The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

Abstract

The GGDEF response regulator WspR couples the chemosensory Wsp pathway to the overproduction of acetylated cellulose and cell attachment in the Pseudomonas fluorescens SBW25 wrinkly spreader (WS) genotype. Here, it is shown that WspR is a diguanylate cyclase (DGC), and that DGC activity is elevated in the WS genotype compared to that in the ancestral smooth (SM) genotype. A structure-function analysis of 120 wspR mutant alleles was employed to gain insight into the regulation and activity of WspR. Firstly, 44 random and defined pentapeptide insertions were produced in WspR, and the effects determined using assays based on colony morphology, attachment to surfaces and cellulose production. The effects of mutations within WspR were interpreted using a homology model, based on the crystal structure of Caulobacter crescentus PleD. Mutational analyses indicated that WspR activation occurs as a result of disruption of the interdomain interface, leading to the release of effector-domain repression by the N-terminal receiver domain. Quantification of attachment and cellulose production raised significant questions concerning the mechanisms of WspR function. The conserved RYGGEEF motif of WspR was also subjected to mutational analysis, and 76 single amino acid residue substitutions were tested for their effects on WspR function. The RYGGEEF motif of WspR is functionally conserved, with almost every mutation abolishing function.

Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens. III. Mutational Origins of Wrinkly Spreader Diversity

“…A functionally compromised WspF methylesterase results in constitutive activation of the WspR DGC (Goymer et al 2006), overproduction of c-di-GMP (Malone et al 2007), and enhanced production of adhesive substances (Spiers et al 2002(Spiers et al , 2003Spiers and Rainey 2005), most importantly, of a partially acetylated cellulose polymer (Spiers et al 2002). The net effect is to cause daughter cells to remain attached after cell division.…”

“…In LSWS, the wss-encoded enzymes are constitutively active as a consequence of overproduction of bis-(39-5-)-cyclic dimeric guanosine monophosphate (c-di-GMP), an allosteric activator of cellulose (Ross et al 1987;Goymer et al 2006;Malone et al 2007). Overproduction of c-di-GMP results in constitutive production of polymer, which, in conjunction with a similarly overproduced proteinaceous adhesin (Spiers et al 2003), causes daughter cells to remain attached after cell division.…”

“…When phosphorylated (at Asp67), the C-terminal DGC domain of WspR becomes functionally active and this leads to the joining of two molecules of GTP in a head-to-tail manner, resulting in the formation of c-di-GMP (Malone et al 2007). …”

Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads

“…WspC constitutively antagonises WspF, and in wild-type SBW25 the activities of the two are balanced, preventing the activation of WspR and allowing the Wsp complex to oscillate between active and inactive states. WspR is a di-guanylate cyclase (DGC) response regulator, and the phosphorylated active form, WspR-P, synthesizes c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate) from GTP [69]. In this model, we hypothesised that mutations in WspR which stimulated DGC activity without requiring phosphorylation, or mutations inhibiting WspF function, would result in an increase in c-di-GMP production.…”

“…Interestingly, no naturally occurring WspR mutants have been identified yet, despite the fact that engineered WspR mutants like WspR19 were found to show the predicted phenotype [60,[69][70][71]. Mutations in other operons leading to the activation of the AwsR and MwsR DGCs can also induce the WS phenotype [47,72].…”