BlihIA-A Novel Type I Restriction-Modification System from Bacillus licheniformis Is Sensitive to In Vitro Inhibition...

Type I restriction-modification (RMI) systems play a crucial role in bacterial defense against mobile elements by distinguishing self and foreign DNA through sequence-specific methylation and cleavage. Here, we characterize BlihIA, a novel RMI system from Bacillus licheniformis DSM13 which features redundancy in its hsdS gene copies. Using ONT sequencing, we identify the bipartite recognition site of BlihIA as RTAC(N)₅GCT. We demonstrate the system's activity both in vivo through efficiency of plaquing (EOP) assay and in vitro in a nuclease reaction with purified BlihIA complex. Notably, mutation of the recognition site abolished in vitro DNA cleavage, confirming sequence specificity. Furthermore, we show that the antirestriction protein ArdB from plasmid R64 effectively prevents DNA cleavage by BlihIA, suggesting a direct mechanism of inhibition. This study provides the first functional characterization of a novel RM system BlihIA, extending the diversity of RM systems in Bacillus species and suggesting potential applications for improving genetic transformation in industrial strains.