Genomic Discovery of Taxon-Specific Molecular Markers for <em>Lactobacillaceae</em> Genera

Background: Members of the family Lactobacillaceae, comprising 36 genera, play vital roles in food fermentation (e.g., wine, yogurt, and cheese production) and contribute significantly to human health through their probiotic properties. Despite their importance, species from different genera are primarily distinguished by phylogenetic clustering and genomic similarity matrices, and no consistent molecular, biochemical, or physiological traits are known that are uniquely found in species from different genera. Methods: To address this limitation, we conducted comprehensive phylogenomic and comparative analyses of protein sequences from 410 publicly available Lactobacillaceae genomes. Results: Based on these analyses, we identified 167 novel conserved signature indels (CSIs) in proteins involved in diverse cellular functions, each specific to a particular genus within the Lactobacillaceae family. These taxon-specific CSIs serve as robust molecular markers for genus-level differentiation and have potential applications in functional and diagnostic studies. Using these markers and the AppIndels.com server, we successfully predicted the genus-level affiliation of 111 uncharacterized Lactobacillus isolates. Structural analysis of representative CSIs from four genera revealed their consistent location in surface-exposed protein loops, suggesting possible roles in genus-specific protein–protein or protein–ligand interactions. Conclusions: The identified CSIs provide novel molecular markers for the robust differentiation of species from different Lactobacillaceae genera, offering new tools for exploring the functional traits unique to each genus.