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•In-depth proteome analysis was achieved for low- and subnanogram biological samples.•NanoLC with 30-μm-i.d. column and Orbitrap Fusion Lumos MS provided greatest coverage.•This is a ...promising analytical platform for proteomic analysis of single mammalian cells.
One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5ng to 50ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ∼3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ∼95% for 0.5ng samples and by ∼42% for 2ng samples. Using the best combination of the above variables, we were able to identify >3000 proteins from 10ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC–MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.
The atmospheric flux of methane from the oceans is largely mitigated through microbially mediated sulphate-coupled methane oxidation, resulting in the precipitation of authigenic carbonates. Deep-sea ...carbonates are common around active and palaeo-methane seepage, and have primarily been viewed as passive recorders of methane oxidation; their role as active and unique microbial habitats capable of continued methane consumption has not been examined. Here we show that seep-associated carbonates harbour active microbial communities, serving as dynamic methane sinks. Microbial aggregate abundance within the carbonate interior exceeds that of seep sediments, and molecular diversity surveys reveal methanotrophic communities within protolithic nodules and well-lithified carbonate pavements. Aggregations of microbial cells within the carbonate matrix actively oxidize methane as indicated by stable isotope FISH-nanoSIMS experiments and (14)CH4 radiotracer rate measurements. Carbonate-hosted methanotrophy extends the known ecological niche of these important methane consumers and represents a previously unrecognized methane sink that warrants consideration in global methane budgets.
The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including ...methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for β-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.
The sulfate-methane transition zone (SMTZ) is a widespread feature of continental margins, representing a diffusion-controlled interface where there is enhanced microbial activity. SMTZ microbial ...activity is commonly associated with the anaerobic oxidation of methane (AOM), which is carried out by syntrophic associations between sulfate-reducing bacteria and methane-oxidizing archaea. While our understanding of the microorganisms catalyzing AOM has advanced, the diversity and ecological role of the greater microbial assemblage associated with the SMTZ have not been well characterized. In this study, the microbial diversity above, within, and beneath the Santa Barbara Basin SMTZ was described. ANME-1-related archaeal phylotypes appear to be the primary methane oxidizers in the Santa Barbara Basin SMTZ, which was independently supported by exclusive recovery of related methyl coenzyme M reductase genes (mcrA). Sulfate-reducing Deltaproteobacteria phylotypes affiliated with the Desulfobacterales and Desulfosarcina-Desulfococcus clades were also enriched in the SMTZ, as confirmed by analysis of dissimilatory sulfite reductase (dsr) gene diversity. Statistical methods demonstrated that there was a close relationship between the microbial assemblages recovered from the two horizons associated with the geochemically defined SMTZ, which could be distinguished from microbial diversity recovered from the sulfate-replete overlying horizons and methane-rich sediment beneath the transition zone. Comparison of the Santa Barbara Basin SMTZ microbial assemblage to microbial assemblages of methane seeps and other organic matter-rich sedimentary environments suggests that bacterial groups not typically associated with AOM, such as Planctomycetes and candidate division JS1, are additionally enriched within the SMTZ and may represent a common bacterial signature of many SMTZ environments worldwide.
Rationale
Bulk and position‐specific stable isotope characterization of nitrous oxide represents one of the most powerful tools for identifying its environmental sources and sinks. Constraining ...14N15N18O and 15N14N18O will add two new dimensions to our ability to uniquely fingerprint N2O sources.
Methods
We describe a technique to measure six singly and doubly substituted isotopic variants of N2O, constraining the values of δ15N, δ18O, ∆17O, 15N site preference, and the clumped isotopomers 14N15N18O and 15N14N18O. The technique uses a Thermo MAT 253 Ultra, a high‐resolution multi‐collector gas source isotope ratio mass spectrometer. It requires 8–10 hours per sample and ~10 micromoles or more of pure N2O.
Results
We demonstrate the precision and accuracy of these measurements by analyzing N2O brought to equilibrium in its position‐specific and clumped isotopic composition by heating in the presence of a catalyst. Finally, an illustrative analysis of biogenic N2O from a denitrifying bacterium suggests that its clumped isotopic composition is controlled by kinetic isotope effects in N2O production.
Conclusions
We developed a method for measuring six isotopic variants of N2O and tested it with analyses of biogenic N2O. The added isotopic constraints provided by these measurements will enhance our ability to apportion N2O sources.
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
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
such as
and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.
Some archaea, known as anaerobic methanotrophs, are capable of converting methane into carbon dioxide when they are growing syntopically with sulfate-reducing bacteria. This partnership is the primary mechanism for methane removal in ocean sediments; however, there is still much to learn about how this syntrophy works. Previous studies have failed to identify the metabolic intermediate, such as hydrogen or formate, that is passed between partners. However, recent analysis of methanotrophic archaea has suggested that the syntrophy is formed through direct electron transfer. In this research, we analyzed the genomes of multiple partner bacteria and showed that they also contain the genes necessary to perform extracellular electron transfer, which are absent in related bacteria that do not form syntrophic partnerships with anaerobic methanotrophs. This genomic evidence shows a possible mechanism for direct electron transfer from methanotrophic archaea into the metabolism of the partner bacteria.
Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in ...biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved.
Syntrophy and mutualism play a central role in carbon and nutrient cycling by microorganisms. Yet our ability to recognize these partnerships in nature or to effectively study their behavior in ...culture has been hindered by the inherent interdependence of syntrophic associations, their dynamic behavior, and their frequent existence at thermodynamic limits. Now solutions to these challenges are emerging in new methodologies. These include: comparative metagenomics and transcriptomics; discovery-based methods such as Magneto-FISH; and metabolic substrate tracking using stable isotopes coupled either with density gradient separation (SIP) or with FISH-SIMS. These novel approaches are redefining the way we study microbial mutualism and are making intimate microbial associations accessible to both identification and characterization in their native habitats.
Over the past years, metagenomics has revolutionized our view of microbial diversity. Moreover, extracting near-complete genomes from metagenomes has led to the discovery of known metabolic traits in ...unsuspected lineages. Genome-resolved metagenomics relies on assembly of the sequencing reads and subsequent binning of assembled contigs, which might be hampered by strain heterogeneity or low abundance of a target organism. Here we present a complementary approach, metagenome marker gene mining, and use it to assess the global diversity of archaeal methane metabolism through the
gene. To this end, we have screened 18,465 metagenomes for the presence of reads matching a database representative of all known mcrA proteins and reconstructed gene sequences from the matching reads. We use our mcrA dataset to assess the environmental distribution of the
and reconstruct and analyze a draft genome belonging to the 'Lake Pavin cluster', an uncultivated environmental clade of the
. Analysis of the 'Lake Pavin cluster' draft genome suggests that this organism has a more restricted capacity for hydrogenotrophic methylotrophic methanogenesis than previously studied
, with only genes for growth on methanol present. However, the presence of the soluble subunits of methyltetrahydromethanopterin:CoM methyltransferase (
provide hypothetical pathways for methanol fermentation, and aceticlastic methanogenesis that await experimental verification. Thus, we show that marker gene mining can enhance the discovery power of metagenomics, by identifying novel lineages and aiding selection of targets for in-depth analyses. Marker gene mining is less sensitive to strain heterogeneity and has a lower abundance threshold than genome-resolved metagenomics, as it only requires short contigs and there is no binning step. Additionally, it is computationally cheaper than genome resolved metagenomics, since only a small subset of reads needs to be assembled. It is therefore a suitable approach to extract knowledge from the many publicly available sequencing projects.
Archaeal anaerobic methanotrophs ("ANME") and sulfate-reducing Deltaproteobacteria ("SRB") form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing ...modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b-SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b-SEEP-SRB1g consortia incubated with a headspace containing CH
and
N
revealed differences in cellular
N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in
N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.
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