Patricia L. Tavormina

Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin

ABSTRACT Methane vents are of significant geochemical and ecological importance. Notable progress has been made toward understanding anaerobic methane oxidation in marine sediments; however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs and distantly related to established methanotrophic clades. Water column-associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry.

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.

Distributions of putative aerobic methanotrophs in diverse pelagic marine environments.

Aerobic methane oxidization in the pelagic ocean serves an important role in limiting methane release to the atmosphere, yet little is known about the identity and distribution of bacteria that mediate this process. The distribution of putative methane-oxidizing marine groups, OPU1, OPU3 and Group X, was assessed in different ocean provinces using a newly developed fingerprinting method (monooxygenase intergenic spacer analysis (MISA)) in combination with pmoA clone library analysis and quantitative PCR (qPCR). The distribution of these three distinct monooxygenase groups, previously reported from pelagic marine environments, was examined in 39 samples including active methane seeps in the Gulf of Mexico and Santa Monica Bay, submarine canyon heads along the California continental margin, an oligotrophic subtropical gyre and areas proximal to a hydrothermal vent in the North Fiji back-arc basin. OPU1 and OPU3 were widely and similarly distributed within the meso- and bathypelagic zone (110 to ∼2000 m water depth) and showed a >50-fold greater abundance near methane seeps relative to non-seep sites. In contrast, Group X was predominantly recovered from samples along the California margin, at both seep and non-seep sites. All three phylotypes were below detection in the epipelagic zone to depths of 100 m. Several additional deeply branching monooxygenase sequences were also identified in this study, indicating the presence of uncharacterized groups of microorganisms potentially involved in the cycling of methane or ammonium.

Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor

Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The D-ESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

Starvation and recovery in the deep‐sea methanotroph Methyloprofundus sedimenti

In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane‐derived carbon into the food web remain poorly described, due to a lack of cultured representative deep‐sea methanotrophic prokaryotes. Here, the response of the deep‐sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup‐specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane‐derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea.

Colonial Tube-Dwelling Ciliates Influence Methane Cycling and Microbial Diversity within Methane Seep Ecosystems

In a variety of marine ecosystems, microbial eukaryotes play important ecological roles; however, our knowledge of their importance in deep-sea methane seep ecosystems is limited. Microbial eukaryotes have the potential to influence microbial community composition and diversity by creating habitat heterogeneity, and may contribute to carbon cycling through grazing or symbiotic associations with microorganisms. In this study, we characterized the distribution, substrate variability and ecology of a particular group of microbial eukaryotes, known as folliculinid ciliates, at methane seeps along the eastern Pacific margin. Folliculinid ciliates were recently recognized as an abundant and ecologically important component of hydrothermal vent ecosystems, but their ecology in methane seeps has not been examined. Folliculinid ciliates inhabited methane seeps from Costa Rica to Oregon, suggesting a broad distribution in the eastern Pacific. Using phylogenetic analyses of the 18S rRNA gene, two different species of folliculinid were identified. Folliculinids occupied a range of physical substrates, including authigenic carbonate rocks, shells of dead vesicomyid clams, polychaete tubes and gastropod shells. Molecular analysis of folliculinid associated microorganisms (16S rRNA and particulate methane monooxygenase) revealed that these ciliates not only influence overall microbial diversity, but also and have a specific relationship with bacteria in the ‘Deep sea-2’ methanotroph clade. Natural δ13C isotope signatures of folliculinids (-35‰) and their 13C-enrichment patterns in shipboard 13CH4 stable isotope-probing experiments indicated these ciliates and their associated microbes are involved in cycling methane-derived carbon. Folliculinids were significantly enriched in 13C after the addition of 13CH4 over short-term (3-8 day) incubations. Together, these results suggest that folliculinid ciliates represent a previously overlooked contributor to the ecology and biogeochemical cycling of deep-sea methane seep ecosystems.

Members of the methanotrophic genus Methylomarinum inhabit inland mud pots

Proteobacteria capable of converting the greenhouse gas methane to biomass, energy, and carbon dioxide represent a small but important sink in global methane inventories. Currently, 23 genera of methane oxidizing (methanotrophic) proteobacteria have been described, although many are represented by only a single validly described species. Here we describe a new methanotrophic isolate that shares phenotypic characteristics and phylogenetic relatedness with the marine methanotroph Methylomarinum vadi. However, the new isolate derives from a terrestrial saline mud pot at the northern terminus of the Eastern Pacific Rise (EPR). This new cultivar expands our knowledge of the ecology of Methylomarinum, ultimately towards a fuller understanding of the role of this genus in global methane cycling.

Aerobic and Anaerobic Methanotrophic Communities Associated with Methane Hydrates Exposed on the Seafloor: A High-Pressure Sampling and Stable Isotope-Incubation Experiment

High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.