SummaryThe wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss . Both polar and non-polar mutations in the sixth gene of the wss operon ( wssF ) or adjacent downstream genes ( wssGHIJ ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa , which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.

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The capacity for phenotypic evolution is dependent upon complex webs of functional interactions that connect genotype and phenotype. Wrinkly spreader (WS) genotypes arise repeatedly during the course of a model Pseudomonas adaptive radiation. Previous work showed that the evolution of WS variation was explained in part by spontaneous mutations in wspF, a component of the Wsp-signaling module, but also drew attention to the existence of unknown mutational causes. Here, we identify two new mutational pathways (Aws and Mws) that allow realization of the WS phenotype: in common with the Wsp module these pathways contain a di-guanylate cyclase-encoding gene subject to negative regulation. Together, mutations in the Wsp, Aws, and Mws regulatory modules account for the spectrum of WS phenotypegenerating mutations found among a collection of 26 spontaneously arising WS genotypes obtained from independent adaptive radiations. Despite a large number of potential mutational pathways, the repeated discovery of mutations in a small number of loci (parallel evolution) prompted the construction of an ancestral genotype devoid of known (Wsp, Aws, and Mws) regulatory modules to see whether the types derived from this genotype could converge upon the WS phenotype via a novel route. Such types-with equivalent fitness effects-did emerge, although they took significantly longer to do so. Together our data provide an explanation for why WS evolution follows a limited number of mutational pathways and show how genetic architecture can bias the molecular variation presented to selection.

Our results suggest that NE plays an important role in the in vivo pathogenesis and may serve as a therapeutic target for inflammation, mucus hypersecretion, and structural lung damage and indicate that additional rehydration strategies may be required for effective treatment of airway mucus obstruction in CF.