pilA Gene Contributes to Virulence, Motility, Biofilm Formation, and Interspecific Competition of Bacteria in Acidovorax citrulli .

Acidovorax citrulli , the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli . In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65 - Δ pilA and Aac5 - Δ pilA ) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-Δ pilA also completely lost twitching motility, whereas Aac5-Δ pilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-Δ pilA and WT pslb65, but Aac5-Δ pilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-Δ pilA and Aac5-Δ pilA were significantly lower than in the WT strains; Aac5-Δ pilA was more strongly competitive than pslb65-Δ pilA , and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-Δ pilA and Aac5-Δ pilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.