Transcriptional Regulation of Carnitine Catabolism in Pseudomonas aeruginosa by CdhR

Mapping the caiX promoter and its CdhR binding site. (A) Transcriptional fusions of lacZ to the upstream region of caiX start at bp +3 from the caiX transcriptional start site and end at base pairs marked in the figure. P. aeruginosa PA14 strains carrying each construct were grown in MOPS with 20 mM pyruvate and 20 µg·ml⁻¹ gentamicin, with (black bars) or without (white bars) 1 mM carnitine. β-Galactosidase activities for these caiX transcriptional fusions are reported in Miller units. Error bars represent standard deviation from three biological replicates, and results are representative of three independent experiments. (B) EMSA with biotin-labeled caiX promoter DNA probe alone (lane 1) or with increasing concentrations of purified MBP-CdhR (lanes 2 and 3). An unlabeled (cold) caiX probe was used to compete for binding of MBP-CdhR from the labeled probe (lane 4). An unrelated dhc DNA probe was used to show specificity of MBP-CdhR binding to caiX (lanes 5 to 8).

The CdhR binding site and key residues for CdhR-dependent induction. (A) A DNase I footprinting assay was performed by taking the caiX UAS end labeled with ³²P and adding increasing concentrations of MBP-CdhR, followed by DNase I treatment and nondenaturing 5% polyacrylamide TBE gel. The first lane of the gel is the A+G sequencing ladder, and the nanomolar concentration of MBP-CdhR is marked. (B) The caiX enhancer site was mutated by changing two bases at a time (in red and underlined) in the caiX distal binding site to adenosines and fused to lacZ. The P. aeruginosa PA14 wild type carrying each of the plasmids was grown in MOPS with 20 mM pyruvate at 20 µg·ml⁻¹, with or without 1 mM carnitine for 4 h, and then β-galactosidase activity was reported as Miller units. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed using a two-way analysis of variance (ANOVA) with a Sidak’s multiple-comparison posttest comparing each mutant’s pyruvate to carnitine. Abbreviations: P, pyruvate; C, carnitine; n.s., not significant; *, P < 0.05; ***, P < 0.001.

caiX transcription is repressed by glucose and glycine betaine. P. aeruginosa PA14 with P_caiX-lacZ (pJAM22) was grown in MOPS medium with 20 mM pyruvate and 20 µg·ml⁻¹ gentamicin, with or without 1 mM carnitine. Glucose or glycine betaine was added at the millimolar concentrations noted. Cultures were induced for 4 h prior to measurement of β-galactosidase activity. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed using a one-way ANOVA with a Dunnett’s multiple-comparison posttest comparing each condition to the condition with carnitine alone. Bars denote that all data underneath are different from carnitine alone with the same statistical certainty. Abbreviations: Glu, glucose; GB, glycine betaine; ***, P < 0.001; ****, P < 0.0001.

CdhR binds but does not regulate cbcXWV expression. (A) EMSA with a biotin-labeled (biot-) cbcX upstream region and purified MBP-CdhR in increasing concentrations. (B) Relative expression of cbcX was calculated based on the expression in WT pyruvate normalized to the rplU transcript using qRT-PCR. Three biological samples were run in triplicate, and the graph represents the mean values and standard deviation. Data were analyzed using a two-way ANOVA with a Tukey’s multiple comparison posttest comparing all strains and conditions to each other. Ends of the bars denote the comparison groups shown. Abbreviations: Pyr, pyruvate; Carn, carnitine; n.s., not significant; ***, P < 0.001.

GbdR binds the caiX-cdhR intergenic region but does not induce transcription of caiX. (A) EMSAs were performed with increasing concentrations of purified MBP-GbdR with either the biotin-labeled cdhR wild-type probe or a mutant binding site probe. The mutated probe has the distal half-site CG residues (in relation to cdhR) changed to AA. (B) β-Galactosidase assay with a caiX-lacZ reporter plasmid (pJAM22) in wild-type, ΔcdhR, ΔgbdR, or ΔcdhR ΔgbdR strains of both PA14 (14) and PAO1 (1) grown in MOPS, 20 mM pyruvate, and 20 µg·ml⁻¹ gentamicin. Induced cultures have an additional 1 mM carnitine. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed with two-way ANOVA with a Tukey’s multiple-comparison test comparing all mutants and conditions within a given strain. (PA14 was not compared to PAO1.) Abbreviations: ***, P < 0.001 compared to WT with pyruvate; #, P < 0.01 compared to WT with carnitine.

CdhR promotes cdhR expression, and GbdR dampens basal repression. (A) WT, ΔcdhR ΔgbdR, and ΔcdhR ΔgbdR strains in both PA14 (14) and PAO1 (1) backgrounds carrying a cdhR-lacZ translational plasmid reporter (pJAM135) were grown in MOPS with 20 mM pyruvate and 20 µg·ml⁻¹ gentamicin, with or without 1 mM carnitine, and β-galactosidase activity was reported as fold change over WT pyruvate. Data were analyzed using a two-way ANOVA with a Sidak’s multiple-comparison posttest comparing to the WT pyruvate condition within each strain. (PA14 was not compared to PAO1.) *, P < 0.05. (B) PAO1 WT, ΔcdhR, and ΔgbdR strains, all with the translational fusion cdhR-yfp integrated at the attTn7 site, were grown on MOPS agar pads with 20 mM pyruvate and with or without 1 mM carnitine. Cells were imaged under phase-contrast and YFP fluorescence every 10 min at 32°C. Data were analyzed using a one-way ANOVA with a Dunnett’s multiple-comparison posttest comparing each time point within a strain to pyruvate at time zero (t = 0). ***, P < 0.001.