Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales.

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).

Variable regions of the glyS, infB and rplB genes usable as novel genetic markers for identification and phylogenetic purposes of genera belonging to the family Propionibacteriaceae.

No common, unique genetic markers applicable to classification and phylogenetics for significant genera within the Propionibacteriaceae family have been suggested yet. Therefore, the aim of the study was to propose those genes in the genera Acidipropionibacterium, Cutibacterium, Propionibacterium and Pseudopropionibacterium. These genera were recently elicited from the genus Propionibacterium through whole genomic analyses. Three housekeeping genes, glyS, infB and rplB, were selected from many others according to the requirements for appropriate classification/phylogenetic markers. Concrete fragments of the genes were amplified using specific primers in most of the type (14) and 11 wild strains (originating from dairy products, human skin and the crop of a laying hen) recently classified into the genus Propionibacterium. Sequences obtained from amplicons were used to perform gene statistics and phylogenetic analyses with respect to applicability in classification, typing and phylogeny. The 16S rRNA gene sequences, still considered relevant in spite of its proven shortcomings as a basic tool for evaluation of bacterial phylogeny, were used as a baseline for comparative analyses. The statistics of the gene sequences revealed that the variable regions of all three genes have higher resolution capabilities among strains examined compared to the 16S rRNA gene analysis. Phylogenetic analyses based on individual gene sequences and their concatenate enabled to distinguish clusters of species belonging to the genera Acidipropionibacterium, Cutibacterium and Propionibacterium, which corresponds with a recently reported genomic study. Thus, the crucial importance of this study is the economically advantageous classification and typing of propionibacterial isolates and strains through the three gene regions in contrast to the requirement for whole genomic assays.