Abstract
Vibrio alginolyticus is a prevalent pathogen in both humans and marine species, exhibiting high adaptability to various adverse environmental conditions. Our previous studies have shown that ΔrpoN formed three enhanced biofilm types, including spectacular surface-attached biofilm (SB), scattered pellicle biofilm (PB), and colony rugosity. However, the precise mechanism through which rpoN regulates biofilm formation has remained unclear. Based on the critical role of Vibrio exopolysaccharide (VPS) in biofilm formation, several genes related to the production and regulation of VPS were characterized in V. alginolyticus. Our findings from mutant strains indicated that VPS has complete control over the formation of rugose colony morphology and PB, while it only partially contributes to SB formation. Among the four transcriptional regulators of the vps gene cluster, vpsR and VA3545 act as promoters, whereas VA3546 and VA2703 function as repressors. Through transcriptome analysis and c-di-GMP concentration determination, VA0356 and VA3580 which encoded diguanylate cyclase were found to mediate the ΔrpoN biofilm formation. As a central regulator, rpoN governed biofilm formation through two regulatory pathways. Firstly, it directly bound to the upstream region of VA4206 to regulate the expression of the vps gene cluster (VA4206-VA4196). Secondly, it directly and indirectly modulated c-di-GMP synthesis gene VA3580 and VA0356, respectively, thereby affecting c-di-GMP concentration and subsequently influencing the expression of vps transcription activators vpsR and VA3545. Under conditions promoting SB formation, ΔrpoN was unable to thrive below the liquid level due to significantly reduced activities of three catalytic enzymes (ACK, ADH, and ALDH) involved in pyruvate metabolism, but tended to reproduce in air-liquid interface, a high oxygen niche compared to the liquid phase. In conclusion, both exopolysaccharide synthesis and oxygen-related metabolism contributed to ΔrpoN biofilm formation. The role of RpoN-mediated hypoxic metabolism and biofilm formation were crucial for comprehending the colonization and pathogenicity of V. alginolyticus in hosts, providing a novel target for treating V. alginolyticus in aquatic environments and hosts.