Characterization of the NspS-MbaA signaling system controlling biofilm formation from polyamine input to phenotypic output

Biofilm formation plays a critical role in the infectious cycle of the bacterial pathogen, Vibrio cholerae. The polyamine, norspermidine, has a positive influence on biofilm formation while spermidine has the opposite effect. These influences occur through a signaling system comprised of the periplasmic polyamine binding protein, NspS, and the transmembrane phosphodiesterase, MbaA. In the absence of NspS, MbaA acts as a phosphodiesterase cleaving the pro-biofilm second messenger, cyclic di-GMP, indicating that NspS may interact with and inhibit phosphodiesterase activity and biofilm repression by MbaA. In this study, I have shown that several nspS ligand-binding pocket mutants have altered sensitivity to norspermidine and spermidine as indicated by vps transcription and biofilm assays. I have also shown using thermal shift assays that these mutants are capable of binding both polyamines. LC-MS/MS analyses indicate that the NspS-MbaA signaling pathway is a high specificity c-di-GMP signaling system. Additionally, a system for reproducible demonstration of the NspS-MbaA interaction was developed which may subsequently be used in future experiments to study the NspS and MbaA interaction. Altogether, this study provides a thorough investigation of the steps involved in polyamine signal input to phenotypic output for a novel polyamine responsive signaling system controlling V. cholerae biofilm formation.