Biochemical Characterization of LsGajA: A Key Nuclease for Gabija Defense in Lactic Acid Bacteria

Lactic acid bacteria (LAB), as important probiotics, face challenges in applications from bacteriophage infection and the instability of foreign genetic elements. Although Gabija systems and their GajA nuclease components have been characterized in model bacteria, their distribution, biochemical properties, and defensive functions in LAB remain largely unexplored. Here, we provide the first systematic characterization of a naturally occurring Gabija system from Ligilactobacillus salivarius Ren and clarify its distribution among LAB. Approximately 9.3% of LAB strains encode the Gabija system, which exists as a gajA-gajB gene cluster. We found that the Gabija system originated independently in different bacterial lineages. The GajA of L. salivarius Ren (LsGajA) was purified and exhibited non-specific nuclease activity that could efficiently cleave various nucleic acid substrates, including plasmids and linear double-stranded DNA (dsDNA). This activity displayed a temperature-dependent profile, with high activity observed from 45 to 60 °C and at pH 8.0. Mg²⁺ markedly enhanced its degradative nuclease activity, whereas high concentrations of dNTPs inhibited DNA cleavage. LsGajA exhibited substrate-dependent differences in cleavage efficiency, indicating that substrate origin and associated physicochemical features may influence its activity. Additionally, we demonstrate that LsGajA exhibits exceptional stability as a nuclease, retaining activity under a wide range of conditions. The LsGabija system significantly enhanced the ability to reject foreign plasmids and provided strong resistance to the bacteriophage T5 in Escherichia coli. This study provides the first systematic biochemical and functional characterization of the Gabija system in LAB, advancing our understanding of this prokaryotic defense system and highlighting its potential for industrial applications.