Function and transcription of the lon gene in Xanthomonas campestris pv. campestris

The Gram-negative bacterium Xanthomonas campestris pv. campestris is the etiological agent of black rot, a widespread and destructive disease affecting cruciferous plants. In this study, a lon mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The lon gene encodes an ATP-dependent protease implicated in protein quality control and stress adaptation across various bacterial species. Functional analysis revealed that lon disruption in X. campestris pv. campestris resulted in diminished extracellular protease activity, reduced virulence, and heightened sensitivity to puromycin and elevated temperatures. Complementation with the wild-type lon allele restored most phenotypes, except thermotolerance, which was only partially recovered. Interestingly, lon overexpression in wild-type cells compromised growth under heat stress, indicating that lon dosage is critical for thermal adaptation. Promoter activity assays indicated that lon expression is subject to catabolite repression and is induced by heat shock. Additionally, analysis of upstream regions of lon and multiple experimentally validated heat-inducible genes revealed a conserved motif similar to the σ³²-binding site in Escherichia coli, suggesting a conserved σ³²-mediated regulatory mechanism. This work provides the functional and regulatory characterization of lon in X. campestris pv. campestris, underscoring its integral role in stress resilience and pathogenicity.