural gas affects the ratio of sulfate-reducing Firmicutes to deltaproteobacteria. In only one aquifer, the methanogenic archaea dominate the sulfate-reducers. This aquifer was used to store town gas (containing at least 50% H 2 ) around 50 years ago. The observed decrease of sulfates in this aquifer could be related to stimulation of subsurface sulfate-reducers. These results suggest that the composition of the microbial communities is impacted by decades old transient gas storage activity. The tremendous stability of these gas-impacted deep subsurface microbial ecosystems suggests that in situ biotic methanation projects in geological reservoirs may be sustainable over time., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the short-lived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

Propionate is a crucial intermediate during methane fermentation. Investigating the effects of different kinds of inhibitors on the propionate-degrading microbial community is necessary to develop countermeasures for improving process stability. In the present study, under inhibitory conditions (acetate, propionate, sulfide, and ammonium addition), the dynamic changes of the propionate-degrading microbial community from a mesophilic chemostat fed with propionate as the sole carbon source were investigated using high-throughput sequencing of 16S rRNA. Sulfide and/or ammonia inhibited specific species in the microbial community. Compared with Syntrophobacter, Smithella was more resistant to inhibition by sulfide and/or ammonia. However, Syntrophobacter demonstrated greater tolerance than Smithella under acid inhibition conditions. Some genera that had close phylogenetic relationships and similar functions showed similar responses to different inhibitors.

Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO 2 ) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

While paint particles are an important part of the microplastic sphere, they have, as yet, received much less research coverage, particularly regarding microplastic-microbiological interactions. This study investigated the biofilm communities of a variety of paint particles from brackish sediment using 16S rRNA gene sequencing. Paint particle biofilm communities appear to be distinct from natural (water and sediment), non-synthetic particle (cellulose) and common microplastic biofilm communities. Notably, there appears to be 1 group of sulphate-reducing bacteria from the Desulfobacteraceae family, Desulfatitalea tepidiphilia, that dominate certain paint biofilms. Of the 8 investigated paint-associated communities, four paints displayed this high Desulfobacteraceae presence. However, it is currently unclear from the chemical analysis performed of the paint surface chemistry (ATR FT-IR spectroscopy, Raman spectroscopy, SEM-EDX) what the drivers behind this might be. As such, this study provides important insights as the first to analyse microplastic-paint biofilm communities and paves the way for future research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.