Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1

Abstract

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

Para-Aminobenzoic Acid, Calcium, and c-di-GMP Induce Formation of Cohesive, Syp-Polysaccharide-Dependent Biofilms in Vibrio fischeri

“…Because a role for c-di-GMP in syp -dependent biofilm formation has not been previously reported, we sought to determine if the ability of pABA to induce biofilm formation is reliant on c-di-GMP. To test this possibility, we overexpressed the gene for a c-di-GMP phosphodiesterase (PDE), VF_0087, which we have previously shown to be effective in decreasing c-di-GMP levels of strain KB2B1 ( 26 ). First, we asked if overexpression of VF_0087 abolished or diminished the pABA-induced increase in c-di-GMP using the biosensor.…”

OpaR exerts a dynamic control over c-di-GMP homeostasis andcpsAexpression inV. parahaemolyticusthrough its regulation of ScrC and the trigger phosphodiesterase TpdA

“…Furthermore, we also hypothesized that the hierarchical regulation of the P tpdA promoter by ScrC, VP0117 and ScrG could be explained by differences in the levels of c-di-GMP in genetic backgrounds lacking these PDEs. To evaluate c-di-GMP levels in the genetic backgrounds of interest we used a genetic biosensor previously shown to be able to detect differences in c-di-GMP levels in Vibrio cholerae, Vibrio parahaemolyticus and Vibrio fischeri (25, 30, 31). In this c-di-GMP biosensor the production of two fluorescent proteins, Amcyan and TurboRFP is controlled by a constitutive promoter (30).…”

OpaR Exerts a Dynamic Control over c-di-GMP Homeostasis and cpsA Expression in Vibrio parahaemolyticus through Its Regulation of ScrC and the Trigger Phosphodiesterase TpdA

“…Furthermore, we also hypothesized that the hierarchical regulation of the P tpdA promoter by ScrC, VP0117, and ScrG could be explained by differences in c-di-GMP levels in genetic backgrounds which lacked these PDEs. To evaluate c-di-GMP levels in the genetic backgrounds of interest, we used a genetic biosensor previously shown to be able to detect differences in c-di-GMP levels in V. cholerae , V. parahaemolyticus , and Vibrio fischeri ( 27 , 32 , 33 ). In this c-di-GMP biosensor, the production of two fluorescent proteins, AmCyan and TurboRFP, is controlled by a constitutive promoter ( 32 ).…”