PcsR2 Is a LuxR-Type Regulator That Is Upregulated on Wheat Roots and Is Unique to Pseudomonas chlororaphis

Abstract

LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30-84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated ∼10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.

Keywords: LuxR solos; Pseudomonas; interkingdom signaling; phenazine; plant-microbe interactions.

FIGURE 1

Amino acid sequence alignments of PcsR2 and other representative LuxR-type transcriptional regulators. Sequence alignments and amino acid identity comparisons were performed using Clustal-omega, and Boxshade was used to highlight the degree of amino acid identity (90–100%). The blue and black lines above the sequences indicate the signal-binding and helix-turn-helix domains of QS LuxRs, respectively. Red asterisks indicate the positions of nine conserved amino acids in QS LuxRs. The two residue positions boxed in red are the most common residues that differ between QS LuxRs (such as LuxR and TraR) and the LuxR solo homologs from plant-associated species (such as PipR, PsoR, NesR, XccR, and OxyR). The PcsR2 sequence also varies in these two positions (L₅₇, I₆₁ instead of W₅₇, Y₆₁ in canonical LuxRs; positions numbered as in TraR). Included in this table with PcsR2 are QS LuxR homologs: LuxR in Vibrio fischeri and TraR in Agrobacterium tumefaciens, and LuxR solos: LesR in Lysobacter enzymogenes; SmoR in Stenotrophomonas maltophilia; PipR in Pseudomonas sp. strain GM79; PsoR in P. fluorescens and P. protegens; NesR in Sinorhizobium meliloti; XccR in Xanthomonas campestris pv. campestris; OryR in X. oryzae pv. oryzea.

FIGURE 2

Expression of pcsR2 and the downstream operon were upregulated on wheat roots. (A) Growth curves of strains grown in AB + CAA at 28°C, for 48 h, with agitation. Populations were enumerated via serial dilution and colony counts (CFU). (B–D) Expression of pcsR2 and two genes in operon 2 (Pchl3084_4801, Pchl3084_4803) in 30–84WT, 30–84ΔpcsR2, or 30–84ΔpcsR2(pGT2PcsR2) when strains were grown on wheat roots as compared to within planktonic culture (AB + CAA) for 16 h. Expression levels were measured via qRT-PCR (where relative gene expression is expressed as ΔΔCt, with rpoD as internal control) and data are the mean and standard error of nine replicates. Gene expression levels were compared by strain using a Student’s t-test. Asterisks indicate whether treatments are significantly different (P < 0.05).

FIGURE 3

PcsR2 is involved in plant-microbe interactions. (A,B) Expression of the transcriptional pcsR2 GFP reporter (pGT2PpcsR2:gfp) in 30–84WT and 30–84ΔpcsR2 when incubated in the presence of different concentrations of root or leaf macerate. 30–84WT and 30–84ΔpcsR2 carrying the reporter plasmid were incubated in 96 well plates for 12 h in AB medium with glucose (AB + G) without or with varying concentrations (1–20%) of root macerate (RM) (A) or Leaf macerate (LM) (B). GFP fluorescence is expressed as relative fluorescence units (RFU) (fluorescence units standardized to population density). (C,D) Growth 30–84WT, 30–84ΔpcsR2, and 30–84ΔpcsR2(pGT2PcsR2) in AB medium without a carbon (C) or nitrogen (D) source and with 80% root macerate (AB-C + M or AB-N + M) or without it (AB-C or AB-N). Population density was measured spectrophotometrically (OD₆₂₀) at 24 h of growth. NI means no insert control plasmid. Data are the means and standard errors of three replicates. Letters indicate whether treatments are significantly different (P < 0.05, ANOVA and Tukey HSD).

FIGURE 4

Phenazine production and growth of 30–84WT(NI), 30–84ΔpcsR2(NI), and 30–84ΔpcsR2(pGT2PcsR2) and 30–84WT(pGT2PcsR2) in different media. Strains were grown in (A,D) AB + CAA, (B,E) LB, and (C,F) PPMD at 28°C, for 48 h, with agitation. (A–C) Phenazines were quantified spectrophotometrically (OD₃₆₇) and (D–F) growth curves were calculated spectrophotometrically (OD₆₂₀). NI means no insert control plasmid. Data are the means and standard errors of at least three replicates; some error bars do not exceed the size of symbol. Letters indicate whether treatments are significantly different (P < 0.05, ANOVA and Tukey HSD).

FIGURE 5

Biofilm traits of 30–84WT(NI), 30–84ΔpcsR2(NI), and 30–84ΔpcsR2(pGT2PcsR2). (A) Spectrophotometric quantification of surface-attached biofilms by the crystal violet staining method (at OD₅₄₀) 24 and 48 h. (B) Quantification of extracellular (eDNA) when strains were grown in static culture after 24 and 48 h. (C) Image of biofilm matrix and mass of cells and hydrated matrix when strains grown in static culture measured by weight after 48 and 72 h. All figures use the same labeling for each strain. NI means no insert control plasmid. For all experiments, bacteria were grown in AB + CAA. Data are the means and standard errors of at least three replicates. Letters indicate whether treatments are significantly different (P < 0.05, ANOVA and Tukey HSD).

FIGURE 6

Gene expression of phenazine-regulators and in 30–84WT(NI), 30–84ΔpcsR2(NI), and 30–84ΔpcsR2(pGT2PcsR2) and phenazine production by 30–84WT, and 30–84ΔpcsR2 with and without additional copies of some phenazine regulatory proteins in trans. (A) Relative gene expression of some phenazine regulatory genes measured by qRT-qPCR (ΔΔCt method with rpoD as internal control) after 24 h in AB + CAA. Relative fold change of these genes is compared to 30–84WT expression levels (which are set to 1). (B–E) Phenazine production of strains with and without additional copies of phzR, pip, rpeB, or rpoS, via expression the following plasmids in trans: pGT2Ptac:phzR, pUCPip, pUCRpeB, pUCRpoS, respectively. NI means no insert control plasmid (pGT2 or pUCP20). Images above graphs are of cultures after 48 h growth in AB + CAA when phenazines were extracted and quantified spectrophotometrically (OD₃₆₇). Data are the means and standard errors of five replicates. Letters indicate whether treatments are significantly different (P < 0.05, ANOVA and Tukey HSD).