Desulfurobacterium indicum sp. nov., a thermophilic sulfur-reducing bacterium from the Indian Ocean.

A novel sulfur-reducing bacterium, strain K6013T, was isolated from a sulfide sample collected at a depth of 2771 m from a high-temperature hydrothermal vent in the Indian Ocean. Cells were Gram-stain-negative, anaerobic, motile rods (0.9-2.2×0.4-0.6 µm). The strain grew at NaCl concentrations ranging from 1 to 4.5 % (w/v) (optimum 2.5 %), at pH 5 to 8 (optimum pH 6), and at temperatures between 40 and 75 °C (optimum 65 °C). K6013T was an obligate chemolithoautotroph, using thiosulfate, sulfur and nitrate as terminal electron acceptors in the presence of H2 but not sulfate, sulfite nor nitrite. The major cellular fatty acids were C16 : 0 (17.4 %), C18 : 1ω7c/C18 : 1ω6c (ummed feature 8, 37.91 %), C18 : 0 (18.29 %) and C14 : 0 3-OH/iso-C16: 1I (summed feature 2, 8.56 %). The DNA G+C content was 38.2 mol%. The results of phylogenetic 16S rRNA gene sequence analyses indicated that K6013T represents a member of the genus Desulfurobacterium within the class Aquificae, with highest sequence similarity of 96.93 % to Desulfurobacterium atlanticum SL22T. On the basis of genotypic and phenotypic data, K6013T is considered to represent a novel species of the genus Desulfurobacterium, for which the name Desulfurobacterium indicum sp. nov. is proposed, with the type strain K6013T (=DSM 101677T=MCCC 1A01868T).

Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus.

A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in Thermococcus kodakarensis, consistently correlated with the TK0664 gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced pyrF by TK0664 on our suicide vector and tested T. barophilus strain sensitivity to 6-MP before and after transformation. Wild-Type (WT) T. barophilus is less sensitive to 6-MP than WT T. kodakarensis, and an increase of cell resistance was achieved after deletion of the T. barophilusTERMP_00517 gene homologous to T. kodakarensisTK0664. Results confirmed the natural resistance of T. barophilus to 6-MP and show that TK0664 can confer sensitivity. This new counterselection system vastly improves genetic manipulations in T. barophilus MP, with a strong decrease in false positives to <15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., polB and rnhB).