[Phylogenetic diversity of culturable bacteria in the ancient salt deposits of the Yipinglang Salt Mine, P. R. China]

Abstract

The microbial diversity of cultivable bacteria, isolated from the ancient salt deposits from the Yipinglang Salt Mine (YPL) in the Yunnan Province, P. R. China,was investigated by using conventional culture-dependent method and phylogenetic analyses based on 16S rRNA gene sequence comparisons. 38 bacteria strains were isolated from the brine, halite and saline soil samples on MBA (marine broth agar 2216, Difco) and ISP 2 (International Streotomyces Project medium 2) media supplemented with 0.5-3.5 mol/L NaCl. The genomic DNAs of the isolates were extracted and their 16S rRNA genes were amplified by PCR using bacterial universal primers. The resulting 16S rRNA gene sequences were compared with sequences obtained from public databases to find the most closely related species. Phylogenetic analyses were performed using the software packages MEGA after multiple alignment of sequence data by CLUSTAL X. The evolutional instances (corrected by Kimura's 2-parameter model) were calculated and clustering was performed with the neighbor-joining method. The results showed that the isolates are members of twenty-four genera (Acinetobacter, Agromyces, Arthrobacter, Bacillus, Brevundimonas, Chromohalobacter, Dietzia, Erythrobacter, Exiguobacterium, Halomonas, Idiomarina, Kocuria, Marinobacter, Micrococcus, Paracoccus, Planomicrbium, Porphyrobacter, Pseudomonas, Psychrobacter, Roseivivax, Saccharospirillum, Salegentibactor, Salinicoccus, Streptomyces) of seventeen families (Alteromonadaceae, Bacillaceae, Caulobacteraceae, Flavobacteriaceae, Halomonadaceae, Idiomarinaceae, Microbacteriaceae, Micrococcaceae, Moraxellaceae, Planococcaceae, Pseudomonadaceae, Rhodobacteraceae, Dietziaceae, Saccharospirillaceae, Sphingomonadaceae, Staphylococcaceae, Streptomycetaceae) in four major phylogenetic groups (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria). The most abundant and diverse isolates were within the phyla of Proteobacteria (47.3%; Gamma-Proteobacteria, 31.5%; Alpha-Proteobacteria, 15.8%) and Firmicutes (34.2%). The phylogenetic distance matrix results suggested that out of 38 isolates 32 are different strains of 27 known species, and that at least 3 stains represent new species within 3 characterized genera. Y3 (Accession No. EF177665) and Y25 (EF177670) represent new species of the genera Idiomarina and Saccharospirillum, respectively. Y15 (DQ837380), Y16 (EF177680) and Y22 (EF177689) represent a new species of the genus Salinicoccus. And strain Y21 (EF177692) may represent a novel species of a possible new genus of the family Staphylococcaceae. The results presented above shown that there are abundant bacterial species diversity and phylogenetic diversity in the ancient salt deposits from the Yipinglang Salt Mine.