Characterization and evaluation of tandem repeats for the identification of Geobacillus

Abstract

Taxonomy of thermophilic, endospore-forming bacteria has evoked a great interest over the past few years. Although a number of taxonomic markers were previously evaluated, their sequences in Geobacillus were too conservative, and identification of more variable markers is needed. Repetitive DNA is one of the promising variable targets in the development of the taxon-specific genotyping and identification schemes in bacteria. The aim of our study was to evaluate the possibility of using repetitive DNA in the taxonomy of Geobacillus. In this paper, we report the analysis of perfect tandem repeats of geobacilli. We focused on the long repeats (with a motif length of ≥20 nucleotides). This choice was based on the assumption that these motifs can be used for the construction of oligonucleotides — primers and probes. Thirty-three Geobacillus genus-specific motifs were identified in our work, fifteen of them were species-specific and fifteen — species cluster -specific. Three of them were genus-, but not species- or species cluster-specific. Some of the motifs were used for the construction of the primer pairs. The primers were validated by PCR. Out of 12 designed primer pairs, 11 were genus-specific and 4 — species-specific. Species-specific primers were successfully constructed for the phylogenetically defined species Geobacillus thermodenitrificans and Geobacillus toebii.

GeobacillusBacteria: Potential Commercial Applications in Industry, Bioremediation, and Bioenergy Production

“…In total, 33 Geobacillus genus-specific motifs with length over 20 nucleotides were determined: 3 were genus-specific, 15-species-specific, and 15-species cluster-specific. Motifs have been used for the construction of the genus-and species-specific (G. thermodenitrificans and G. toebii) primer pairs [44]. Rep-PCR molecular method based on the usage of outwardly facing oligonucleotide PCR primers complementary to interspersed repetitive sequences is widely applied in the characterization of different groups of bacteria.…”