Novel Substrates for Kinases Involved in the Biosynthesis of Inositol Pyrophosphates and Their Enhancement of ATPase Activity of a Kinase.

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo-inositol polyphosphates-scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5-from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

Cryobacterium roopkundense sp. nov., a psychrophilic bacterium isolated from glacial soil.

Strain RuGl7(T) was isolated from a soil sample collected at the periphery of the glacial Lake Roopkund in the Himalayan mountain range, India. Cells of RuGl7(T) were Gram-positive, aerobic, rod-shaped, motile and grew optimally between 15 and 18 degrees C. Cells of RuGl7(T) contained 2,4-diaminobutyric acid in the cell-wall peptidoglycan and the major menaquinones were MK-10, MK-11 and MK-12. The polar lipids present were diphosphatidylglycerol and phosphatidylglycerol and an unknown lipid and the major fatty acid was anteiso-C(15 : 0). Based on the above characteristics, strain RuGl7(T) was assigned to the genus Cryobacterium. Strain RuGl7(T) shared a 16S rRNA gene sequence similarity of 97.0 and 99.0 % with Cryobacterium psychrotolerans JCM 13925( T) and Cryobacterium psychrophilum JCM 1463(T), respectively. However, DNA-DNA relatedness values between strain RuGl7(T) and C. psychrotolerans and C. psychrophilum were 28 and 23 %, respectively. Furthermore, strain RuGl7(T) exhibited several phenotypic and genotypic differences when compared with C. psychrotolerans , C. psychrophilum and Cryobacterium mesophilum. Based on these differentiating characteristics, strain RuGl7(T) was identified as a novel species of the genus Cryobacterium for which the name Cryobacterium roopkundense sp. nov. is proposed. The type strain is RuGl7( T) (=DSM 21065(T)=JCM 15131(T)).

Arthrobacter antarcticus sp. nov., isolated from an Antarctic marine sediment.

A bacterial strain, SPC26(T), was isolated from a sediment sample of the Southern Ocean off Antarctica. The strain was Gram-staining- and catalase-positive and contained lysine and alanine in the cell-wall peptidoglycan. The major cellular fatty acids were anteiso-C15:0 (54.92 %), iso-C15:0 (11.47 %), anteiso-C17:0 (6.48 %) and anteiso-C15:1 (6.38 %) and the major menaquinones were MK-8, MK-9 and MK-10. The major polar lipids were phosphatidylethanolamine and diphosphatidylglycerol. The G+C content was 68 ± 0.5 mol%. Based on 16S rRNA gene sequence similarities, the nearest phylogenetic neighbours of strain SPC26(T) were identified as Arthrobacter gangotriensis Lz1y(T) (98.8 %), A. sulfureus DSM 20167(T) (98.6 %), A. psychrophenolicus DSM 15454(T) (97.9 %) and A. kerguelensis KGN15(T) (97.5). With these strains, strain SPC26(T) exhibited DNA-DNA relatedness values of 36, 21, 12 and 10 %, respectively. Therefore, on the basis of 16S rRNA gene sequence comparisons, phylogenetic analysis, phenotypic characteristics and DNA-DNA relatedness, it is proposed that strain SPC26(T) represents a novel species of Arthrobacter, for which the name Arthrobacter antarcticus sp. nov. is proposed, with strain SPC26(T) (=LMG 24542(T) =NCCB 100228(T)) as the type strain.

Predominance of Roseobacter, Sulfitobacter, Glaciecola and Psychrobacter in seawater collected off Ushuaia, Argentina, Sub-Antarctica.

Bacterial diversity in sub-Antarctic seawater, collected off Ushuaia, Argentina, was examined using a culture independent approach. The composition of the 16S rRNA gene libraries from seawater and seawater contaminated with the water soluble fraction of crude oil was statistically different (P value 0.001). In both libraries, clones representing the Alphaproteobacteria, Gammaproteobacteria, the Cytophaga-Flavobacterium-Bacteroidetes group and unculturable bacteria were dominant. Clones associated with the genera Roseobacter, Sulfitobacter, Staleya, Glaciecola, Colwellia, Marinomonas, Cytophaga and Cellulophaga were common to both the libraries. However, clones associated with Psychrobacter, Arcobacter, Formosa algae, Polaribacter, Ulvibacter and Tenacibaculum were found only in seawater contaminated with hydrocarbons (Table 1). Further, the percentage of clones of Roseobacter, Sulfitobacter and Glaceicola was high in seawater (43%, 90% and 12% respectively) compared to seawater contaminated with hydrocarbons (35%, 4% and 9% respectively). One of the clones F2C63 showed 100% similarity with Marinomonas ushuaiensis a bacterium identified by us from the same site.

Marinomonas ushuaiensis sp. nov., isolated from coastal sea water in Ushuaia, Argentina, sub-Antarctica.

A Gram-negative, rod-shaped, psychrophilic, motile, non-spore-forming bacterium, strain U1T, was isolated from Ushuaia located at the southernmost tip of Argentina. On the basis of 16S rRNA gene sequence similarity, strain U1T was found to be closely related to Marinomonas communis (DSM 5604T) and Marinomonas primoryensis (IAM 15010T). At the DNA-DNA level, however, the values for similarity were 41 and 25 %, respectively. The major fatty acids present were iso-C(16 : 0), C(16 : 1)omega7c, iso-C(17 : 1) and C(18 : 1)omega7c and the G+C content of the DNA was 43.6 mol%. All of the above characteristics support the affiliation of strain U1T to the genus Marinomonas. Furthermore, on the basis of phenotypic features, chemotaxonomic characteristics and phylogenetic analysis of the 16S rRNA gene sequence, it appears that strain U1T is distinct from the four Marinomonas species with validly published names. Strain U1T, therefore, represents a novel species, for which the name Marinomonas ushuaiensis sp. nov. is proposed. The type strain of M. ushuaiensis is U1T (=MTCC 6143T=DSM 15871T=JCM 12170T).