PpoR binds 3-oxo-C6-HSL. E. coli M15 (pRep4) containing either pQEPpoR or pQE30 were grown in LB in the presence of various AHLs (1 μM) added separately and protein expression induced with IPTG (1 μM). After 3.5 hours of growth post induction, AHLs were extracted from the cell pellets and visualized by TLC overlaid with A. tumefaciens NTL4 (pZLR4). The standards used are synthetic AHLs. In the figure, the lanes are marked as follows; S – AHL standards, 1 – AHL extracted from E. coli/pQE30 cell pellets grown with 3-oxo-C6-HSL supplementation and 2 – AHL extracted from E. coli/pQEPpoR cell pellets grown with 3-oxo-C6-HSL supplementation.

Root colonization assay of P. putida WCS358 wild type and mutants. Colonization assays were performed as described previously (Steindler et al. 2008). The data presented are from one experiment. Anova analysis in combination with Dunnett's multiple comparison test revealed a significant difference between the mean values of wild type & IBE5 as well as between wild type & WCS358PPOR at P < 0.01 significance level [F(4,45) = 2.870].

3-oxo-C6-HSL measurement produced by P. putida WCS358 and by the ppoR mutant WCS358PPOR. AHLs were extracted from spent supernatants and levels were measured using a biosensor as described by Steindler et al., [15]. AHL levels were measured using a volume of extract corresponding to an amount of 5 × 10⁸ cfu as described in the Methods section. 3-oxo-C6-HSL level is proportional to β-galactosidase activity (Miller Units); β-gal refers to β-galactosidase; OC6 refers to 0.05 μM of synthetic 3-oxo-C6-HSL used for the experiment and EA refers to ethyl acetate added as control in the experiment. β-galactosidase activity (Miller Units) represented as bars were obtained from one such experiment; similar values were obtained from additional experiments carried out with AHLs extracted independently from P. putida WCS358 and WCS358PPOR strains.

ppoR promoter activities in wild type and various mutant strains of P. putida WCS358 and RD8MR3. Bacterial cultures were started with an initial inoculum of 5 × 10⁶ CFU per ml in 20 ml of minimal medium (M9-Cas) and β-galactosidase activities were measured at different stages of growth. The growth curves of different mutants and the wild type strain are indicated in each graph. All experiments were performed in triplicate and the mean values of each time point along with standard deviations are shown in each graph. All the graphs were plotted using SigmaPlot version10. (a) ppoR promoter activity in P. putida WCS358 wild type, gacA (IBE1), psrA (M17), rpoS (MKO1) and ppoR (WCS358PPOR) using plasmid pPpoR2. (b) ppoR promoter activity in P. putida RD8MR3 wild type and ppoR (RD8MR3PPOR) mutants using plasmid pPpoR1. β-gal, β-galactosidase; OD600, optical density at 600 nm; MU, Miller Units.

Alignment showing similarity of deduced sequence of PpoR to its orthologs. Multiple sequence alignment was performed using the ClustalW2 program (Thompson et al. 1994). The protein sequences used for the alignment are as follows; P. putida KT2440 (AAN70220.1), P. putida F1 (ABQ80629.1), P. putida RD8MR3 (this study; accession number FM992078), P. putida GB-1 (ABZ00528.1), P. putida WCS358 (this study; accession number FM992077) and P. putida W619 (ACA71296.1). The amino acids that are conserved in QS LuxR family proteins are indicated in bold [3]. In the alignment, all identical amino acids (*), similar amino acids (:) and completely different amino acids (.) at a particular position are indicated. Also indicated are the regions of the protein sequence of PpoR of P. putida KT2440 that constitutes the AHL binding domain (bold line from 17 to 162 amino acids; PFAM 03472) and the DNA binding domain (dashed line from 176 to 213 amino acids; PFAM 00196).

RT-PCR analysis to validate expression of genes in P. putida WCS358. Total RNA isolations were carried out from bacterial cultures grown in minimal M9 medium using Ribopure RNA isolation kit (Ambion) and DNase treatment was carried out. cDNA synthesis was done using AMV Reverse Transcriptase (Promega) and second strand synthesis performed using Go Taq Flexi polymerase (Promega). RT-PCR analysis was performed with RNA obtained from two independent isolations and the figure shows results of one such experiment. (a) Agarose gel showing RT-PCR products for the genes PP_0170, PP_0233 and PP_0235. RT_PCR for 16S rRNA was carried out from the same RNA samples as control to ensure that equal amounts of RNA were taken. A. RT-PCR on RNA sample from P. putida WCS358 containing pBBR vector alone and B. RT-PCR on RNA sample from P. putida WCS358 containing pBBRPpoR. (b) Graph showing normalized fold difference of genes when compared to 16S rRNA expression levels. The gel image containing bands was analyzed by the ImageJ software and the bars indicate the fold increase in the intensity of the bands in PpoR++ strain (P. putida WCS358 containing pBBRPpoR) when compared to wild type (P. putida WCS358 containing pBBR vector alone).

β-Galactosidase assays showing expression profile of ppoR and the QS system genes of P. putida WCS358 and RD8MR3. Bacterial cultures were started with an initial inoculum of 5 × 10⁶ CFU per ml in 20 ml of minimal medium (M9-Cas) and β-Galactosidase activities were measured at different stages of growth. The growth curves of different mutants and the wild type strain are indicated in each graph. All experiments were performed in triplicate and the mean values of each time point along with standard deviations are shown in each graph. All the graphs were plotted using SigmaPlot version10.0. (a, b, and c) ppuI, ppuR and rsaL promoter activities of P. putida WCS358 in wild type and WCS358PPOR using plasmids pPUI220, pPUR220 and pRSA220. Paired t-test analysis of ppuI promoter activities revealed a significant difference between the mean values of wild type and WCS358PPOR at 7 hours of growth (p value 0.0184; t = 7.268 df = 2) at P < 0.05 significance level. (d) pprI promoter activity in P. putida RD8MR3 wild type and RD8MR3PPOR with the plasmid pMPpprIprom. (e) ppoR promoter activity in P. putida WCS358 wild type, ppuI knock-out (IBE5), ppuR (IBE2) and rsaL (IBE3) mutants with the plasmid pPpoR2. Anova analysis of sample means followed by Dunnett's multiple comparison test revealed that there is a significant difference between the means of wild type and IBE5 at P < 0.05 significance level at 4, 6 and 24 hours growth [F(3,8) = 6.278, F(3,8) = 22.97 and F(3,8) = 16.37 respectively] (f) ppoR promoter activity in P. putida RD8MR3 wild type, pprI (RD8MR3PPRI) and pprR (RD8MR3PPRR) mutants with the plasmid pPpoR1. β-gal, β-galactosidase; OD600, optical density at 600 nm; MU, Miller Units.