A Toxin-Based Counter-Selection System for Markerless Gene Deletion and High-Density Tn5 Transposon Mutagenesis in Pectobacterium brasiliense

Pectobacterium brasiliense is a highly pathogenic bacterium responsible for potato tuber soft rot, blackleg, and aerial stem rot. Elucidation of its pathogenic mechanisms is essential for disease control; however, genetic tools for this organism remain limited. A toxin-based counter-selection system was developed to enable markerless gene deletion and high-density Tn5 transposon mutagenesis. The vmi480 toxin derived from the pLP12 plasmid was used as a counter-selection marker to construct the suicide vector pKV2. Using this system, the recA gene in the P. brasiliense SM strain was successfully deleted. The resulting recA mutant served as the parental strain for the construction of a Tn5 insertion library comprising over 12,000 mutants through conjugation with the RHO3 helper strain carrying pKV2-LLtnp. In this derivative plasmid, the vmi480 toxin was replaced with a hyperactive Tn5 transposase under the control of an outward-facing lac promoter to reduce polar effects. High-throughput tuber inoculation assays enabled the identification of pathogenicity-deficient mutants, including insertions in the type II secretion system, a key virulence determinant that mediates the secretion of plant cell wall-degrading enzymes. This platform supports functional genomics and facilitates the investigation of virulence mechanisms in this economically important pathogen.