Abstract: Two Gram-positive, rod-shaped, moderately halophilic bacteria were isolated from a deep-sea carbonate rock at a methane cold seep in Kuroshima Knoll, Japan. These bacteria, strains IS-Hb4(T) and IS-Hb7(T), were spore-forming and non-motile. They were able to grow at temperatures as low as 9 degrees C and hydrostatic pressures up to 30 MPa. Based on high sequence similarity of their 16S rRNA genes to those of type strains of the genus Halobacillus, from 96.4 % (strain IS-Hb7(T) to Halobacillus halophilus NCIMB 9251(T)) to 99.4 % (strain IS-Hb4(T) to Halobacillus dabanensis D-8(T)), the strains were shown to belong to this genus. DNA-DNA relatedness values of 49.5 % and 1.0-33.0 %, respectively, were determined between strains IS-Hb4(T) and IS-Hb7(T) and between these strains and other Halobacillus type strains. Both strains showed the major menaquinone MK7 and L-orn-D-Asp cell-wall peptidoglycan type. Straight-chain C(16 : 0), unsaturated C(16 : 1)omega7c alcohol and C(18 : 1)omega7c and cyclopropane C(19 : 0) cyc fatty acids were predominant in both strains. The DNA G+C contents of IS-Hb4(T) and IS-Hb7(T) were respectively 43.3 and 42.1 mol%. Physiological and biochemical analyses combined with DNA-DNA hybridization results allowed us to place strains IS-Hb4(T) (=JCM 14154(T)=DSM 18394(T)) and IS-Hb7(T) (=JCM 14155(T)=DSM 18393(T)) in the genus Halobacillus as the respective type strains of the novel species Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov.
Abstract: DNA base composition expressed as mol% of guanine plus cytosine (% GC) or GC content is a key parameter of bacterial taxonomy and genomic analyses. Direct chemical determination methods such as HPLC as well as indirect methods based on physical properties of deoxyribonucleic acid (DNA), melting point (T(m)), and buoyant density (B(d)) have been conventionally applied to determine the GC content. However, these methods require relatively large amounts of sample DNA, time, and labor. We have developed a protocol to determine the GC content by fine separation of nucleosides with CZE. Genomic DNAs with known GC content from 23 bacterial strains were determined by CE at the optimized conditions of 27 degrees C, 20 kV in 50 mM of NaHCO(3) (pH 9.0) and 70 mM SDS added. Nucleosides from <1 microg of DNA hydrolyzed with nuclease-P1 and bacterial alkaline phosphatase were separated in a 75 microm wide and 80 cm long silica capillary. The nucleoside peak areas were determined at 254 nm in less than 12 min. The CE-based determination of GC content requires only small amounts of DNA, and thus should be applicable to environmental genomics (metagenomics), as >90% of environmental micro-organisms are nonculturable and produce only small amounts of genomic DNA.
