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Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle. Multicellular magnetotactic bacteria (MMB) are unique in having no unicellular stage in their life cycle. This study characterizes the genomics and physiology of MMB consortia at single cell resolution, revealing that individual cells are not genetically identical and exhibit significant metabolic differentiation, highlighting their level of complexity. [ABSTRACT FROM AUTHOR]

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration. [ABSTRACT FROM AUTHOR]

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic (ANME) archaea in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it. A unique syntrophic partnership between anaerobic methanotrophic archaea and sulfate-reducing bacteria exists in deep-sea ecosystems, where they live in multicellular consortia. This study reveals a possible evolutionary trajectory of the bacterial partner towards adaptation to this syntrophic lifestyle. [ABSTRACT FROM AUTHOR]

Salt marshes are dynamic, highly productive ecosystems positioned at the interface between terrestrial and marine systems. They are exposed to large quantities of both natural and anthropogenic carbon input, and their diverse sediment-hosted microbial communities play key roles in carbon cycling and remineralization. To better understand the effects of natural and anthropogenic carbon on sediment microbial ecology, several sediment cores were collected from Little Sippewissett Salt Marsh (LSSM) on Cape Cod, MA, USA and incubated with either Spartina alterniflora cordgrass or diesel fuel. Resulting shifts in microbial diversity and activity were assessed via bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Both Spartina and diesel amendments resulted in initial decreases of microbial diversity as well as clear, community-wide shifts in metabolic activity. Multi-stage degradative frameworks shaped by fermentation were inferred based on anabolically active lineages. In particular, the metabolically versatile Marinifilaceae were prominent under both treatments, as were the sulfate-reducing Desulfovibrionaceae, which may be attributable to their ability to utilize diverse forms of carbon under nutrient limited conditions. By identifying lineages most directly involved in the early stages of carbon processing, we offer potential targets for indicator species to assess ecosystem health and highlight key players for selective promotion of bioremediation or carbon sequestration pathways. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor. A comparative genomics study of anaerobic methanotrophic (ANME) archaea reveals the genetic "parts list" associated with the repeated evolutionary transition between methanogenic and methanotrophic metabolism in the archaeal domain of life. [ABSTRACT FROM AUTHOR]

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community's metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach. [ABSTRACT FROM AUTHOR]

Summary: Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural–functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7‐day in situ incubations with a non‐canonical amino acid that was incorporated into new biomass, samples were resin‐embedded and analysed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence‐activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from ~60% in the top centimetre to 9.4%–22.4% between 2 and 10 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to putative sulfate‐reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non‐quartz minerals including rutile, orthoclase and plagioclase. Microbe–mineral relationships appear to be dynamic and context‐dependent arbiters of biogeochemical cycling. [ABSTRACT FROM AUTHOR]

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts. [ABSTRACT FROM AUTHOR]

Enzyme stability and activity at elevated temperatures are important aspects in biotechnological industries, such as the conversion of plant biomass into biofuels. In order to reduce the costs and increase the efficiency of biomass conversion, better enzymatic processing must be developed. Hot springs represent a treasure trove of underexplored microbiological and protein chemistry diversity. Herein, we conduct an exploratory study into the diversity of hot spring biomass-degrading potential. We describe the taxonomic diversity and carbohydrate active enzyme (CAZyme) coding potential in 71 publicly available metagenomic datasets from 58 globally distributed terrestrial geothermal features. Through taxonomic profiling, we detected a wide diversity of microbes unique to varying temperature and pH ranges. Biomass-degrading enzyme potential included all five classes of CAZymes and we described the presence or absence of genes encoding 19 glycosyl hydrolases hypothesized to be involved with cellulose, hemicellulose, and oligosaccharide degradation. Our results highlight hot springs as a promising system for the further discovery and development of thermo-stable biomass-degrading enzymes that can be applied toward generation of renewable biofuels. This study lays a foundation for future research to further investigate the functional diversity of hot spring biomass-degrading enzymes and their potential utility in biotechnological processing. [ABSTRACT FROM AUTHOR]

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L -azidohomoalanine (AHA) and L -homopropargylglycine (HPG); L -methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools. [ABSTRACT FROM AUTHOR]

The shallow-sea hydrothermal vents at White Point (WP) in Palos Verdes on the southern California coast support microbial mats and provide easily accessed settings in which to study chemolithoautotrophic sulfur cycling. Previous studies have cultured sulfuroxidizing bacteria from the WP mats; however, almost nothing is known about the in situ diversity and activity of the microorganisms in these habitats. We studied the diversity, micron-scale spatial associations and metabolic activity of the mat community via sequence analysis of 16S rRNA and aprA genes, fluorescence in situ hybridization (FISH) microscopy and sulfate reduction rate (SRR) measurements. Sequence analysis revealed a diverse group of bacteria, dominated by sulfur cycling gamma-, epsilon-, and deltaproteobacterial lineages such as Marithrix, Sulfurovum, and Desulfuromusa. FISH microscopy suggests a close physical association between sulfur-oxidizing and sulfur-reducing genotypes, while radiotracer studies showed low, but detectable, SRR. Comparative 16S rRNA gene sequence analyses indicate the WP sulfur vent microbial mat community is similar, but distinct from other hydrothermal vent communities representing a range of biotopes and lithologic settings. These findings suggest a complete biological sulfur cycle is operating in the WP mat ecosystem mediated by diverse bacterial lineages, with some similarity with deep-sea hydrothermal vent communities. [ABSTRACT FROM AUTHOR]

This paper describes a microfluidics-based workflow for genetically targeted isolation and cultivation of microorganisms from complex clinical samples. Data sets from high-throughput sequencing suggest the existence of previously unidentified bacterial taxa and functional genes with high biomedical importance. Obtaining isolates of these targets, preferably in pure cultures, is crucial for advancing understanding of microbial genetics and physiology and enabling physical access to microbes for further applications. However, the majority of microbes have not been cultured, due in part to the difficulties of both identifying proper growth conditions and characterizing and isolating each species. We describe a method that enables genetically targeted cultivation of microorganisms through a combination of microfluidics and on-and off-chip assays. This method involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a "chip wash" technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures. We validated this targeted approach by cultivating a bacterium, here referred to as isolate microfluidicus 1, from a human cecal biopsy. Isolate microfluidicus 1 is, to our knowledge, the first successful example of targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project's "Most Wanted" list, and, to our knowledge, the first cultured representative of a previously unidentified genus of the Ruminococcaceae family. [ABSTRACT FROM AUTHOR]

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, has been suggested to have been a central part of the global biogeochemical nitrogen cycle since the oxygenation of Earth. The cultivation of several ammonia-oxidizing archaea (AOA) as well as the discovery that archaeal ammonia monooxygenase (amo)-like gene sequences are nearly ubiquitously distributed in the environment and outnumber their bacterial counterparts in many habitats fundamentally revised our understanding of nitrification. Surprising insights into the physiological distinctiveness of AOA are mirrored by the recognition of the phyloge-netic uniqueness of these microbes, which fall within a novel archaeal phylum now known as Thaumarchaeota. The relative importance of AOA in nitrification, compared to ammonia-oxidizing bacteria (AOB), is still under debate. This minireview provides a synopsis of our current knowledge of the diversity and physiology of AOA, the factors controlling their ecology, and their role in carbon cycling as well as their potential involvement in the production of the greenhouse gas nitrous oxide. It emphasizes the importance of activity-based analyses in AOA studies and formulates priorities for future research. [ABSTRACT FROM AUTHOR]

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called “marine” group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as “Candidatus Nitrosotenuis uzonensis”, is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, “Candidatus N. uzonensis” also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in “Ca. N. uzonensis”, which potentially include genetic determinants of ecological niche differentiation. [ABSTRACT FROM AUTHOR]