Abstract:[Background] Strain JSM 102089 was a moderately halophilic, siderophore-producing, facultatively anaerobic, non-motile, endospore-forming and Gram-positive bacterium, isolated from a non-saline cultivated soil sample collected from the Dehang Canyon (28°15′–28°43′N, 109°30′–109°45′E) in Jishou, Hunan, China. The strain was positive for gelatin hydrolysis and urease activity, and exhibited a unique phylogenetic relationship with the members of the family Bacillaceae. [Objective] To find out the phylogenetic status of JSM 102089 and unravel its secondary metabolic potential on the basis of the draft genome. [Methods] The phylogeny of JSM 102089 was primarily analyzed by 16S rRNA gene sequencing and comparison of phenotypic characteristics. Then, the exact phylogenetic status was investigated comprehensively by means of comparative genomics analysis based on whole genome sequences, including comparisons of G+C content, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) estimated values, as well as by phylogenomic analysis. To investigate the secondary metabolic potential of JSM 102089, we used multiple bioinformatics tools such as antiSMASH 7.0, BLASTn and BLASTp for rapid genome-wide identification, annotation, and analysis of biosynthetic gene clusters (BGCs) for secondary metabolites. [Results] The results of the phylogenetic analysis based on 16S rRNA gene sequences showed that JSM 102089 belonged to the family Bacillaceae and was closely related to the type strains of 3 known species of the genus Pseudalkalibacillus, i.e. P. sedimenti FJAT-53715T(16S rRNA gene sequence similarity, 99.72%), P. spartinae FJAT-53046T (99.64%) and P. hwajinpoensis SW-72T (99.59%), followed by Alkalihalobacillus hemicentroti JSM 076093T (98.64%). These 5 strains formed an obvious clade in the phylogenetic tree based on 16S rRNA gene sequences. Specifically, strains JSM 102089, P. sedimenti FJAT-53715T and P. spartinae FJAT-53046T formed a distinct subclade, but JSM 102089 made a separated branch on the outskirt of the subclade. Chemotaxonomic data of JSM 102089 were consistent with its assignment to Bacillaceae, while JSM 102089 could be distinguished from its closest relatives by a number of important characteristics, such as salt tolerance, motility, urease activity and hydrolysis of gelatin. The results of comparative genomics analysis showed that both of the ANI and dDDH estimated values between JSM 102089 and type strains of phylogenetically closely known species of Pseudalkalibacillus and Alkalihalobacillus were well lower than the generally recognized thresholds of bacterial species (ANI, 95%–96%; dDDH, 70%), which strongly supported JSM 102089 representing a potential new species of Bacillaceae. The results of the phylogenomic analysis also showed that JSM 102089 clustered together with P. sedimenti FJAT-53715T and P. spartinae FJAT-53046T but the strain studied made a distinctly separated species cluster, which suggested that JSM 102089 represented a new species of the genus Pseudalkalibacillus. A total of 86 BGCs belonging to 19 function-types including NI-siderophores, type III polyketide synthases (T3PKSs) and terpenes, were mined from the genomes of JSM 102089 and 13 representative strains of Pseudalkalibacillus and Alkalihalobacillus. Most of the BGCs exhibited prominent novelty compared with known BGCs as well as high function diversity between each other. Particularly, two strain-exclusive BGCs of JSM 102089 were relatively unique in structure as well as particularly novel in function. [Conclusion] The combination of the results of phylogenetic analysis based on 16S rRNA gene sequences, comparison of phenotypic characteristics and comparative genomics analysis showed definitely that JSM 102089 cannot be designated to any of the recognized species of Bacillaceae, but represents a potential new species (or new genomospecies) of Pseudalkalibacillus; Most of the BGCs of JSM 102089 and closest representatives of Pseudalkalibacillus and Alkalihalobacillus exhibite prominent novelty compared with known BGCs as well as high function diversity between each other, and parts of the BGCs of JSM 102089 exhibit unique structures and novel functions, which all clearly show that these strains have the biosynthesis potential of a variety of novel secondary metabolites. Therefore, we think JSM 102089 should be a typical example of new-resource microbes, and the systematic taxonomy, salt-tolerant mechanism, secondary metabolism and biotechnological potential of the new strain is worth being further explored. Moreover, it is evident that the phylogeny and taxonomy of the genus Alkalihalobacillus and/or some known Alkalihalobacillus species should be re-examined, and on this a few of well-targeted suggestions are proposed by us.
