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

¹ Division of Molecular Microbiology, Biozentrum, Klingelbergstrasse 50–70, CH-4056 Basel, Switzerland
² Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
³ Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK
⁴ School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Correspondence
P. B. Rainey
p.rainey{at}auckland.ac.nz

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.

Lists of mutant wspR constructs produced by PSM, mutant wspR constructs produced by SOE-PCR, and oligonucleotides used in the study, BLAST alignment data for WspR and PleD, and a figure showing attachment to glass for SM wspR versus WS wspR, are available as supplementary data with the online version of this paper.

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