Bacillus tequilensis sp nov., isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis

A Gram-positive, spore-forming bacillus was isolated from a sample taken from an approximately 2000-year-old shaft-tomb located in the Mexican state of Jalisco, near the city of Tequila. Tentative identification using conventional biochemical analysis consistently identified the isolate as Bacillus subtilis. DNA isolated from the tomb isolate, strain 10b(T), and closely related species was used to amplify a Bacillus-specific portion of the highly conserved 16S rRNA gene and an internal region of the superoxide dismutase gene (sodA(int)). Trees derived from maximum-likelihood methods applied to the sodAint sequences yielded non-zero branch lengths between strain 10b(T) and its closest relative, whereas a comparison of a Bacillus-specific 546 bp amplicon of the 16S rRNA gene demonstrated 99% similarity with B. subtilis. Although the 16S rRNA gene sequences of strain 19b T and B. subtilis were 99 % similar, PFGE of Notl-digested DNA of strain 10b(T) revealed a restriction profile that was considerably different from those of B. subtilis and other closely related species. Whereas qualitative differences in whole-cell fatty acids were not observed, significant quantitative differences were found to exist between strain 1 Ob T and each of the other closely related Bacillus species examined. In addition, DNA-DNA hybridization studies demonstrated that strain 10b(T) had a relatedness value of less than 70% with B. subtilis and other closely related species. Evidence from the sodA(int) sequences, whole-cell fatty acid profiles and PFGE analysis, together with results from DNA-DNA hybridization studies, justify the classification of strain 1 Ob T as representing a distinct species, for which the name Bacillus tequilensis sp. nov. is proposed. The type strain is 10b(T) (=ATCC BAA-819(T) = NCTC 13306(T)).