Discovery and engineering of the antibody response against a prominent skin commensal.

The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8 + T cell response pre-emptively, in the absence of an infection 1 . However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel ß-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high titers of antibody under conditions of physiologic colonization, including a robust IgA response in the nasal mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome.

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

Ultra-deep sequencing of Hadza hunter-gatherers recovers vanishing gut microbes.

The gut microbiome modulates immune and metabolic health. Human microbiome data are biased toward industrialized populations, limiting our understanding of non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing on 351 fecal samples from the Hadza hunter-gatherers of Tanzania and comparative populations in Nepal and California. We recovered 91,662 genomes of bacteria, archaea, bacteriophages, and eukaryotes, 44% of which are absent from existing unified datasets. We identified 124 gut-resident species vanishing in industrialized populations and highlighted distinct aspects of the Hadza gut microbiome related to in situ replication rates, signatures of selection, and strain sharing. Industrialized gut microbes were found to be enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource, expands our understanding of microbes capable of colonizing the human gut, and clarifies the extensive perturbation induced by the industrialized lifestyle.

Ultra-deep Sequencing of Hadza Hunter-Gatherers Recovers Vanishing Gut Microbes.

The gut microbiome is a key modulator of immune and metabolic health. Human microbiome data is biased towards industrialized populations, providing limited understanding of the distinct and diverse non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing and strain cultivation on 351 fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophages, and eukaryotes, 43% of which are absent from existing unified datasets. Analysis of in situ growth rates, genetic pN/pS signatures, high-resolution strain tracking, and 124 gut-resident species vanishing in industrialized populations reveals differentiating dynamics of the Hadza gut microbiome. Industrialized gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource that expands our understanding of microbes capable of colonizing the human gut and clarifies the extensive perturbation brought on by the industrialized lifestyle.

Design, construction, and in vivo augmentation of a complex gut microbiome.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Robust variation in infant gut microbiome assembly across a spectrum of lifestyles.

Infant microbiome assembly has been intensely studied in infants from industrialized nations, but little is known about this process in nonindustrialized populations. We deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with more than 20% of genomes detected in the Hadza infant gut representing novel species. Industrialized infants-even those who are breastfed-have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.

Omics-Inferred Partitioning and Expression of Diverse Biogeochemical Functions in a Low-O2 Cyanobacterial Mat Community.

Cyanobacterial mats profoundly influenced Earth's biological and geochemical evolution and still play important ecological roles in the modern world. However, the biogeochemical functioning of cyanobacterial mats under persistent low-O2 conditions, which dominated their evolutionary history, is not well understood. To investigate how different metabolic and biogeochemical functions are partitioned among community members, we conducted metagenomics and metatranscriptomics on cyanobacterial mats in the low-O2, sulfidic Middle Island sinkhole (MIS) in Lake Huron. Metagenomic assembly and binning yielded 144 draft metagenome assembled genomes, including 61 of medium quality or better, and the dominant cyanobacteria and numerous Proteobacteria involved in sulfur cycling. Strains of a Phormidium autumnale-like cyanobacterium dominated the metagenome and metatranscriptome. Transcripts for the photosynthetic reaction core genes psaA and psbA were abundant in both day and night. Multiple types of psbA genes were expressed from each cyanobacterium, and the dominant psbA transcripts were from an atypical microaerobic type of D1 protein from Phormidium. Further, cyanobacterial transcripts for photosystem I genes were more abundant than those for photosystem II, and two types of Phormidium sulfide quinone reductase were recovered, consistent with anoxygenic photosynthesis via photosystem I in the presence of sulfide. Transcripts indicate active sulfur oxidation and reduction within the cyanobacterial mat, predominately by Gammaproteobacteria and Deltaproteobacteria, respectively. Overall, these genomic and transcriptomic results link specific microbial groups to metabolic processes that underpin primary production and biogeochemical cycling in a low-O2 cyanobacterial mat and suggest mechanisms for tightly coupled cycling of oxygen and sulfur compounds in the mat ecosystem. IMPORTANCE Cyanobacterial mats are dense communities of microorganisms that contain photosynthetic cyanobacteria along with a host of other bacterial species that play important yet still poorly understood roles in this ecosystem. Although such cyanobacterial mats were critical agents of Earth's biological and chemical evolution through geological time, little is known about how they function under the low-oxygen conditions that characterized most of their natural history. Here, we performed sequencing of the DNA and RNA of modern cyanobacterial mat communities under low-oxygen and sulfur-rich conditions from the Middle Island sinkhole in Lake Huron. The results reveal the organisms and metabolic pathways that are responsible for both oxygen-producing and non-oxygen-producing photosynthesis as well as interconversions of sulfur that likely shape how much O2 is produced in such ecosystems. These findings indicate tight metabolic reactions between community members that help to explain the limited the amount of O2 produced in cyanobacterial mat ecosystems.

Airborne Bacteria in Earth's Lower Stratosphere Resemble Taxa Detected in the Troposphere: Results From a New NASA Aircraft Bioaerosol Collector (ABC).

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Some taxa were more abundant in the flight samples than the ground samples, including OTUs from families Lachnospiraceae, Ruminococcaceae and Erysipelotrichaceae as well as the following genera: Clostridium, Mogibacterium, Corynebacterium, Bacteroides, Prevotella, Pseudomonas, and Parabacteroides. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

Elucidating the impact of microbial community biodiversity on pharmaceutical biotransformation during wastewater treatment.

In addition to removing organics and other nutrients, the microorganisms in wastewater treatment plants (WWTPs) biotransform many pharmaceuticals present in wastewater. The objective of this study was to examine the relationship between pharmaceutical biotransformation and biodiversity in WWTP bioreactor microbial communities and identify taxa and functional genes that were strongly associated with biotransformation. Dilution-to-extinction of an activated sludge microbial community was performed to establish cultures with a gradient of microbial biodiversity. Batch experiments were performed using the dilution cultures to determine biotransformation extents of several environmentally relevant pharmaceuticals. With this approach, because the communities were all established from the same original community, and using sequencing of the 16S rRNA and metatranscriptome, we identified candidate taxa and genes whose activity and transcript abundances associated with the extent of individual pharmaceutical biotransformation and were lost across the biodiversity gradient. Metabolic genes such as dehydrogenases, amidases and monooxygenases were significantly associated with pharmaceutical biotransformation, and five genera were identified whose activity significantly associated with pharmaceutical biotransformation. Understanding how biotransformation relates to biodiversity will inform the design of biological WWTPs for enhanced removal of chemicals that negatively impact environmental health.

Are Oligotypes Meaningful Ecological and Phylogenetic Units? A Case Study of Microcystis in Freshwater Lakes.

Oligotyping is a computational method used to increase the resolution of marker gene microbiome studies. Although oligotyping can distinguish highly similar sequence variants, the resulting units are not necessarily phylogenetically and ecologically informative due to limitations of the selected marker gene. In this perspective, we examine how oligotyping data is interpreted in recent literature, and we illustrate some of the method's constraints with a case study of the harmful bloom-forming cyanobacterium Microcystis. We identified three Microcystis oligotypes from a western Lake Erie bacterial community 16S rRNA gene (V4 region) survey that had previously clustered into one OTU. We found the same three oligotypes and two additional sequence variants in 46 Microcystis cultures isolated from Michigan inland lakes spanning a trophic gradient. In Lake Erie, shifts in Microcystis oligotypes corresponded to spatial nutrient gradients and temporal transitions in bloom toxicity. In the cultures, Microcystis oligotypes showed preferential distributions for different trophic states, but genomic data revealed that the oligotypes identified in Lake Erie did not correspond to toxin gene presence. Thus, oligotypes could not be used for inferring toxic ecotypes. Most strikingly, Microcystis oligotypes were not monophyletic. Our study supports the utility of oligotyping for distinguishing sequence types along certain ecological features, while it stresses that 16S rRNA gene sequence types may not reflect ecologically or phylogenetically cohesive populations. Therefore, we recommend that studies employing oligotyping or related tools consider these caveats during data interpretation.

Genomic and Transcriptomic Resolution of Organic Matter Utilization Among Deep-Sea Bacteria in Guaymas Basin Hydrothermal Plumes.

Microbial chemosynthesis within deep-sea hydrothermal vent plumes is a regionally important source of organic carbon to the deep ocean. Although chemolithoautotrophs within hydrothermal plumes have attracted much attention, a gap remains in understanding the fate of organic carbon produced via chemosynthesis. In the present study, we conducted shotgun metagenomic and metatranscriptomic sequencing on samples from deep-sea hydrothermal vent plumes and surrounding background seawaters at Guaymas Basin (GB) in the Gulf of California. De novo assembly of metagenomic reads and binning by tetranucleotide signatures using emergent self-organizing maps (ESOM) revealed 66 partial and nearly complete bacterial genomes. These bacterial genomes belong to 10 different phyla: Actinobacteria, Bacteroidetes, Chloroflexi, Deferribacteres, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Verrucomicrobia. Although several major transcriptionally active bacterial groups (Methylococcaceae, Methylomicrobium, SUP05, and SAR324) displayed methanotrophic and chemolithoautotrophic metabolisms, most other bacterial groups contain genes encoding extracellular peptidases and carbohydrate metabolizing enzymes with significantly higher transcripts in the plume than in background, indicating they are involved in degrading organic carbon derived from hydrothermal chemosynthesis. Among the most abundant and active heterotrophic bacteria in deep-sea hydrothermal plumes are Planctomycetes, which accounted for seven genomes with distinct functional and transcriptional activities. The Gemmatimonadetes and Verrucomicrobia also had abundant transcripts involved in organic carbon utilization. These results extend our knowledge of heterotrophic metabolism of bacterial communities in deep-sea hydrothermal plumes.

Unraveling the Physiological Roles of the Cyanobacterium Geitlerinema sp. BBD and Other Black Band Disease Community Members through Genomic Analysis of a Mixed Culture.

Black band disease (BBD) is a cyanobacterial-dominated polymicrobial mat that propagates on and migrates across coral surfaces, necrotizing coral tissue. Culture-based laboratory studies have investigated cyanobacteria and heterotrophic bacteria isolated from BBD, but the metabolic potential of various BBD microbial community members and interactions between them remain poorly understood. Here we report genomic insights into the physiological and metabolic potential of the BBD-associated cyanobacterium Geitlerinema sp. BBD 1991 and six associated bacteria that were also present in the non-axenic culture. The essentially complete genome of Geitlerinema sp. BBD 1991 contains a sulfide quinone oxidoreductase gene for oxidation of sulfide, suggesting a mechanism for tolerating the sulfidic conditions of BBD mats. Although the operon for biosynthesis of the cyanotoxin microcystin was surprisingly absent, potential relics were identified. Genomic evidence for mixed-acid fermentation indicates a strategy for energy metabolism under the anaerobic conditions present in BBD during darkness. Fermentation products may supply carbon to BBD heterotrophic bacteria. Among the six associated bacteria in the culture, two are closely related to organisms found in culture-independent studies of diseased corals. Their metabolic pathways for carbon and sulfur cycling, energy metabolism, and mechanisms for resisting coral defenses suggest adaptations to the coral surface environment and biogeochemical roles within the BBD mat. Polysulfide reductases were identified in a Flammeovirgaceae genome (Bacteroidetes) and the sox pathway for sulfur oxidation was found in the genome of a Rhodospirillales bacterium (Alphaproteobacteria), revealing mechanisms for sulfur cycling, which influences virulence of BBD. Each genomic bin possessed a pathway for conserving energy from glycerol degradation, reflecting adaptations to the glycerol-rich coral environment. The presence of genes for detoxification of reactive oxygen species and resistance to antibiotics suggest mechanisms for combating coral defense strategies. This study builds upon previous research on BBD and provides new insights into BBD disease etiology.

Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity.

The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita's core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

Genomic and transcriptomic evidence for scavenging of diverse organic compounds by widespread deep-sea archaea.

Microbial activity is one of the most important processes to mediate the flux of organic carbon from the ocean surface to the seafloor. However, little is known about the microorganisms that underpin this key step of the global carbon cycle in the deep oceans. Here we present genomic and transcriptomic evidence that five ubiquitous archaeal groups actively use proteins, carbohydrates, fatty acids and lipids as sources of carbon and energy at depths ranging from 800 to 4,950 m in hydrothermal vent plumes and pelagic background seawater across three different ocean basins. Genome-enabled metabolic reconstructions and gene expression patterns show that these marine archaea are motile heterotrophs with extensive mechanisms for scavenging organic matter. Our results shed light on the ecological and physiological properties of ubiquitous marine archaea and highlight their versatile metabolic strategies in deep oceans that might play a critical role in global carbon cycling.

Identification and analysis of the bacterial endosymbiont specialized for production of the chemotherapeutic natural product ET-743.

Ecteinascidin 743 (ET-743, Yondelis) is a clinically approved chemotherapeutic natural product isolated from the Caribbean mangrove tunicate Ecteinascidia turbinata. Researchers have long suspected that a microorganism may be the true producer of the anticancer drug, but its genome has remained elusive due to our inability to culture the bacterium in the laboratory using standard techniques. Here, we sequenced and assembled the complete genome of the ET-743 producer, Candidatus Endoecteinascidia frumentensis, directly from metagenomic DNA isolated from the tunicate. Analysis of the ∼ 631 kb microbial genome revealed strong evidence of an endosymbiotic lifestyle and extreme genome reduction. Phylogenetic analysis suggested that the producer of the anti-cancer drug is taxonomically distinct from other sequenced microorganisms and could represent a new family of Gammaproteobacteria. The complete genome has also greatly expanded our understanding of ET-743 production and revealed new biosynthetic genes dispersed across more than 173 kb of the small genome. The gene cluster's architecture and its preservation demonstrate that the drug is likely essential to the interactions of the microorganism with its mangrove tunicate host. Taken together, these studies elucidate the lifestyle of a unique, and pharmaceutically important microorganism and highlight the wide diversity of bacteria capable of making potent natural products.

Genomic and transcriptomic analyses of the facultative methanotroph Methylocystis sp. strain SB2 grown on methane or ethanol.

A minority of methanotrophs are able to utilize multicarbon compounds as growth substrates in addition to methane. The pathways utilized by these microorganisms for assimilation of multicarbon compounds, however, have not been explicitly examined. Here, we report the draft genome of the facultative methanotroph Methylocystis sp. strain SB2 and perform a detailed transcriptomic analysis of cultures grown with either methane or ethanol. Evidence for use of the canonical methane oxidation pathway and the serine cycle for carbon assimilation from methane was obtained, as well as for operation of the complete tricarboxylic acid (TCA) cycle and the ethylmalonyl-coenzyme A (EMC) pathway. Experiments with Methylocystis sp. strain SB2 grown on methane revealed that genes responsible for the first step of methane oxidation, the conversion of methane to methanol, were expressed at a significantly higher level than those for downstream oxidative transformations, suggesting that this step may be rate limiting for growth of this strain with methane. Further, transcriptomic analyses of Methylocystis sp. strain SB2 grown with ethanol compared to methane revealed that on ethanol (i) expression of the pathway of methane oxidation and the serine cycle was significantly reduced, (ii) expression of the TCA cycle dramatically increased, and (iii) expression of the EMC pathway was similar. Based on these data, it appears that Methylocystis sp. strain SB2 converts ethanol to acetyl-coenzyme A, which is then funneled into the TCA cycle for energy generation or incorporated into biomass via the EMC pathway. This suggests that some methanotrophs have greater metabolic flexibility than previously thought and that operation of multiple pathways in these microorganisms is highly controlled and integrated.

Novel hydrocarbon monooxygenase genes in the metatranscriptome of a natural deep-sea hydrocarbon plume.

Particulate membrane-associated hydrocarbon monooxygenases (pHMOs) are critical components of the aerobic degradation pathway for low molecular weight hydrocarbons, including the potent greenhouse gas methane. Here, we analysed pHMO gene diversity in metagenomes and metatranscriptomes of hydrocarbon-rich hydrothermal plumes in the Guaymas Basin (GB) and nearby background waters in the deep Gulf of California. Seven distinct phylogenetic groups of pHMO were present and transcriptionally active in both plume and background waters, including several that are undetectable with currently available polymerase chain reaction (PCR) primers. The seven groups of pHMOs included those related to a putative ethane oxidizing Methylococcaceae-like group, a group of the SAR324 Deltaproteobacteria, three deep-sea clades (Deep sea-1/symbiont-like, Deep sea-2/PS-80 and Deep sea-3/OPU3) within gammaproteobacterial methanotrophs, one clade related to Group Z and one unknown group. Differential abundance of pHMO gene transcripts in plume and background suggests niche differentiation between groups. Corresponding 16S rRNA genes reflected similar phylogenetic and transcriptomic abundance trends. The novelty of transcriptionally active pHMOs we recovered from a hydrocarbon-rich hydrothermal plume suggests there are significant gaps in our knowledge of the diversity and function of these enzymes in the environment.

Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics.

Chemolithotrophy is a pervasive metabolic lifestyle for microorganisms in the dark ocean. The SAR324 group of Deltaproteobacteria is ubiquitous in the ocean and has been implicated in sulfur oxidation and carbon fixation, but also contains genomic signatures of C1 utilization and heterotrophy. Here, we reconstructed the metagenome and metatranscriptome of a population of SAR324 from a hydrothermal plume and surrounding waters in the deep Gulf of California to gain insight into the genetic capability and transcriptional dynamics of this enigmatic group. SAR324's metabolism is signified by genes that encode a novel particulate hydrocarbon monooxygenase (pHMO), degradation pathways for corresponding alcohols and short-chain fatty acids, dissimilatory sulfur oxidation, formate dehydrogenase (FDH) and a nitrite reductase (NirK). Transcripts of the pHMO, NirK, FDH and transporters for exogenous carbon and amino acid uptake were highly abundant in plume waters. Sulfur oxidation genes were also abundant in the plume metatranscriptome, indicating SAR324 may also utilize reduced sulfur species in hydrothermal fluids. These results suggest that aspects of SAR324's versatile metabolism (lithotrophy, heterotrophy and alkane oxidation) operate simultaneously, and may explain SAR324's ubiquity in the deep Gulf of California and in the global marine biosphere.