Julie Robidart

Metabolic versatility of the Riftia pachyptila endosymbiont revealed through metagenomics

The facultative symbiont of Riftia pachyptila, named here Candidatus Endoriftia persephone, has evaded culture to date, but much has been learned regarding this symbiosis over the past three decades since its discovery. The symbiont population metagenome was sequenced in order to gain insight into its physiology. The population genome indicates that the symbionts use a partial Calvin-Benson Cycle for carbon fixation and the reverse TCA cycle (an alternative pathway for carbon fixation) that contains an unusual ATP citrate lyase. The presence of all genes necessary for heterotrophic metabolism, a phosphotransferase system, and dicarboxylate and ABC transporters indicate that the symbiont can live mixotrophically. The metagenome has a large suite of signal transduction, defence (both biological and environmental) and chemotaxis mechanisms. The physiology of Candidatus Endoriftia persephone is explored with respect to functionality while associated with a eukaryotic host, versus free-living in the hydrothermal environment.

Ecogenomic sensor reveals controls on N2-fixing microorganisms in the North Pacific Ocean.

Nitrogen-fixing microorganisms (diazotrophs) are keystone species that reduce atmospheric dinitrogen (N2) gas to fixed nitrogen (N), thereby accounting for much of N-based new production annually in the oligotrophic North Pacific. However, current approaches to study N2 fixation provide relatively limited spatiotemporal sampling resolution; hence, little is known about the ecological controls on these microorganisms or the scales over which they change. In the present study, we used a drifting robotic gene sensor to obtain high-resolution data on the distributions and abundances of N2-fixing populations over small spatiotemporal scales. The resulting measurements demonstrate that concentrations of N2 fixers can be highly variable, changing in abundance by nearly three orders of magnitude in less than 2 days and 30 km. Concurrent shipboard measurements and long-term time-series sampling uncovered a striking and previously unrecognized correlation between phosphate, which is undergoing long-term change in the region, and N2-fixing cyanobacterial abundances. These results underscore the value of high-resolution sampling and its applications for modeling the effects of global change.

Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation

Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.

Coupling Metabolite Flux to Transcriptomics: Insights Into the Molecular Mechanisms Underlying Primary Productivity by the Hydrothermal Vent Tubeworm Ridgeia piscesae

Deep-sea hydrothermal vents host highly productive ecosystems. Many of these communities are dominated by vestimentiferan tubeworms that house endosymbiotic chemoautotrophic bacteria that provide the hosts with their primary nutritional needs. Rates of carbon fixation by these symbioses are also among the highest recorded. Despite the breadth of physiological and biochemical research on these associations, the underlying molecular mechanisms that regulate host and symbiont metabolite flux and carbon fixation are largely unknown. Here we present metabolite flux and transcriptomics data from shipboard high-pressure respirometry experiments in which we maintained Ridgeia piscesae tubeworms at conditions comparable to those in situ. Host trophosome was used for cDNA library construction and sequencing. Of the 19,132 clones sequenced, 10,684 represented unique expressed sequence tags (ESTs). The highest proportions of genes are involved with translation, ribosomal structure and biogenesis, cellular processing, and signal transduction. There was moderate representation of genes involved in metabolite exchange and acid-base regulation. These data represent the first concomitant surveys of metabolite flux rates and gene expression for a chemoautotrophic symbiosis during net autotrophy, and they suggest that—in the case of Ridgeia piscesae—host-symbiont interactions such as cell cycle regulation may play a significant role in maintaining physiological poise during high productivity.

Characterizing microbial community and geochemical dynamics at hydrothermal vents using osmotically driven continuous fluid samplers.

Microbes play a key role in mediating aquatic biogeochemical cycles. However, our understanding of the relationships between microbial phylogenetic/physiological diversity and habitat physicochemical characteristics is restrained by our limited capacity to concurrently collect microbial and geochemical samples at appropriate spatial and temporal scales. Accordingly, we have developed a low-cost, continuous fluid sampling system (the Biological OsmoSampling System, or BOSS) to address this limitation. The BOSS does not use electricity, can be deployed in harsh/remote environments, and collects/preserves samples with daily resolution for >1 year. Here, we present data on the efficacy of DNA and protein preservation during a 1.5 year laboratory study as well as the results of two field deployments at deep-sea hydrothermal vents, wherein we examined changes in microbial diversity, protein expression, and geochemistry over time. Our data reveal marked changes in microbial composition co-occurring with changes in hydrothermal fluid composition as well as the temporal dynamics of an enigmatic sulfide-oxidizing symbiont in its free-living state. We also present the first data on in situ protein preservation and expression dynamics highlighting the BOSSs potential utility in meta-proteomic studies. These data illustrate the value of using BOSS to study relationships among microbial and geochemical phenomena and environmental conditions.

Single-taxon field measurements of bacterial gene regulation controlling DMSP fate

The ‘bacterial switch’ is a proposed regulatory point in the global sulfur cycle that routes dimethylsulfoniopropionate (DMSP) to two fundamentally different fates in seawater through genes encoding either the cleavage or demethylation pathway, and affects the flux of volatile sulfur from ocean surface waters to the atmosphere. Yet which ecological or physiological factors might control the bacterial switch remains a topic of considerable debate. Here we report the first field observations of dynamic changes in expression of DMSP pathway genes by a single marine bacterial species in its natural environment. Detection of taxon-specific gene expression in Roseobacter species HTCC2255 during a month-long deployment of an autonomous ocean sensor in Monterey Bay, CA captured in situ regulation of the first gene in each DMSP pathway (dddP and dmdA) that corresponded with shifts in the taxonomy of the phytoplankton community. Expression of the cleavage pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the demethylation pathway was greater in the presence of a mixed diatom and dinoflagellate community. These field data fit the prevailing hypothesis for bacterial DMSP gene regulation based on bacterial sulfur demand, but also suggest a modification involving oxidative stress response, evidenced as upregulation of catalase via katG, when DMSP is demethylated.

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia Orbicularis (Bivalvia: Lucinidae)

ABSTRACT The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.

A microarray for assessing transcription from pelagic marine microbial taxa

Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world’s oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions.

Linking Hydrothermal Geochemistry to Organismal Physiology: Physiological Versatility in Riftia pachyptila from Sedimented and Basalt-hosted Vents

Much of what is known regarding Riftia pachyptila physiology is based on the wealth of studies of tubeworms living at diffuse flows along the fast-spreading, basalt-hosted East Pacific Rise (EPR). These studies have collectively suggested that Riftia pachyptila and its chemoautotrophic symbionts are physiologically specialized, highly productive associations relying on hydrogen sulfide and oxygen to generate energy for carbon fixation, and the symbionts nitrate reduction to ammonia for energy and biosynthesis. However, Riftia also flourish in sediment-hosted vents, which are markedly different in geochemistry than basalt-hosted systems. Here we present data from shipboard physiological studies and global quantitative proteomic analyses of Riftia pachyptila trophosome tissue recovered from tubeworms residing in the EPR and the Guaymas basin, a sedimented, hydrothermal vent field. We observed marked differences in symbiont nitrogen metabolism in both the respirometric and proteomic data. The proteomic data further suggest that Riftia associations in Guaymas may utilize different sulfur compounds for energy generation, may have an increased capacity for energy storage, and may play a role in degrading exogenous organic carbon. Together these data reveal that Riftia symbionts are far more physiologically plastic than previously considered, and that -contrary to previous assertions- Riftia do assimilate reduced nitrogen in some habitats. These observations raise new hypotheses regarding adaptations to the geochemical diversity of habitats occupied by Riftia, and the degree to which the environment influences symbiont physiology and evolution.

Non-cyanobacterial nifH phylotypes in the North Pacific Subtropical Gyre detected by flow-cytometry cell sorting

In contrast to cyanobacteria, the significance of bacteria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur as contamination from reagents and other sources, complicating the detection and interpretation of environmental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell populations sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photoheterotrophic non-cyanobacterial nifH phylotypes previously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations, and was detected in water column samples using quantitative PCR (qPCR), with 60% of samples detected in the > 10 μm size fraction in addition to the 0.2-10 μm size fraction. A novel cluster 3-like nifH sequence was also recovered from discrete cell sorts and detected by qPCR in environmental samples. This approach enables the detection of rare nifH phylotypes, identifies possible associations with larger cells or particles and offers a possible solution for distinguishing reagent contaminants from real microbial community components.