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

Abstract

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 asBacillus subtilis. DNA isolated from the tomb isolate, strain 10b^T, and closely related species was used to amplify aBacillus-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 thesodA_intsequences yielded non-zero branch lengths between strain 10b^Tand its closest relative, whereas a comparison of aBacillus-specific 546 bp amplicon of the 16S rRNA gene demonstrated 99 % similarity withB. subtilis. Although the 16S rRNA gene sequences of strain 10b^TandB. subtiliswere 99 % similar, PFGE ofNotI-digested DNA of strain 10b^Trevealed a restriction profile that was considerably different from those ofB. subtilisand other closely related species. Whereas qualitative differences in whole-cell fatty acids were not observed, significant quantitative differences were found to exist between strain 10b^Tand each of the other closely relatedBacillusspecies examined. In addition, DNA–DNA hybridization studies demonstrated that strain 10b^Thad a relatedness value of less than 70 % withB. subtilisand other closely related species. Evidence from thesodA_intsequences, whole-cell fatty acid profiles and PFGE analysis, together with results from DNA–DNA hybridization studies, justify the classification of strain 10b^Tas representing a distinct species, for which the nameBacillus tequilensissp. nov. is proposed. The type strain is 10b^T(=ATCC BAA-819^T=NCTC 13306^T).