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 phenotype-generating 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.

BACKGROUND:Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.RESULTS:Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.CONCLUSIONS:P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

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.

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 majority of phenotypic and ecological diversity on the planet has arisen during successive adaptive radiations, that is, periods in which a single lineage diverges rapidly to generate multiple niche-specialist types. Microbiologists tend not to think of bacteria as undergoing adaptive radiation, but there is no reason to exclude them from this general statement – in fact, rapid generation times and large population sizes suggest that bacteria may be particularly prone to bouts of rapid ecological diversification. Indeed, there is evidence from both experimental bacterial populations (Korona et al., 1994; Rainey & Travisano, 1998) and natural populations (Stahl et al., 2002). This being so, insight into the evolutionary emergence of diversity requires an understanding of the causes of adaptive radiation.

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.

ABSTRACT Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla + but Mot − phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5′ region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.

Pseudomonas fluorescens SBW25 is a gram-negative, fluorescent pseudomonad that is able to colonise the roots and leaves of a wide range of plants. PfSBW25 has been widely adopted as a model organism for investigations into the molecular basis of plant colonisation, plant growth promotion and biocontrol, and is also used as a model organism in studies of biofilm formation and experimental evolution. The 6.7 Mb genome of PfSBW25 is currently being sequenced at the Sanger Institute. We have developed two in vivo expression technology (IVET) systems for P. fluorescens SBW25, and have used IVET as a functional genomics approach to identify and characterise more than a hundred plant-induced genes from P. fluorescens SBW25. The predicted functions of many of these genes can be assigned to broad functional categories such as nutrient acquisition, surface colonisation, stress tolerance, antibiotic biosynthesis, protein secretion and transcriptional regulation. We have begun the process of analysing the function and expression of some of these plant-induced genes in depth, focusing in particular on genes encoding a rhizosphere-induced type III protein secretion system (TTSS) that is similar to the Hrp TTSS of P. syringae. Functional genomic analyses of P. fluorescens SBW25 have provided insight into the biology and ecology of plant-colonising Pseudomonas, and into the utility and limitations of functional genomic techniques in understanding the biology of bacteria in complex environments.

The 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.

Pseudomonas fluorescens SBW25 is a gram-negative, fluorescent pseudomonad that is able to colonise the roots and leaves of a wide range of plants. PfSBW25 has been widely adopted as a model organism for investigations into the molecular basis of plant colonisation, plant growth promotion and biocontrol, and is also used as a model organism in studies of biofilm formation and experimental evolution. The 6.7 Mb genome of PfSBW25 is currently being sequenced at the Sanger Institute. We have developed two in vivo expression technology (IVET) systems for P. fluorescens SBW25, and have used IVET as a functional genomics approach to identify and characterise more than a hundred plant-induced genes from P. fluorescens SBW25. The predicted functions of many of these genes can be assigned to broad functional categories such as nutrient acquisition, surface colonisation, stress tolerance, antibiotic biosynthesis, protein secretion and transcriptional regulation. We have begun the process of analysing the function and expression of some of these plant-induced genes in depth, focusing in particular on genes encoding a rhizosphere-induced type III protein secretion system (TTSS) that is similar to the Hrp TTSS of P. syringae. Functional genomic analyses of P. fluorescens SBW25 have provided insight into the biology and ecology of plant-colonising Pseudomonas, and into the utility and limitations of functional genomic techniques in understanding the biology of bacteria in complex environments.

ABSTRACT 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,phe- notypic 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 con- nect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both struc- tured 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. T HE diversity of life poses a myriad,of problems,for