Diversity and evolutionary dynamics of universal stress proteins in the Liquorilactobacillus genus.

The genus Liquorilactobacillus comprises lactic acid bacteria frequently associated with liquid fermentation environments, where they are exposed to multiple stressors such as pH fluctuations, ethanol accumulation, osmotic pressure, and nutrient limitation. Universal stress proteins (USP) are key molecular components that enable bacterial adaptation and persistence under these challenging conditions. In this study, we investigated the diversity, structural features, and evolutionary dynamics of USP genes across 13 type strains of Liquorilactobacillus species. A total of 65 usp-encoding genes were identified, ranging from three to six copies per genome, all predicted to be cytoplasmic and exhibiting the conserved Usp fold. Phylogenetic analysis revealed that Usp diversification resulted primarily from ancestral gene duplication events followed by gene losses, with no evidence of recent lineage-specific expansions. Approximately half of the proteins retained a canonical ATP-binding motif, while others lacked it or contained degenerate variants, suggesting an evolutionary trajectory from an ancestral non-ATP-binding form toward a gain-of-function motif. Codon-level selection analysis indicated strong purifying selection acting on most sites, with substitutions concentrated in loop regions that may contribute to functional divergence. A focused analysis of Liquorilactobacillus vini revealed structural and sequence heterogeneity among its five Usp paralogs, correlating with differential stress-response expression patterns. Overall, our findings demonstrate that Usp evolution in Liquorilactobacillus reflects a balance between conservation and diversification, highlighting their central role in stress adaptation and ecological success in dynamic fermentation environments.