Methane-Fueled Syntrophy through Extracellular Electron Transfer: Uncovering the Genomic Traits Conserved within Diverse Bacterial Partners of Anaerobic Methanotrophic Archaea

The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors

Geochemical, metagenomic and metaproteomic insights into trace metal utilization by methane-oxidizing microbial consortia in sulphidic marine sediments

Microbes have obligate requirements for trace metals in metalloenzymes that catalyse important biogeochemical reactions. In anoxic methane- and sulphide-rich environments, microbes may have unique adaptations for metal acquisition and utilization because of decreased bioavailability as a result of metal sulphide precipitation. However, micronutrient cycling is largely unexplored in cold (≤ 10°C) and sulphidic (> 1 mM ΣH_(2)S) deep-sea methane seep ecosystems. We investigated trace metal geochemistry and microbial metal utilization in methane seeps offshore Oregon and California, USA, and report dissolved concentrations of nickel (0.5–270 nM), cobalt (0.5–6 nM), molybdenum (10–5600 nM) and tungsten (0.3–8 nM) in Hydrate Ridge sediment porewaters. Despite low levels of cobalt and tungsten, metagenomic and metaproteomic data suggest that microbial consortia catalysing anaerobic oxidation of methane (AOM) utilize both scarce micronutrients in addition to nickel and molybdenum. Genetic machinery for cobalt-containing vitamin B_(12) biosynthesis was present in both anaerobic methanotrophic archaea (ANME) and sulphate-reducing bacteria. Proteins affiliated with the tungsten-containing form of formylmethanofuran dehydrogenase were expressed in ANME from two seep ecosystems, the first evidence for expression of a tungstoenzyme in psychrophilic microorganisms. Overall, our data suggest that AOM consortia use specialized biochemical strategies to overcome the challenges of metal availability in sulphidic environments

Reconstructing a hydrogen-driven microbial metabolic network in Opalinus Clay rock

The Opalinus Clay formation will host geological nuclear waste repositories in Switzerland. It is expected that gas pressure will build-up due to hydrogen production from steel corrosion, jeopardizing the integrity of the engineered barriers. In an in situ experiment located in the Mont Terri Underground Rock Laboratory, we demonstrate that hydrogen is consumed by microorganisms, fuelling a microbial community. Metagenomic binning and metaproteomic analysis of this deep subsurface community reveals a carbon cycle driven by autotrophic hydrogen oxidizers belonging to novel genera. Necromass is then processed by fermenters, followed by complete oxidation to carbon dioxide by heterotrophic sulfate-reducing bacteria, which closes the cycle. This microbial metabolic web can be integrated in the design of geological repositories to reduce pressure build-up. This study shows that Opalinus Clay harbours the potential for chemolithoautotrophic-based system, and provides a model of microbial carbon cycle in deep subsurface environments where hydrogen and sulfate are present

Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea

Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F_(420)-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments

Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea

Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments

Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativaL.) cultivar Bura Rata and their degradation during recovery from salinity stress

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress

Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples (Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples

Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples (Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples