Diversity and phylogeny of culturable spore-forming Bacilli isolated from marine sediments

Members of the genus Bacillus and related genera are ubiquitous in nature. However, Bacillus species isolated from marine sediments have attracted less interest respect to their terrestrial relatives. Here, we report the phylogenetic diversity of a collection of 96 Bacilli, isolated from 17 distinct stations of 5 oceanographic campaigns. The diversity was analysed by phenotypic and molecular approaches based on the amplified rDNA restriction analysis (ARDRA), amplification of the internal transcribed spacers (ITS-PCR) and on 16S rRNA sequencing. Intra-specific polymorphism was efficiently detected by biochemical analysis and ARDRA while results of ITS-PCR were in agreement with 16S rRNA sequencing. The identification results assigned 68% of the isolates to the species B. subtilis, B. licheniformis, B. pumilus and B. cereus. Phylogenetic analysis allowed the separation of 9 isolates in a clade that may represent a group of obligate marine Bacillus since they clustered with B. firmus, B. foraminis and marine isolates with metal oxidation and bioaccumulation capabilities. The remaining isolates showed a close affiliation to the genera Virgibacillus, Gracilibacillus and Paenibacillus. The widespread of Bacilli and their high diversity level observed in this work point out the need of more extensive studies to understand their distribution and ecology in deep-sea environments.