Sulfurimonas paralvinellae sp. nov., a novel mesophilic, hydrogen- and sulfur-oxidizing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent polychaete nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. and emended description of the genus Sulfurimonas

A novel mesophilic bacterium, strain GO25T, was isolated from a nest of hydrothermal vent polychaetes, Paralvinella sp., at the Iheya North field in the Mid-Okinawa Trough. Cells were motile short rods with a single polar flagellum. Growth was observed between 4 and 35 °C (optimum 30 °C; 13–16 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.1). The isolate was a facultatively anaerobic chemolithoautotroph capable of growth using molecular hydrogen, elemental sulfur or thiosulfate as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source and elemental sulfur, thiosulfate or yeast extract as the sole sulfur source. Strain GO25T represents the first deep-sea epsilonproteobacterium capable of growth by both hydrogen and sulfur oxidation. Nitrate or molecular oxygen (up to 10 % partial pressure) could serve as the sole electron acceptor to support growth. Metabolic products of nitrate reduction shifted in response to the electron donor provided. The G+C content of genomic DNA was 37.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurimonas and was most closely related to Sulfurimonas autotrophica OK10T (96.3 % sequence similarity). DNA–DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurimonas autotrophica OK10T. On the basis of the physiological and molecular properties of the novel isolate, the name Sulfurimonas paralvinellae sp. nov. is proposed, with strain GO25T (=JCM 13212T=DSM 17229T) as the type strain. Thiomicrospira denitrificans DSM 1251T (=ATCC 33889T) is phylogenetically associated with Sulfurimonas autotrophica OK10T and Sulfurimonas paralvinellae GO25T. Based on the phylogenetic relationship between Thiomicrospira denitrificans DSM 1251T, Sulfurimonas autotrophica OK10T and Sulfurimonas paralvinellae GO25T, we propose the reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. (type strain DSM 1251T=ATCC 33889T). In addition, an emended description of the genus Sulfurimonas is proposed.

Identification of a Thiomicrospira denitrificans-Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea

ABSTRACT Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.

Sulfuricurvum kujiense gen. nov., sp. nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity

A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1T, was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan. The cells were motile, curved rods and had a single polar flagellum. Optimum growth occurred in a low-strength salt medium at pH 7·0 and 25 °C. It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite. Oxygen also served as the electron acceptor under the microaerobic condition (O2 in the head space 1 %). It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources. The DNA G+C content of strain YK-1T was 45 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the ‘Epsilonproteobacteria’, albeit with low homology (90 %). On the basis of physiological and phylogenetic data, strain YK-1T should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen. nov., sp. nov. is proposed. The type strain is YK-1T (=JCM 11577T=MBIC 06352T=ATCC BAA-921T).

Molecular Characterization of Bacterial Populations in Petroleum-Contaminated Groundwater Discharged from Underground Crude Oil Storage Cavities

ABSTRACT Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 106 microorganisms ml−1, a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the Proteobacteria; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to Thiomicrospira denitrificans (88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to Arcobacterspp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the Proteobacteria grow as major populations in the petroleum-contaminated cavity groundwater.