CdgH-mediated c-di-GMP signaling orchestrates biofilm development via the transcription factors VpsT homologs and VpsR in Vibrio alginolyticus

Vibrio alginolyticus is an opportunistic pathogen impacting both aquaculture and public health. The second messenger cyclic-di-GMP (c-di-GMP) regulates bacterial transitions between motility and biofilm formation, as well as the expression of secretion systems, thereby enhancing environmental persistence, competitiveness, and virulence. However, the c-di-GMP regulatory network in V. alginolyticus remains poorly characterized, limiting our understanding of its pathogenic and adaptive mechanisms. Here, using phenotypic assays combined with LC-MS/MS, we demonstrate that overexpression of the diguanylate cyclase CdgH elevates intracellular c-di-GMP levels, promoting biofilm formation, rugose colony morphology and Congo Red (CR) binding. RNA-seq and molecular analyses revealed that the EPS synthesis cluster vpsQ-wcaJ is essential for c-di-GMP-mediated biofilm formation. V. alginolyticus encodes three VpsT-like transcriptional regulators (VA3545, VA3546 and VA2703). We identified the VA3545 and the regulator VpsR as key positive regulators of biofilm formation, while VpsT exhibited opposing effects. Surface plasmon resonance (SPR) assays confirmed c-di-GMP binding to VA3545 and VpsR, with the conserved R residue in the c-di-GMP-binding pocket W[F/L/M][T/S]R being critical for VA3545 function. Furthermore, elevated c-di-GMP levels suppressed swarming motility by downregulating flagellar synthesis genes. Notably, unlike V. cholerae and Pseudomonas aeruginosa, high c-di-GMP inversely regulated the expression of core conserved genes associated with type II and type VI secretion systems (T2SS/T6SS) in V. alginolyticus. Collectively, our findings reveal that transcription factors including VpsT homologs and VpsR play unique roles in biofilm development and c-di-GMP signaling exerts distinctive control over secretion systems, providing new insights into the pathogenicity and environmental adaptation of V. alginolyticus.