NspS, A PotD1 Homolog, Acts as a Spermidine Signal Sensor, Not a Transporter, in Vibrio Cholera

Biofilm formation is important for the survival of Vibrio cholerae in its natural aquatic environments and within the human intestinal tract. We have previously shown that the absence of PotD1, the periplasmic binding protein for the spermidine ABC transporter, leads to an increase in biofilm formation. In addition, spermidine reduces biofilm formation through its interaction with NspS, a homolog of PotD1. Due to its similarity to PotD1, NspS has been annotated as a polyamine transporter. In this study, we seek to establish whether NspS is capable of transporting spermidine into the cell. We show that the absence of NspS did not lead to a loss of spermidine inside the cell, indicating that NspS does not function as a transporter. Polyamine analysis of wild-type, ?nspS, ?potD1, ?nspS?potD1, and the ?nspS?potD2?potD1 strains suggested that there are no high affinity transporters present in the cell in addition to PotD1. Biofilm assays showed that the biofilm cell densities of ?nspS?potD1 mutant were intermediate between the ?nspS and ?potD1 single mutants, implicating neither NspS nor PotD1 are epistatic over the other. Our results suggest that NspS plays a role in spermidine signaling, but not transport in V. cholerae.