Jillian M. Petersen

Hydrogen is an energy source for hydrothermal vent symbioses

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Pathways of Carbon and Energy Metabolism of the Epibiotic Community Associated with the Deep-Sea Hydrothermal Vent Shrimp Rimicaris exoculata

BACKGROUND The shrimp Rimicaris exoculata dominates the faunal biomass at many deep-sea hydrothermal vent sites at the Mid-Atlantic Ridge. In its enlarged gill chamber it harbors a specialized epibiotic bacterial community for which a nutritional role has been proposed. METHODOLOGY/PRINCIPAL FINDINGS We analyzed specimens from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge by complementing a 16S rRNA gene survey with the analysis of genes involved in carbon, sulfur and hydrogen metabolism. In addition to Epsilon- and Gammaproteobacteria, the epibiotic community unexpectedly also consists of Deltaproteobacteria of a single phylotype, closely related to the genus Desulfocapsa. The association of these phylogenetic groups with the shrimp was confirmed by fluorescence in situ hybridization. Based on functional gene analyses, we hypothesize that the Gamma- and Epsilonproteobacteria are capable of autotrophic growth by oxidizing reduced sulfur compounds, and that the Deltaproteobacteria are also involved in sulfur metabolism. In addition, the detection of proteobacterial hydrogenases indicates the potential for hydrogen oxidation in these communities. Interestingly, the frequency of these phylotypes in 16S rRNA gene clone libraries from the mouthparts differ from that of the inner lining of the gill chamber, indicating potential functional compartmentalization. CONCLUSIONS Our data show the specific association of autotrophic bacteria with Rimicaris exoculata from the Snake Pit hydrothermal vent field, and suggest that autotrophic carbon fixation is contributing to the productivity of the epibiotic community with the reductive tricarboxylic acid cycle as one important carbon fixation pathway. This has not been considered in previous studies of carbon fixation and stable carbon isotope composition of the shrimp and its epibionts. Furthermore, the co-occurrence of sulfur-oxidizing and sulfur-reducing epibionts raises the possibility that both may be involved in the syntrophic exchange of sulfur compounds, which could increase the overall efficiency of this epibiotic community.

Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma‐ and epsilonproteobacteria at four Mid‐Atlantic Ridge hydrothermal vent fields

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.

Convergent and divergent evolution of metabolism in sulfur-oxidizing symbionts and the role of horizontal gene transfer.

Symbioses between marine invertebrates and autotrophic sulfur-oxidizing bacteria have evolved from multiple lineages within the Gammaproteobacteria in a striking example of convergent evolution. These GammaSOX symbionts all perform the same basic function: they provide their hosts with nutrition through the fixation of CO(2) into biomass using reduced sulfur compounds as an energy source. However, our review of recent -omics based studies and genome mining for this study revealed that the GammaSOX symbionts diverge in many other metabolic capabilities and functions, and we show how these divergences could reflect adaptations to different hosts and habitat conditions. Our phylogenetic analyses of key metabolic genes in GammaSOX symbionts revealed that these differed markedly from 16S rRNA phylogenies. We hypothesize that horizontal gene transfer (HGT) would explain many of these incongruencies, and conclude that HGT may have played a significant role in shaping the metabolic evolution of GammaSOX symbionts.

Origins and Evolutionary Flexibility of Chemosynthetic Symbionts From Deep-Sea Animals

Bathymodiolin mussels dominate hydrothermal vent and cold seep communities worldwide. Symbiotic associations with chemosynthetic sulfur- and methane-oxidizing bacteria that provide for their nutrition are the key to their ecological and evolutionary success. The current paradigm is that these symbioses evolved from two free-living ancestors, one methane-oxidizing and one sulfur-oxidizing bacterium. In contrast to previous studies, our phylogenetic analyses of the bathymodiolin symbionts show that both the sulfur and the methane oxidizers fall into multiple clades interspersed with free-living bacteria, many of which were discovered recently in metagenomes from marine oxygen minimum zones. We therefore hypothesize that symbioses between bathymodiolin mussels and free-living sulfur- and methane-oxidizing bacteria evolved multiple times in convergent evolution. Furthermore, by 16S rRNA sequencing and fluorescence in situ hybridization, we show that close relatives of the bathymodiolin symbionts occur on hosts belonging to different animal phyla: Raricirrus beryli, a terebellid polychaete from a whale-fall, and a poecilosclerid sponge from a cold seep. The host range within the bathymodiolin symbionts is therefore greater than previously recognized, confirming the remarkable flexibility of these symbiotic associations.

Genetic Connectivity between North and South Mid-Atlantic Ridge Chemosynthetic Bivalves and Their Symbionts

Transform faults are geological structures that interrupt the continuity of mid-ocean ridges and can act as dispersal barriers for hydrothermal vent organisms. In the equatorial Atlantic Ocean, it has been hypothesized that long transform faults impede gene flow between the northern and the southern Mid-Atlantic Ridge (MAR) and disconnect a northern from a southern biogeographic province. To test if there is a barrier effect in the equatorial Atlantic, we examined phylogenetic relationships of chemosynthetic bivalves and their bacterial symbionts from the recently discovered southern MAR hydrothermal vents at 5°S and 9°S. We examined Bathymodiolus spp. mussels and Abyssogena southwardae clams using the mitochondrial cytochrome c oxidase subunit I (COI) gene as a phylogenetic marker for the hosts and the bacterial 16S rRNA gene as a marker for the symbionts. Bathymodiolus spp. from the two southern sites were genetically divergent from the northern MAR species B. azoricus and B. puteoserpentis but all four host lineages form a monophyletic group indicating that they radiated after divergence from their northern Atlantic sister group, the B. boomerang species complex. This suggests dispersal of Bathymodiolus species from north to south across the equatorial belt. 16S rRNA genealogies of chemoautotrophic and methanotrophic symbionts of Bathymodiolus spp. were inconsistent and did not match the host COI genealogy indicating disconnected biogeography patterns. The vesicomyid clam Abyssogena southwardae from 5°S shared an identical COI haplotype with A. southwardae from the Logatchev vent field on the northern MAR and their symbionts shared identical 16S phylotypes, suggesting gene flow across the Equator. Our results indicate genetic connectivity between the northern and southern MAR and suggest that a strict dispersal barrier does not exist.

Geochemical constraints on the diversity and activity of H2-oxidizing microorganisms in diffuse hydrothermal fluids from a basalt- and an ultramafic-hosted vent

Mixing processes of reduced hydrothermal fluids with oxygenated seawater and fluid-rock reactions contribute to the chemical signatures of diffuse venting and likely determine the geochemical constraints on microbial life. We examined the influence of fluid chemistry on microbial diversity and activity by sampling diffuse fluids emanating through mussel beds at two contrasting hydrothermal vents. The H(2) concentration was very low at the basalt-hosted Clueless site, and mixing models suggest O(2) availability throughout much of the habitat. In contrast, effluents from the ultramafic-hosted Quest site were considerably enriched in H(2) , while O(2) is likely limited to the mussel layer. Only two different hydrogenase genes were identified in clone libraries from the H(2) -poor Clueless fluids, but these fluids exhibited the highest H(2) uptake rates in H(2) -spiked incubations (oxic conditions, at 18 °C). In contrast, a phylogenetically diverse H(2) -oxidizing potential was associated with distinct thermal conditions in the H(2) -rich Quest fluids, but under oxic conditions, H(2) uptake rates were extremely low. Significant stimulation of CO(2) fixation rates by H(2) addition was solely illustrated in Quest incubations (P-value <0.02), but only in conjunction with anoxic conditions (at 18 °C). We conclude that the factors contributing toward differences in the diversity and activity of H(2) oxidizers at these sites include H(2) and O(2) availability.

Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the southern Gulf of Mexico

Chemosynthetic life was recently discovered at Chapopote, an asphalt hydrocarbon seep in the southern Gulf of Mexico. Preliminary morphological analyses indicated that one tubeworm and two mussel species colonize Chapopote. Our molecular analyses identified the tubeworm as Escarpia sp., and the mussels as Bathymodiolus heckerae and B. brooksi. Comparative 16S rRNA analysis and FISH showed that all three species harbour intracellular sulfur-oxidizing symbionts highly similar or identical to those found in the same host species from northern Gulf of Mexico (nGoM). The mussels also harbour methane-oxidizing symbionts, and these shared highly similar to identical 16S rRNA sequences to their nGoM conspecifics. We discovered a novel symbiont in B. heckerae, which is closely related to hydrocarbon-degrading bacteria of the genus Cycloclasticus. In B. heckerae, we found key genes for the use of aromatic compounds, and its stable carbon isotope values were consistently higher than B. brooksi, indicating that the novel symbiont might use isotopically heavy aromatic hydrocarbons from the asphalt seep. This discovery is particularly intriguing because until now only methane and reduced sulfur compounds have been shown to power cold-seep chemosynthetic symbioses. The abundant hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition.

Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels

Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable ‘arsenal’ of TRGs. DOI: http://dx.doi.org/10.7554/eLife.07966.001

The gill chamber epibiosis of deep-sea shrimp Rimicaris exoculata: an in-depth metagenomic investigation and discovery of Zetaproteobacteria

The gill chamber of deep-sea hydrothermal vent shrimp Rimicaris exoculata hosts a dense community of epibiotic bacteria dominated by filamentous Epsilonproteobacteria and Gammaproteobacteria. Using metagenomics on shrimp from the Rainbow hydrothermal vent field, we showed that both epibiont groups have the potential to grow autotrophically and oxidize reduced sulfur compounds or hydrogen with oxygen or nitrate. For carbon fixation, the Epsilonproteobacteria use the reductive tricarboxylic acid cycle, whereas the Gammaproteobacteria use the Calvin-Benson-Bassham cycle. Only the epsilonproteobacterial epibionts had the genes necessary for producing ammonium. This ability likely minimizes direct competition between epibionts and also broadens the spectrum of environmental conditions that the shrimp may successfully inhabit. We identified genes likely to be involved in shrimp-epibiont interactions, as well as genes for nutritional and detoxification processes that might benefit the host. Shrimp epibionts at Rainbow are often coated with iron oxyhydroxides, whose origin is intensely debated. We identified 16S rRNA sequences and functional genes affiliated with iron-oxidizing Zetaproteobacteria, which indicates that biological iron oxidation might play a role in forming these deposits. Fluorescence in situ hybridizations confirmed the presence of active Zetaproteobacteria in the R. exoculata gill chamber, thus providing the first evidence for a Zetaproteobacteria-invertebrate association.