Taxonomic description and genome sequence of Halobacillus marinus sp. nov., a novel strain isolated from Chilika Lake, India.

moderately halophilic spore forming, motile, Gram-positive, rod-shaped bacterial strain designated as KGW1T was isolated from water sample of Chilika Lake and characterized taxonomically using polyphasic approach. The strain grew in the presence of 0-25% (w/v) NaCl in marine salt agar media, hydrolyzes casein, and gelatin and shows presence of alkaline proteases. The major cell wall menaquinone was MK7 and major cellular fatty acids were anteiso-C15:0 (44.89%), anteiso-C17:0 (6.18%), isoC15:0 (19.38%), and iso-C16:0 (7.39%). Several chemotaxonomic features conform the isolate be a member of genus Halobacillus. The isolate KGW1T contained A1γ meso-Dpm-direct type of peptidoglycan which is different from its phylogenetically closest neighbours. The 16S rRNA gene sequence based phylogenetic analysis also revealed the strain KGW1T was affiliated to the genus Halobacillus and sequence similarity between the isolated strain and the type strains of Halobacillus species were found closest to, H. dabanensis D-8 DSM 18199T (99.08%) and H. faecis IGA7-4 DSM 21559T (99.01%), H. trueperi SL-5 DSM 10404T (98.94%). The in silico DDH showed that the values in a range of 14.2-17.5% with the most closest strain H. dabanensis D-8 DSM 18199T and other type strains of the genus Halobacillus for which whole genome sequence is reported. DNA-DNA relatedness between strain KGW1T and the closest type strain Halobacillus trueperi DSM 10404T was 11.75% (± 1.15). The draft genome sequence includes 3,683,819 bases and comprises of 3898 predicted coding sequences with a G + C content of 46.98%. Thus, the significant distinctiveness supported by phenotypic and genotypic data with its closest neighbors and other closely related species confirm the strain KGW1T to be classified as a novel species within the genus Halobacillus, for which the name Halobacillus marinus sp. nov. is proposed. The type strain is KGW1T (= DSM 29522 = JCM 30443).

Halobacillus salicampi sp. nov., a moderately halophilic bacterium isolated from a solar saltern sediment.

A Gram-positive, moderately halophilic bacterium, designated strain TGS-15(T), was isolated from the sediment of a solar saltern pond located in Shinan, Korea. Strain TGS-15(T) was found to be a strictly aerobic, non-motile rod which can grow at pH 6.0-10.0 (optimum, pH 9.0), at 20-35 °C (optimum, 28 °C) and at salinities of 1-20 % (w/v) NaCl (optimum, 9 % NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain TGS-15(T) belongs to the genus Halobacillus, with sequence similarity of 98.5-96.0 % to known type strains, showing high sequence similarity to Halobacillus locisalis MSS-155(T) (98.5 %), Halobacillus faecis IGA7-4(T) (98.2 %) and Halobacillus alkaliphilus FP5(T) (98.0 %), and less than 98.0 % sequence similarity to other currently recognised type strains of the genus. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and an unidentified lipid. The cell wall peptidoglycan was found to be based on L-Orn-D-Asp, the predominant isoprenoid quinone was identified as menaquinone-7 (MK-7) and the major fatty acids were identified as anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:1 ω7c alcohol. The DNA G+C content of this novel isolate was determined to be 45.3 mol %. Levels of DNA:DNA relatedness between strain TGS-15(T) and the type strains of 13 other species of the genus ranged from 52 to 9 %. On the basis of the polyphasic analysis conducted in this study, strain TGS-15(T) is concluded to represent a novel species of the genus Halobacillus, for which the name Halobacillus salicampi sp. nov. is proposed. The type strain is TGS-15(T) (=KACC 18264(T) = NBRC 110640(T)).

Halobacillus sediminis sp. nov., a moderately halophilic bacterium isolated from a solar saltern sediment.

A Gram-staining-positive, moderately halophilic bacterium, designated strain NGS-2T, was isolated from sediment of a solar saltern pond located in Shinan, Korea. Strain NGS-2T was a strictly aerobic, non-motile rod that grew at pH 5.0-10.0 (optimum, pH 8.0), at 10-30 °C (optimum, 28 °C) and in the presence of 1-20 % (w/v) NaCl (optimum, 10 % NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain NGS-2T belonged to the genus Halobacillus, with sequence similarity of 98.4-95.8 % to existing type strains, showing the highest sequence similarity to Halobacillus dabanensis D-8T (98.4 %), H. litoralis SL-4T (98.4 %), H. trueperi SL-5T (98.2 %), H. faecis IGA7-4T (98.2 %), H. profundi IS-Hb4T (98.1 %) and H. mangrovi MS10T (98.0 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl-N-methylethanolamine and an unknown glycolipid. The cell-wall peptidoglycan was based on l-Orn-d-Asp, the predominant isoprenoid quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C15: 0 and anteiso-C17: 0. The DNA G+C content of the novel isolate was 45.0 mol%. Levels of DNA-DNA relatedness between strain NGS-2T and the type strains of 12 other species of the genus ranged from 32 to 3 %. On the basis of the polyphasic analysis conducted in this study, strain NGS-2T represents a novel species of the genus Halobacillus, for which the name Halobacillus sediminis sp. nov. is proposed. The type strain is NGS-2T ( = KACC 18263T = NBRC 110639T).

Bioenergetics of the moderately halophilic bacterium Halobacillus halophilus: composition and regulation of the respiratory chain.

In their natural environments, moderately halophilic bacteria are confronted not only with high salinities but also with low oxygen tensions due to the high salinities. The growth of H. halophilus is strictly aerobic. To analyze the dependence of respiration on the NaCl concentration and oxygen availability of the medium, resting cell experiments were performed. The respiration rates were dependent on the NaCl concentration of the growth medium, as well as on the NaCl concentration of the assay buffer, indicating regulation on the transcriptional and the activity level. Respiration was accompanied by the generation of an electrochemical proton potential (Δµ(H+)) across the cytoplasmic membrane whose magnitude was dependent on the external pH. Genes encoding proteins involved in respiration and Δµ(H+) generation, such as a noncoupled NADH dehydrogenase (NDH-2), complex II, and complex III, were identified in the genome. In addition, genes encoding five different terminal oxidases are present. Inhibitor profiling revealed the presence of NDH-2 and complex III, but the nature of the oxidases could not be resolved using this approach. Expression analysis demonstrated that all the different terminal oxidases were indeed expressed, but by far the most prominent was cta, encoding cytochrome caa3 oxidase. The expression of all of the different oxidase genes increased at high NaCl concentrations, and the transcript levels of cta and qox (encoding cytochrome aa3 oxidase) also increased at low oxygen concentrations. These data culminate in a model of the composition and variation of the respiratory chain of H. halophilus.

Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, Halobacillus sp. LY6.

A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na(2)SO(4) or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30-80°C), pH (6.0-12.0), and NaCl concentration (0-25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca(2+) and Cu(2+), but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.