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

¹ Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA
² Department of Biology, Texas Wesleyan University, Fort Worth, TX 76105, USA
³ National Research Institute of Fisheries, Food Processing Division, Yokohama, Kanagawa 236-8648, Japan
⁴ Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA
⁵ Division of Microbiology (HFT-250), National Center for Toxicological Research, 3900 NCTR Rd, Jefferson, AR 72079, USA

Correspondence
Mark E. Hart
mark.hart{at}fda.hhs.gov

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 sodA_int 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 10b^T and B. subtilis were 99 % similar, PFGE of NotI-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 10b^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 10b^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).

A comparison of phenotypic characteristics of Bacillus tequilensis sp. nov. 10b^T and other Bacillus species is available as supplementary material in IJSEM Online.

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