A cyclic-di-GMP receptor required for bacterial exopolysaccharide production

Bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide

Phenotypic comparison of Δ mutation or overexpression of diguanylate cyclase on pellicle production and mRNA levels

<p><b>Copyright information:</b></p><p>Taken from "A cyclic-di-GMP receptor required for bacterial exopolysaccharide production"</p><p></p><p>Molecular Microbiology 2007;65(6):1474-1484.</p><p>Published online Jan 2007</p><p>PMCID:PMC2170427.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A and B. Pellicle formation in static culture as revealed by crystal violet staining for (A) PA14, PA14Δ, PA14Δ and PA14Δ harbouring vector control or induced for expression of diguanylate cyclases , or ; (B) PA14Δ, PA14ΔΔ, PA14ΔΔ and PA14ΔΔ. C. Expression of was determined by measuring β-galactosidase levels using a reporter construct in strains PA14, PA14Δ, PA14Δ and PA14Δ harbouring vector control or induced for expression of diguanylate cyclases , or . D. Quantification of PEL polysaccharide by binding to Congo red was normalized to total protein for strains PA14 pMMB-, PA14Δ pMMB-, PA14Δ and PA14ΔΔ

Residues required for c-di-GMP binding are required for PEL polysaccharide synthesis

<p><b>Copyright information:</b></p><p>Taken from "A cyclic-di-GMP receptor required for bacterial exopolysaccharide production"</p><p></p><p>Molecular Microbiology 2007;65(6):1474-1484.</p><p>Published online Jan 2007</p><p>PMCID:PMC2170427.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> Complementation of the Δ mutation in PA14Δ pDN19- with pMMB containing full-length and indicated point mutations. Quantification of PEL polysaccharide binding to Congo red was normalized to total protein for each strain. Pellicle formation for each stain was tested in static culture and revealed by crystal violet staining. PelD and mutant derivatives were fused at their C-termini to HA tags. Total-cell extracts were prepared, loaded equally and separated on SDS-PAGE, transferred to PVDF and PelD-HA was detected with anti-HA antibody and chemiluminescence