Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae

Abstract

The signaling molecule cyclic di-GMP (c-di-GMP) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of c-di-GMP have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of c-di-GMP are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize c-di-GMP to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyze c-di-GMP to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing c-di-GMP levels, we followed a time course of overexpression of either the V. campbellii diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases c-di-GMP levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on c-di-GMP levels plateaus at 30 minutes, indicating tight adaptive control of c-di-GMP levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to c-di-GMP, leads to higher baseline levels of c-di-GMP and continuously increasing c-di-GMP through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on c-di-GMP levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high c-di-GMP concentrations, mediating this negative feedback on c-di-GMP levels. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of c-di-GMP to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.