dctA, dctB, and dctD contribute to the utilization of C4-dicarboxylates, carbon, nitrogen, as well as virulence in Acidovorax citrulli

Bacterial fruit blotch (BFB), a destructive bacterial disease triggered by Acidovorax citrulli, affects cucurbit crops like watermelon and melon. The absorption and use of carbon sources are foundational for bacteria to successfully colonize host plants. C4-dicarboxylates are critical carbon and energy substances, and their transport is completed by the C4-dicarboxylate transport system (Dct) which plays an important role in typical bacterial metabolism. However, the role of dct genes have not been determined for A. citrulli. To clarify the biological roles of the Dct system-related genes in A. citrulli, we developed dctA1, dctA2, dctB, and dctD deletion mutants, as well as dctA1A2 double deletion mutant, with their corresponding complementary strains in the A. citrulli wild-type strain Aac5 in this study. The functions of Dct-related genes in A. citrulli were analyzed through phenotype assays, including pathogenicity, C4-dicarboxylates utilization, carbon and nitrogen utilization, biofilm formation, swimming motility, and qRT-PCR analysis. Compared to the wild-type strain, the pathogenicity, utilization of C4-dicarboxylates, growth ability in vivo and in vitro, and seed adhesion ability of the mutant strains were significantly limited, while the biofilm formation ability was significantly improved. Additionally, the utilization of select carbon sources (glucose, maltose, and sucrose) and nitrogen sources ((NH₄)₂SO₄, NH₄Cl, CH₄N₂O, and KNO₃) was significantly enhanced. qRT-PCR results demonstrated that the deletion of Dct-related genes resulted in significant downregulation of the expression of T3SS-related genes (hrpG and hrpE), the pili-related genes (pilA and pilN), and some flagellum-related genes (fliC, flhC, and flhD). These findings suggested that Dct-related genes were involved in C4-dicarboxylate utilization, carbon and nitrogen use, and the pathogenicity of A. citrulli.