Abstract:Objective To reveal the phylogenetic diversity of 12 marine siderophore-producing bacteria isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea, and to unravel the components, functions, genetic diversity and genetic evolution of siderophore biosynthetic gene clusters (BGCs) of the isolates as well as representatives of Microbulbifer.Methods The phenotypic characteristics as well as the siderophore-producing activity of the strains were observed by conventional methods. The phylogenetic diversity (including taxon, species, and genetic diversity) of the strains was analyzed based on 16S rRNA gene sequences. Then, the exact phylogenetic status of the representative strain JSM ZJ756 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. We then used multiple bioinformatic stools including antiSMASH 7.0, BLASTn, BLASTp, and MEGA 11 for rapid identification, annotation, and sequence alignment of BGCs, thus exploring the components, functions, genetic diversity, and genetic evolution of siderophore BGCs.Results All the 12 isolates were Gram-negative, aerobic, non-sporulating and slightly halophilic rods with siderophore-producing activity. The results of phylogenetic analyses based on 16S rRNA gene sequences as well as whole-genome sequences showed that all the 12 strains belonged to Microbulbifer, representing 6 to 8 species and forming 4 clades with Microbulbifer type strains in the phylogenetic tree. Among them, strain JSM ZJ756 should be a new member of Microbulbifer zhoushanensis. JSM ZJ756 and 9 representative strains of Microbulbifer each carried one copy of NI-siderophore BGCs, and 8 out of the 10 NI-siderophore BGCs shared the similarities ≤40% with known BGCs. According to the types and the similarities of known BGCs with the NI-siderophore BGCs identified in this study, the 10 NI-siderophore BGCs could be categorized into 5 functional subtypes: Ochrobactin (JSM ZJ756 and 2 type strains, JYFX01000060.1, similarity of 28%), Vibrioferrin (M. mangrove DD-13T, AB082123.1, 100%; M. epialgicus DSM 18651T, CP005094.1, 85%), Putrebactin (M. agarilyticus GP101, NIBS01000001.1, 40%), Aerobactin (M. echini JCM 30400T, AB199785.1, 22%), and unknown functional subtype (3 type strains including M. variabilis ATCC 700307T). The results of BLASTn and BLASTp analyses showed that the core biosynthetic gene sequences of the NI-siderophore BGCs identified were unique, and encoded novel proteins. The results of genetic evolution analysis showed that the core biosynthetic genes of the NI-siderophore BGCs found exhibited high genetic diversity. In the phylogenetic tree based on core biosynthetic gene sequences, 9 out of the 10 NI-siderophore BGCs were grouped to 3 clades, but M. agarilyticus GP101 exhibited an independent evolution path. The comparative analysis revealed the genetic evolution of NI-siderophore BGCs being consistent with that predicted based on the 16S rRNA sequences.Conclusion All the 12 siderophore-producing strains isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea belong to Microbulbifer, demonstrating high phylogenetic diversity, and JSM ZJ756 is a new member of M. zhoushanensis. The NI-siderophore BGCs of JSM ZJ756 and representative strains of Microbulbifer are novel and showcase high diversity, which indicates that those strains possess high potential of producing a variety of novel siderophores. Moreover, strong positive correlations exist between the biological functions and genetic evolution of the NI-siderophore BGCs and the phylogeny of JSM ZJ756 and 9 representative strains of Microbulbifer. Therefore, we hypothesize that the 12 marine siderophore-producing bacteria and the representatives of Microbulbifer are typical new-resource microbes, and the taxonomy of Microbulbifer, and the metabolic mechanism and genetic evolution as well as the biotechnological potential of the NI-siderophore BGCs are worth being further explored.
