Genomic Discovery of Robust Molecular Markers Differentiating Lactobacillaceae Genera and Providing Novel Tools for Functional Insights

Background: Members of the family Lactobacillaceae, encompassing 23 distinct genera, play essential roles in food fermentation processes such as wine, yogurt, and cheese production and contribute significantly to human health through their probiotic properties. Despite their importance, accurately distinguishing between Lactobacillaceae genera has been challenging due to the absence of reliable biochemical or molecular markers. Currently, these genera are primarily differentiated based on phylogenetic relationships. Methods: To address this limitation, we have performed comprehensive phylogenomic and comparative analyses of protein sequences from 411 publicly available Lactobacillaceae genomes. Results: The results of these analyses have identified 171 novel conserved signature indels (CSIs), within proteins involved in diverse cellular functions, which are specific for the species from different Lactobacillaceae genera. The taxon-specificities of these CSIs make them robust molecular markers for differentiation of Lactobacillaceae genera and for functional insights. Using these taxon-specific CSIs and the AppIndels.com server, we were able to successfully predict the taxonomic affiliation of 112 uncharacterized genomes of Lactobacillus isolates, demonstrating the practical utility of these CSIs for genus-level identification and classification. Structural analyses on representative CSIs specific for Lactobacillaceae genera reported here show that all examined CSIs are located in surface-exposed loops of proteins, suggesting their potential roles in genus-specific functional traits, such as interaction with specific proteins and ligands, host interactions, or environmental adaptations. Conclusions: The CSIs identified here not only provide reliable tools for diagnostic and taxonomic studies but also open new avenues for exploring the functional diversity and biotechnological potential of species from different Lactobacillaceae genera.