| In this study, the culturable halophilic/halotolerant bacteria in non-saline soil(froest and farming soil) collected from Dehang Canyon(28°15?-28°43?N, 109°30?-109°45?E) in Jishou, Hunan, PR China, were isolated by using culture-dependent method. After purificaiton, the biodiversity of the isolates was investigated by using phylogenetic analysis based on 16 S r RNA gene sequences. The ability of the isolates exhibiting antibacterial activities was then detected on eight sensitive microorganisms by using a agar diffusion method. Furhter more, some strains representing potential new taxa were studies by means of the polyphasic method to establish their precise taxonomic positions.A total of 294 halophilic/halotolerant bacterium strains were isolated from the samples on a number of media(humic acid agar, ISP 2 and 5, marine agar 2216 and nutrient agar) supplemented with 5~20%(w/v) Na Cl. Based on the results of some morphological characteristics and physiological/biochemical tests, 134 isolates were selected for further study. The results of salt tolerance test shouwed that out of 134 tested strain, 34 are moderately halophilic, 70 slight halophilica, and 30 halotolerant.The phylogenetic analysis results showed that, these 134 strains were members of 36 bacteria species, belonging to 16 genera(Arthrobacter, Brachybacterium, Kocuria, Nocardiopsis, Bacillus, Gracilibacillus, Halobacillus, Oceanobacillus, Piscibacillus, Marinococcus, Thalassobacillus, Virgibacillus, Jeotgalibacillus, Staphylococcus, Salinicoccus, Halomonas) of 7 families(Dermabacteraceae, Micrococcaceae, Nocardiopsaceae, Bacillaceae, Planococcaceae, Staphylococcaceae, Halomonadaceae) in 3 phyla(Proteobacteria, Actinobacteria, Firmicutes). The most abundant and diverse isolates were members of Firmicutes(118 strains, 88.1%), in which the family Bacillaceae was absolutely predominant(109 strains, 81.3%) and the genus was the predominant genus(68 strains, 50.7%). A small number of isolates belonged to the phylum Actinobacteria(9 strains, 6.7%) and Proteobacteria(7 strains, 5.2%). There were obvious genetic divergences between most isolates and their closestly related type strains(sequence similarities, 96.6% to 99.9%). Out of 134 isolates, at least 5 JSM strains(101020, 101024, 101097, 101037, 102055) should were representatives of 4 potential novel species of four recognized genera(Bacillu, Halobacillus, Jeotgalibacillus, Virgibacillus).The ability of 294 isolates exhibiting antibacterial activities was screened by using a agar diffusion method on eight sensitive microorganisms, i.e. three G- bacteria(Escherichia coli, Aerobacter aerogenes, Proteus vulgaris), three G+ bacteria(Bacillus subtilis, Staphylococcus aureus, Sarcina lutea) and two fungi(Candida albican, Asperillus niger). The results showed that, the antimicrobial activities of 109 isolates were positive(37.1%), in which 5 strains exhibited strong antimicrobial activity. The phylogenetic analysis results showed that the 5 antimicrobial activity-positive strains were members of the phyla Proteobacteria, Firmicutes and Actinobacteria, belong to the genera Halobacillus(JSM 102030), Oceanobacillus(JSM 102082), Nocardiopsis(JSM 102066) and Halomonas(JSM 102052, JSM 102054), respectively.The precise taxonomic position of an actinobacteria strain JSM 101020 was investigated by using a polyphasic taxonomic approach. The results indicated that strain JSM 101020 was Gram-stain-positive, non-motile, catalase- and oxidase-positive, endospore-forming, facultatively anaerobic, rod-shaped bacterium. Growth occurred with 1–15 %(w/v) Na Cl(optimum 3–5 %) and at p H 5.5–11.0(optimum p H 7.5) and at 5–40 ?C(optimum 25–30 ?C). With meso-diaminopimelic acid as the diagnostic amino acid in the cell wall peptidoglycan, and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The predominant respiratory quinone was menaquinone 7(MK-7). The major cellular fatty acids were iso-C15:0, anteiso-C15:0, iso-C17:0 and iso-C16:0. The genomic DNA G + C content was 41.6 mol%. A phylogenetic analysis based on 16 S r RNA gene sequences indicated that strain JSM 101020 T belongs to the genus Bacillus, and was most closely related to the type strains of B. halmapalus(16S r RNA gene sequence similarity 98.3%), B. tianshenii YIM M13235T(98.0%), B. zhanjiangensis JSM 099021T(97.6%) and B. horikoshii(97.6%). The results of the phylogenetic analysis and of morphological and chemotaxonomic investigations supported the affiliation of strain JSM 101020 T to the genus Bacillus. However, the light yellow-pigmented colony, the positvie reaction of methyl red test, the ability to reduec nutirate, as well as the ability to grow under anaerobic conditions, differentiated the isolate clearly from its phylogenetic relatives. The combination of phylogenetic analysis, DNA–DNA hybridization, phenotypic characteristics and chemotaxonomic data supported the proposal that strain JSM 101020 T represents a new species of the genus Bacillus, for which the name Bacillus dehangensis sp. nov. is proposed. The type strain was JSM 101020T(= CCTCC AB 2014163 T = CGMCC 1.12948 T = KCTC 33538T).In conclusion, there is a abundant bacterial diversity and some potential new taxa in the non-saline soil from Dehang Canyon in Jishou, China, and that a number of isolates exhibited antibacterial activity. |
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