Fig 1.
Evolutionary predictions and outcomes of experimental evolution for Pseudomonas protegens Pf-5.
(A) Growth under static conditions when oxygen is limiting to growth is expected to result in selection for colonization of the air-liquid interface by increased cell-cell adhesion and surface attachment. Pictured is a wrinkly spreader mutant of Pseudomonas fluorescens SBW25. (B) The cell wall of Pf-5 has several components that could possibly be used to promote cell-cell adhesion and surface attachment, including lipopolysaccharides (LPS), capsular polysaccharides (CPSs), exopolysaccharides (EPSs), adhesive proteins, and incomplete cleavage of the peptidoglycan (PG) layer. (C) The types of adaptive mutations expected are, in decreasing frequency, loss-of-function mutations, promoter mutations, intragenic activating mutations, and double inactivating mutations [28]. (D) Mutational activation of di-guanylate cyclases (DGCs–outlined in black; WspR, AwsR, and MwsR) by loss-of-function mutations in negative regulators (outlined in red; WspF, AwsX, and MwsR) will lead to increased c-di-GMP production, resulting in overexpression of an EPS and reduced motility. In SBW25, these occur in three main pathways, Wsp, Aws, and Mws. (E) Predicted fraction of WS mutations for genes in the main three pathways. Knowledge of the molecular networks allowed for the formulation of a mathematical model that accurately predicted the relative rates of use of the common three pathways (Wsp, Aws, and Mws) [26] and rates for proteins in each pathway. The assumption that the molecular functions of these networks are conserved in Pf-5 allows prediction of the rates of pathways and proteins.
Fig 2.
Predicted mutational targets and proposed molecular effects.
Black represents any inactivating mutation including frame shifts, blue represents in frame inactivating mutations, green represents amino acid substitutions. Numbers refer to amino acid residue numbers in Pf-5. (A) WspA–amino acid substitutions are expected at the tip of the stalk and in-frame deletion of methylation sites. (B) WspF–any inactivating mutation is predicted, amino acid substitutions are predicted only in areas where they disrupt intermolecular interactions. (C) WspE–amino acid substitutions are predicted near the phosphorylation site. (D) WspR–small in frame deletion and amino acid substitutions in the linker is predicted to cause constitutive activation. (E) AwsO–amino acid substitutions disrupting AwsO dimerization is predicted to lead to increased binding to AwsX without the presence of an activating signal. (F) AwsX–any inactivating mutation that keep the reading frame intact and do not interfere with expression of downstream AwsR is predicted. (G) AwsR–amino acid substitutions in the periplasmic region or transmembrane helix that disrupt the interaction with AwsX or to the HAMP linker is predicted. (H) MwsR–mutations are predicted in the interface between the DGC and phosphodiesterase domains and in the most C-terminal of the PAS domains resulting in constitutive activation.
Fig 3.
(A) Forty-three independent mutants of wild type Pseudomonas protegens Pf-5 were isolated after experimental evolution based on their divergent colony morphology and mutations were identified in four operons. Numbers in brackets are the number of independent mutants found. Details are available in S2 Table. (B) Experimental evolution with a Δwsp Δaws Δmws triple deletion mutant resulted in WS types with mutations in the DGC PFL_0087/DgcH.
Fig 4.
Phenotypic characterization of reconstructed mutants.
(A) Motility, colony morphology and air-liquid interface colonization of reconstructed representative mutations. (B) As expected, motility was significantly reduced for all mutants if the c-di-GMP network was activated but was only slightly reduced for the AwsXRO promoter capture. Additionally, motility of the PFL_3078 promoter mutant was not significantly reduced, but this mutant was not expected to have increased c-di-GMP levels (one-way ANOVA, F(12,39) = 63.6, p < 0.0001, pairwise differences assessed with student t-tests with α = 0.05). Four replicates on two separate plates per strain were used.
Fig 5.
Fitness of reconstructed P. protegens Pf-5 WS mutants was measured in pairwise competitions.
(A) Invasion fitness was measured relative a dominant ancestral wild type strain with a 1:100 initial ratio. All mutations were adaptive and can increase from rare to colonize the air-liquid interface. Six independent competitions were performed for each pair. (B) Competition fitness was measured relative the most common WS mutant (WspF V271G) in a 1:1 initial ratio to compare the fitness of different WS mutants and the alternative phenotypic solutions. Four independent competitions were performed for each pair.
Fig 6.
Contribution of pel to WS phenotype and fitness.
(A). Deletion of pel in WS mutants reduces invasion fitness (mean values of intact pel mutants plotted as red triangle in all plots). Fitness of reconstructed P. protegens Pf-5 WS mutants without the pel operon was measured in pairwise competitions. Invasion fitness was measured relative a dominant ancestral wild type strain with a 1:100 initial ratio. Six independent competitions were performed for each pair. (B) Deletion of pel in WS mutants reduces competition fitness. Competition fitness was measured relative the most common WS mutant (WspF V271G) in a 1:1 initial ratio. Four independent competitions were performed for each pair. (C) Deletion of pel in WS mutants did not result in ancestral smooth colony morphology or loss of ability to colonize the air-liquid interface suggesting a secondary EPS component is produced. (D). Deletion of pel did not restore motility showing that Pel overproduction is not the cause of the motility defect in WS mutants. Four replicates on two separate plates per strain were used.
Fig 7.
Diversity of DGCs and biofilm-related genes for seven Pseudomonas species.
(A) Five other Pseudomonas species (P. putida KT2440, P. syringae pv. tomato DC3000, P. savastanoi pv. phaseolicola 1448A, P. aeruginosa PAO1, P. stutzeri ATCC 17588) were chosen based on phylogenetic diversity to extend predictions. Including P. fluorescens SBW25 and P. protegens Pf-5, the seven species encode 251 putative DGCs, divided into 87 different homolog groups of which 8 are present in all genomes. WS mutations in SBW25 have been found affecting 13 of these DGCs (marked with *) with an additional nine that have been detected only in combinations with other mutations. SBW25 and Pf-5 share 33 DGCs with 6 unique for each species. It should be noted that not all DGCs are likely to be catalytically active. (B) Diversity of biofilm-related genes including putative EPSs, LPS modification, cell chaining, adhesins and known regulators. The genomes of the P. aeruginosa strains PA7, UCBPP-PA14 were also included in the analysis and results were in most cases identical to PAO1 (not shown in Fig 7) except for an absence of homologues for EPS genes pelA-D and the DGCs PA2771 in UCBPP-PA14 and PA3343 in PA7. Detailed information is available in S4 Table.
Table 1.
Predictions for other Pseudomonas species.