Co-registered Geochemistry and Metatranscriptomics Reveal Unexpected Distributions of Microbial Activity within a Hydrothermal Vent Field.

Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria-particularly carbon fixation-in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.

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.

Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone.

Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine environment, suggesting widespread potential for oxidation of reduced substrates. Here, we used the well-defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating significantly with reduced environmental oxygen concentrations and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene-utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO-encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affiliated phylogenetically with the recently described Cu-MMO 'pXMO', and Group Z clustered with Cu-MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced molecules and advance our understanding of the microorganisms involved in methane cycling in the OMZ environment.

A hydrothermal seep on the Costa Rica margin: middle ground in a continuum of reducing ecosystems.

Upon their initial discovery, hydrothermal vents and methane seeps were considered to be related but distinct ecosystems, with different distributions, geomorphology, temperatures, geochemical properties and mostly different species. However, subsequently discovered vents and seep systems have blurred this distinction. Here, we report on a composite, hydrothermal seep ecosystem at a subducting seamount on the convergent Costa Rica margin that represents an intermediate between vent and seep ecosystems. Diffuse flow of shimmering, warm fluids with high methane concentrations supports a mixture of microbes, animal species, assemblages and trophic pathways with vent and seep affinities. Their coexistence reinforces the continuity of reducing environments and exemplifies a setting conducive to interactive evolution of vent and seep biota.

Bromeliad catchments as habitats for methanogenesis in tropical rainforest canopies.

Tropical epiphytic plants within the family Bromeliaceae are unusual in that they possess foliage capable of retaining water and impounded material. This creates an acidic (pH 3.5-6.5) and anaerobic (<1 ppm O(2)) environment suspended in the canopy. Results from a Costa Rican rainforest show that most bromeliads (n = 75/86) greater than ~20 cm in plant height or ~4-5 cm tank depth, showed presence of methanogens within the lower anoxic horizon of the tank. Archaea were dominated by methanogens (77-90% of recovered ribotypes) and community structure, although variable, was generally comprised of a single type, closely related to either hydrogenotrophic Methanoregula or Methanocella, a specific clade of aceticlastic Methanosaeta, or Methanosarcina. Juvenile bromeliads, or those species, such as Guzmania, with shallow tanks, generally did not possess methanogens, as assayed by polymerase chain reaction specific for methanogen 16S rRNA genes, nor did artificial catchments (~100 ml volume), in place 6-12 months prior to sample collection. Methanogens were not detected in soil (n = 20), except in one case, in which the dominant ribotype was different from nearby bromeliads. Recovery of methyl coenzyme M reductase genes supported the occurrence of hydrogenotrophic and aceticlastic methanogens within bromeliad tanks, as well as the trend, via QPCR analysis of mcrA, of increased methanogenic capacity with increased plant height. Methane production rates of up to 300 nmol CH(4) ml tank water(-1) day(-1) were measured in microcosm experiments. These results suggest that bromeliad-associated archaeal communities may play an important role in the cycling of carbon in neotropical forests.

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 approximately 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.

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

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.