In the modern era, molecular genetic techniques are crucial in ecological studies, as well as in the classification, typing, and phylogenetic analysis of prokaryotes. These techniques are mainly aimed at whole genome comparisons and PCR-derived experiments, including amplifying the 16S rRNA and other various housekeeping genes used in taxonomy, as well as MLST (multilocus sequence typing) and MLSA (multilocus sequence analysis) of different taxonomic bacterial groups. The gene encoding threonine-tRNA ligase (thrS) is a gene potentially applicable as an identification and phylogenetic marker in bacteria. It is widely distributed in bacterial genomes and is subject to evolutionary selection pressure due to its important function in protein synthesis. In this study, specific primers were used to amplify a thrS gene fragment (~740 bp) in 36 type and 30 wild strains classified under family Bifidobacteriaceae. The full-length gene has not yet been considered as a possible identification, classification, and phylogenetic marker in bifidobacteria. The thrS sequences revealed higher sequence variability (82.7% of pairwise identities) among members of the family than that shown by 16S rRNA gene sequences (96.0%). Although discrepancies were found between the thrS-derived and previously reported whole genome phylogenetic analyses, the main phylogenetic groups of bifidobacteria were properly assigned. Most wild strains of bifidobacteria were better differentiated based on their thrS sequences than on their 16S rRNA gene identities. Phylogenetic confidence of the evaluated gene with respect to other alternative genetic markers widely used in taxonomy of bifidobacteria (fusA, GroELhsp60, pyrG, and rplB genes) was confirmed using the localized incongruence difference - Templeton analysis.

Czech University of Life Sciences Faculty of Agrobiology Food and Natural Resources Department of Microbiology Nutrition and Dietetics Kamýcká 129 165 00 Czechia

Czech University of Life Sciences Faculty of Agrobiology Food and Natural Resources Department of Microbiology Nutrition and Dietetics Kamýcká 129 165 00 Czechia Food Research Institute Prague Radiová 1285 7 102 00 Czechia

Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Czechia Czech University of Life Sciences Faculty of Agrobiology Food and Natural Resources Department of Microbiology Nutrition and Dietetics Kamýcká 129 165 00 Czechia

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