Phorcysia thermohydrogeniphila gen. nov., sp. nov., a thermophilic, chemolithoautotrophic, nitrate-ammonifying bacterium from a deep-sea hydrothermal vent.

A novel hyperthermophilic, anaerobic, chemolithoautotrophic bacterium, designated strain HB-8(T), was isolated from the tube of Alvinella pompejana tubeworms collected from the wall of an actively venting sulfide structure on the East Pacific Rise at 13° N. The cells were Gram-negative rods, approximately 1.0-1.5 µm long and 0.5 µm wide. Strain HB-8(T) grew between 65 and 80 °C (optimum 75 °C), 15 and 35 g NaCl l(-1) (optimum 30 g l(-1)) and pH 4.5 and 8.5 (optimum pH 6.0). Generation time under optimal conditions was 26 min. Growth occurred under chemolithoautotrophic conditions with H(2) as the energy source and CO(2) as the carbon source. Nitrate and sulfur were used as electron acceptors, with concomitant formation of ammonium or hydrogen sulfide, respectively. The presence of lactate, formate, acetate or tryptone in the culture medium inhibited growth. The G+C content of the genomic DNA was 47.8 mol%. Phylogenetic analysis of the 16S rRNA gene and of the alpha subunit of the ATP citrate lyase of strain HB-8(T) indicated that this organism formed a novel lineage within the class Aquificae, equally distant from the type strains of the type species of the three genera that represent the family Desulfurobacteriaceae: Thermovibrio ruber ED11/3LLK8(T), Balnearium lithotrophicum 17S(T) and Desulfurobacterium thermolithotrophum BSA(T). The polar lipids of strain HB-8(T) differed substantially from those of other members of the Desulfurobacteriaceae, and this bacterium produced novel quinones. On the basis of phylogenetic, physiological and chemotaxonomic characteristics, it is proposed that the organism represents a novel genus and species within the family Desulfurobacteriaceae, Phorcysia thermohydrogeniphila gen. nov., sp. nov. The type strain of Phorcysia thermohydrogeniphila is HB-8(T) ( = DSM 24425(T)  = JCM 17384(T)).

Parvibaculum hydrocarboniclasticum sp. nov., a mesophilic, alkane-oxidizing alphaproteobacterium isolated from a deep-sea hydrothermal vent on the East Pacific Rise.

An aerobic, alkane-oxidizing bacterium, designated strain EPR92(T), was isolated from hydrothermal fluids that had been collected from a deep-sea vent on the East Pacific Rise (at 9° 50' N 104° 17' W). The cells of the novel strain were Gram-staining-negative rods that measured approximately 1.4 µm in length and 0.4 µm in width. Strain EPR92(T) grew at 20-40 °C (optimum 35 °C), with1.0-5.0% (w/v) NaCl (optimum 2.5%), and at pH 4.0-8.5 (optimum pH 7.5). The generation time under optimal conditions was 63 min. Strain EPR92(T) grew aerobically in artificial seawater minimal medium with n-alkanes as sole carbon and energy sources, and also in artificial seawater medium supplemented with peptone and yeast extract. The predominant fatty acids were C(18:1)ω7c, C(19:0) cyclo ω8c, 11-methyl C(18:1)ω7c and a putative C(12:0) aldehyde. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and four unidentified aminolipids. The major respiratory quinone was Q-10 and the genomic DNA G+C content was 60.7 mol%. Phylogenetic analyses of the 16S rRNA gene showed that strain EPR92(T) belongs in the class Alphaproteobacteria and the recognized species that were most closely related to the novel strain were identified as Parvibaculum indicum P-31(T) (98.7% sequence similarity) and Parvibaculum lavamentivorans DS-1(T) (95.8%). In DNA-DNA hybridizations, the level of DNA-DNA relatedness observed between strain EPR92(T) and P. indicum P-31(T) was 47.7%, indicating that the two strains do not belong to the same species. Based on the phylogenetic, physiological, chemotaxonomic and genetic evidence, strain EPR92(T) represents a novel species within the genus Parvibaculum, for which the name Parvibaculum hydrocarboniclasticum sp. nov. is proposed. The type strain is EPR92(T) ( = DSM 23209 = JCM 16666(T)).

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents.

BACKGROUND: Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galápagos Rift. RESULTS: Genetic differentiation (F(ST)) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. CONCLUSIONS: Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events.

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap).

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.

Salinisphaera hydrothermalis sp. nov., a mesophilic, halotolerant, facultatively autotrophic, thiosulfate-oxidizing gammaproteobacterium from deep-sea hydrothermal vents, and emended description of the genus Salinisphaera.

A mesophilic, aerobic, facultatively chemolithoautotrophic bacterium, designated strain EPR70(T), was isolated from hydrothermal fluids from diffuse-flow vents on the East Pacific Rise at degrees 50' N 10 degrees 17' W. Cells were Gram-negative rods, approximately 0.8-1.0 microm long and 0.3-0.5 microm wide. Strain EPR70(T) grew at 20-40 degrees C (optimum 30-35 degrees C), 1-25 % NaCl (optimum 2.5 %) and pH 5.0-7.5 (optimum pH 5.5). The shortest generation time observed for strain EPR70(T) was 42 min. Growth occurred under aerobic chemolithoautotrophic conditions in the presence of thiosulfate and CO(2). Strain EPR70(T) grew heterotrophically with acetate or n-alkanes as sole carbon and energy sources, and in complex artificial seawater medium. Nitrate was not used as an electron acceptor. The G+C content of the genomic DNA was 64 mol%. Phylogenetic analysis of the 16S rRNA gene indicated that this organism is a member of the class Gammaproteobacteria, with Salinisphaera shabanensis E1L3A(T) as its closest relative (94 % sequence similarity). On the basis of phylogenetic analyses based on 16S rRNA, rbcL and alkB genes and physiological analysis, it is proposed that the organism represents a novel species within the genus Salinisphaera, for which the name Salinisphaera hydrothermalis sp. nov. is proposed. The type strain is EPR70(T) (=DSM 21483(T) =JCM 15514(T)).

Vertical distribution and diversity of bacteria and archaea in sulfide and methane-rich cold seep sediments located at the base of the Florida Escarpment.

The bacterial and archaeal communities of the sediments at the base of the Florida Escarpment (Gulf of Mexico, USA) were investigated using molecular phylogenetic analysis. The total microbial community DNA of each of three vertical zones (top, middle and bottom) of a sediment core was extracted and the 16S rRNA genes were amplified by PCR, cloned and sequenced. Shannon-Weaver Diversity measures of bacteria were high in all three zones. For the archaea, diversity was generally low, but increased with depth. The archaeal clonal libraries were dominated by representatives of four groups of organisms involved in the anaerobic oxidation of methane (ANME groups). Phylogenetic analysis of bacteria suggests the dominance of epsilon-proteobacteria in the top zone, the epsilon-, delta- and gamma-proteobacteria in the middle zone and the delta-proteobacteria in the bottom zone of the core. Members of the Cytophaga-Flexibacter-Bacteroidetes group, the Chloroflexi/green non-sulfur bacteria, the Gram+ (Firmicutes), the Planctomyces, candidate division WS3 and Fusobacterium were also detected. Our data suggest that the community structure and diversity of microorganisms can shift greatly within small vertical distances, possibly in response to changes in the physical and chemical conditions.

Mercury adaptation among bacteria from a deep-sea hydrothermal vent.

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.

Coupling of bacterial endosymbiont and host mitochondrial genomes in the hydrothermal vent clam Calyptogena magnifica.

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) depends for its nutrition on sulfur-oxidizing symbiotic bacteria housed in its gill tissues. This symbiont is transmitted vertically between generations via the clam's eggs; however, it remains uncertain whether occasionally symbionts are horizontally transmitted or acquired from the environment. If symbionts are transmitted strictly vertically through the egg cytoplasm, inheritance of symbiont lineages should behave as if coupled to the host's maternally inherited mitochondrial DNA. This coupling would be obscured, however, with low rates of horizontal or environmental transfers, the equivalent of recombination between host lineages. Population genetic analyses of C. magnifica clams and associated symbionts from eastern Pacific hydrothermal vents clearly supported the hypothesis of strictly maternal cotransmission. Host mitochondrial and symbiont DNA sequences were coupled in a clam population that was polymorphic for both genetic markers. These markers were not similarly coupled with sequence variation at a nuclear gene locus, as expected for a randomly mating sexual population. Phylogenetic analysis of the two cytoplasmic genes also revealed no evidence for recombination. The tight association between vesicomyid clams and their vertically transmitted bacterial endosymbionts is phylogenetically very young (<50 million years) and may serve as a model for the origin and evolution of eukaryotic organelles.

Thermovibrio ammonificans sp. nov., a thermophilic, chemolithotrophic, nitrate-ammonifying bacterium from deep-sea hydrothermal vents.

A thermophilic, anaerobic, chemolithoautotrophic bacterium was isolated from the walls of an active deep-sea hydrothermal vent chimney on the East Pacific Rise at 9 degrees 50' N. Cells of the organism were Gram-negative, motile rods that were about 1.0 microm in length and 0.6 microm in width. Growth occurred between 60 and 80 degrees C (optimum at 75 degrees C), 0.5 and 4.5% (w/v) NaCl (optimum at 2%) and pH 5 and 7 (optimum at 5.5). Generation time under optimal conditions was 1.57 h. Growth occurred under chemolithoautotrophic conditions in the presence of H2 and CO2, with nitrate or sulfur as the electron acceptor and with concomitant formation of ammonium or hydrogen sulfide, respectively. Thiosulfate, sulfite and oxygen were not used as electron acceptors. Acetate, formate, lactate and yeast extract inhibited growth. No chemoorganoheterotrophic growth was observed on peptone, tryptone or Casamino acids. The genomic DNA G+C content was 54.6 mol%. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the organism was a member of the domain Bacteria and formed a deep branch within the phylum Aquificae, with Thermovibrio ruber as its closest relative (94.4% sequence similarity). On the basis of phylogenetic, physiological and genetic considerations, it is proposed that the organism represents a novel species within the newly described genus Thermovibrio. The type strain is Thermovibrio ammonificans HB-1T (=DSM 15698T=JCM 12110T).