Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting

Abstract

With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments.

Fig. 1

Location of the four sampling sites relative to the oceanographic campaign “CIESM-SUB1”. Station coordinate limits and depths: Station 6 (Palinuro), 39°52.820′ N, 012°46.070′ E, 3581 m; Station 4, 39°32.050′ N, 013°22.280′ E, 3,453 m; Station 2 (Marsili), 39°07.470′ N, 014°06.430′ E, 3,425 m; Station 8, 38°55.900′ N, 013°16.490′ E, 3,460 m.

Fig. 2

Neighbor-joining phylogenetic tree of 49 partial 16S rRNA gene sequences (coordinates in E. coli 52 to 787 n) of marine Bacillales isolates with 27 closest NCBI relatives. Phylogenetic dendrogram was evaluated by performing bootstrap analysis of 1,000 data sets using MEGA 4.1. ARISA type (according to Fig. 2 and Table 2) and the number of isolates per type is indicated. 16S rRNA gene sequence accession numbers of the reference strains are indicated in parentheses.

Fig. 3

Geographic distribution of marine Bacillales in seamount and non-seamount stations.