Project description:The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a time course of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that the PDE CdgC is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name sRNA negative regulator of CdgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner depending on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. 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. These results underscore the importance of negative feedback regulation of cdG 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.

Project description:In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade.IMPORTANCEVibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

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Vibrio cholerae adapts to sessile and motile lifestyles by cyclic di-GMP regulation of cell shape.

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