Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA-DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).

Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Kamýcká 129 165 00 Prague 6 Suchdol Czech Republic

Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Kamýcká 129 165 00 Prague 6 Suchdol Czech Republic Food Research Institute Prague Radiová 1285 7 102 00 Prague 10 Hostivař Czech Republic

Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Krč Czech Republic

Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Krč Czech Republic Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Kamýcká 129 165 00 Prague 6 Suchdol Czech Republic

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