Fur Represses Vibrio cholerae Biofilm Formation via Direct Regulation of vieSAB, cdgD, vpsU, and vpsA-K Transcription
He Gao, Lizhi Ma, Qin Qin, Yue Qiu, Jingyun Zhang, Jie Li, Jing Lou, Baowei Diao, Hongqun Zhao, Qiannan Shi, Yiquan Zhang, Biao Kan
Abstract
Attached Vibrio cholerae biofilms are essential for environmental persistence and infectivity. The vps loci (vpsU, vpsA-K, and vpsL-Q) are required for mature biofilm formation and are responsible for the synthesis of exopolysaccharide. Transcription of vps genes is activated by the signaling molecule bis-(3'-5')-cyclic di-GMP (c-di-GMP), whose metabolism is controlled by the proteins containing the GGDEF or/and EAL domains. The ferric uptake regulator (Fur) plays key roles in the transcription of many genes involved in iron metabolism and non-iron functions. However, its roles in biofilms of vibrios have been not documented. In this study, phenotypic assays demonstrated that Fur, independently of iron, decreases in vivo c-di-GMP levels and inhibits in vitro biofilm formation by V. cholerae. Fur box-like sequences were detected within the promoter-proximal DNA regions of vpsU, vpsA-K, vieSAB and cdgD, suggesting that transcription of these genes may be under the direct control of Fur. Indeed, the results of luminescence, qPCR, EMSA and DNase I footprinting assays demonstrated that Fur binds to the promoter-proximal DNA regions of vpsU, vpsA-K, and cdgD, to repress their transcription, whereas it activates the transcription of vieSAB in a direct manner. The cdgD and vieSAB encode proteins with GGDEF and EAL domains, respectively. Thus, data presented here highlight a new physiological role for Fur that acts as a repressor of V. cholerae biofilm formation, which is mediated by decreasing the production of exopolysaccharide and the intracellular levels of c-di-GMP.