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.

2′-O-methylation of guanosine 18 is a naturally occurring tRNA modification that can suppress immune TLR7 responses.

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Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 10 6 mutants were screened for each of 27 strains carrying dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.functional genomics ͉ gene expression ͉ gene regulation ͉ phytosphere ͉ Pseudomonas fluorescens T he function of bacteria in natural environments is poorly understood. If bacteria were the size of birds and possessed similar morphological complexity, then progress could be made by observation alone. In the absence of readily observable phenotypes, microbiologists have taken to detecting changes in patterns of gene expression. Genes expressed in one environment, but not in another, are likely to encode traits relevant to the former environment but not the latter. Understanding the biological significance of these traits, their contribution to ecological performance, and the regulatory networks that control their expression is central to understanding the function of bacteria in the wild.Pseudomonas fluorescens SBW25 is a rhizosphere colonizing bacterium whose survival at the plant-soil interface depends on ability to integrate a multiplicity of environmental stimuli. Insight into the causes of the bacterium's ecological success have come from application of promoter-trapping strategies, such as IVET (in vivo expression technology) (1), which have led to the identification of genes displaying elevated levels of expression in the plant environment (2-9). Although the biological function and ecological significance of a number of these genes has been determined, significant progress has been hampered because (by definition) the genes of interest express no phenotype in vitro. Progress the...