The ColR/S two-component system is a conserved determinant of host association across Pseudomonas species

Abstract

Members of the bacterial genus Pseudomonas form mutualistic, commensal, and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not host-specific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles. This suggests that conservation of two-component systems, coupled with life-style dependent diversification of the regulon, may play a role in host association and lifestyle transitions.

Fig. 1. ColR is a conserved host association factor across Pseudomonas spp. in plant and murine hosts.

A To test if colR is necessary for rhizosphere colonization, seedlings growing hydroponically in 48-well plates were inoculated with GFP-expressing P. fluorescens WCS365, WCS365 ∆colR, WCS365 ∆colR containing an empty vector (EV), a vector expressing WCS365 colR under its native promoter (WcolR), a vector expressing PAO1 colR under its native promoter (PcolR) or (B) P. aeruginosa PAO1, PAO1 ΔcolR, or PAO1 ΔcolR expressing colR under its native promoter (PcolR). A, B GFP fluorescence values in each plant-containing well were measured 5 days post-inoculation and converted to OD₆₀₀ using a standard curve. Each data point represents the estimated CFU values from a single well containing a single plant. Experiments were repeated three independent times with 10-30 plants per replicate (n = 30–90). Mean +/− standard error is shown and letters indicate significant (p < 0.05) differences as determined by a one-way ANOVA follow by a post-hoc Tukey HSD test. C Light microscopy of P. aeruginosa PAO1 expressing GFP (green) growing for two days on Arabidopsis roots shows that deletion of colR leads to visibly less PAO1 colonization of the root. D, E Wild-type P. aeruginosa LESB58 and a LESB58 ∆colR mutant were injected (~5 ×10⁷ CFU inoculum) into the subcutaneous thin skeletal muscle on the dorsum of mice. CFU counts (E) and abscess lesion size (E) were determined three days post infection. Means +/− standard error are shown; each dot represents the results from one animal. Different letters indicate significant differences at p < 0.05 determined using a one-way ANOVA followed by a Tukey’s HSD test, or using a t-test if only one comparison was made. F Representative images of abscess formation by wild-type LESB58 and the ∆colR mutant.

Fig. 2. ColR regulates a limited number of genes in the rhizosphere that are distinct between Pseudomonas strains.

A PCA plots of transcript count matrices of RNA-Seq analysis comparing gene expression in wild-type P. aeruginosa PAO1 and the PAO1 ΔcolR mutant (left) or P. fluorescens WCS365 and the WCS365 ΔcolR mutant (right). Heat maps and Venn diagrams showing the number of significantly differentially expressed genes (greater than 0.585-fold change, padj <0.01) between wild-type and the colR mutant in the rhizosphere or in minimal media in PAO1 (B) and WCS365 (C). Gene counts higher than row average are shown in yellow, and gene counts lower than row average are shown in blue. D A phylogenetic tree showing orthologous genes across Pseudomonas strains. Genes fell into 5 distinct clusters: Cluster 1. Genes with ColR-dependent expression in both WCS365 and PAO1; Cluster 2. Genes with ColR-dependent expression in WCS365 with ColR-independent orthologs in PAO1; Cluster 3. Genes with ColR-dependent expression in PAO1 with ColR-independent orthologs in WCS365; Cluster 4. Genes with ColR-dependent in WCS365 without orthologs in PAO1; Cluster 5. ColR-dependent expression in PAO1 without orthologs in WCS365. Genes tested for rhizosphere colonization (Fig. 3) are marked with an asterisk.

Fig. 3. Strain-specific ColR-dependent genes are required for both rhizosphere colonization and virulence in a mouse abscess model.

A Wild-type P. aeruginosa PAO1 or PAO1 mutants [∆colR, warA::Tn5 (PA4379), tpbA::Tn5 (PA3885); ∆eptA (PA1927), dgkA::Tn5 (PA3603), pap2::Tn5 (PA4382), or PA4518::Tn5] and B Wild-type P. fluorescens WCS365 or WCS365 mutants [∆colR, ∆warA (LRP86_00786), ∆tpbA (LRP86_02280), or ∆eptA (LRP86_03626)] containing GFP-expressing plasmids were inoculated into the Arabidopsis rhizosphere of hydroponically grown seedlings. Each data point represents the estimated cfu/mL for a single plant. Three independent experiments, each with a minimum of 10 plants, were performed (n > 30). Letters indicate significant differences at p < 0.05 using an ANOVA and Tukey’s HSD. Colors correspond to the gene clusters shown in Fig. 2D. C Genomic arrangement of orthologous genes in P. fluorescens WCS365 and P. aeruginosa PAO1. Orthologous genes are shown in the same color. Red arrows and striped, red boxes indicate putative ColR-binding sites and ColR-dependent transcription. Black arrows indicate ColR- independent transcription. D and E Wild-type P. aeruginosa LESB58 or LESB58 mutants [ΔdgkA (PALES_14321), ΔtpbA (PALES_10921), and Δpap2 (PALES_47610)] were injected into the subcutaneous thin skeletal muscle on the dorsum of mice. Abscess lesion size (D) and CFU counts (E) were determined three days post-infection. Error bars represent standard deviation. Letters indicate significant differences at p < 0.05 as determined by a Mann-Whitney U test; each dot represents the results from one animal.

Fig. 4. The P. aeruginosa PAO1 ∆colR mutant is sensitive to low pH and high iron.

Venn diagrams of the number of total genes in PAO1 (A) and WCS365 (B) that are significantly upregulated in wild-type or a colR mutant in the rhizosphere vs. M9 minimal media (greater than 0.585-fold change, padj < 0.01). C, D GO categories of significantly differentially expressed genes between the rhizosphere and M9 minimal media in wild-type PAO1 (C) and wild-type WCS365 (D). Significantly enriched GO terms were identified using GOfuncR. E–H Growth curves of wild-type PAO1 and PAO1 mutants (∆colR, tpbA::Tn5, dgkA::Tn5 or pap2::Tn5) in LB at pH 5 or 7 with and without the addition of 0.2 mM FeSO₄. Growth curves were performed at least three times with similar results. Single experimental replicates are shown with averages and standard deviations of 6 technical replicates per time point.