Cif is negatively regulated by the TetR family repressor CifR

Abstract

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

FIG. 1.

Cif degrades the epoxide EBH. The degradation of epoxides by EHs creates highly reactive vicinal diols. The colorimetric detection of these diols is outlined in panel A. Generation of the vicinal diol of EBH by Cif can be detected spectrophotometrically through the oxidation of 3-bromo-1,2-propanediol by periodate and subsequent analysis of the redox state of epinephrine added to the reaction. (B) Purified Cif (50 μg) was incubated with 10 mM EBH, and absorbance at 490 nm was monitored. Also shown is a heat-inactivated protein (heat kill) control. Data were normalized to samples containing buffer alone. *, P value of <0.05. Values are presented as specific activity (EBH degraded/min/mg Cif). (C) Dose response of EBH degradation by Cif. Bar 1 shows 10 mM EBH and 50 μg Cif; bar 2, EBH and 25 μg Cif; bar 3, EBH and 12.5 μg Cif; bar 4, EBH and 6.25 μg Cif; and bar 6, EBH and 3 μg Cif. Samples were incubated at 37°C for 15 min. Values are presented as the OD₄₉₀, with the OD₄₉₀ measurements of each condition subtracted from the control condition containing EBH alone.

FIG. 2.

EBH induction of cif gene and Cif protein expression. Wild-type (WT) P. aeruginosa strain PA14 was grown in LB supplemented with increasing concentrations of the epoxide EBH. Expression of the cif gene and Cif protein in response to EBH was assayed using either sqRT-PCR (A) or Western blotting (B), respectively. The expression of rplU, a gene previously shown to be constitutively expressed under most laboratory conditions, served as a control (panel A). Samples for Western blotting were normalized to total protein content (panel B). The graphical interpretation of the Western blot data shown in panel B is the relative Cif expression normalized to the total protein electrophoresed.

FIG. 3.

cif is cotranscribed with morB and PA2933. (A) The cif gene is predicted to belong to a three-gene operon including morB, PA2933, and cif. (B) RNA from the wild type (WT) and the Δcif and ΔPA2931 mutants was assayed using Northern blotting to determine the transcript size detected by the cif-specific probe. A single band at ∼4.2 kb was detected in the ΔPA2931 mutant.

FIG. 4.

cifR negatively regulates cif gene expression. cif gene and Cif protein expression were assayed using either sqRT-PCR or Western blotting, respectively. (A) The wild type (WT), the Δcif mutant, and the ΔPA2931 mutant harboring the empty vector pMQ71 or expressing the PA2931 gene from the plasmid pDPM79 were assayed for cif and rplU gene expression. Deletion of the PA2931 gene resulted in higher levels of cif gene transcript, which was complemented by expression of the histidine-tagged variant of PA2931. (B) Western blot of the WT P. aeruginosa strain PA14, the ΔPA2931 mutant containing the empty vector pMQ71, and the ΔPA2931 mutant carrying a plasmid expressing a histidine-tagged variant of PA2931 from the arabinose-inducible expression plasmid pDPM79. Arabinose was added to a final concentration of 0.2% where indicated.

FIG. 5.

CifR binds to the intergenic region directly upstream of the cif-containing operon. (A) The intergenic region between the cifR and morB genes was amplified using biotinylated primers and used as a probe in EMSAs. (B) EMSA using the morB-cifR intergenic region as a probe and purified CifR. Lane 1, labeled probe; lane 2, probe and 1.15 nM CifR; lane 3, probe, 1.15 nM CifR, and a 20× molar excess of the unlabeled cold competitor; and lane 4, probe, 1.15 nM CifR, and 1 mM EBH. BT, biotinylated. (C) EMSA CifR dose response. Lane 1, probe alone; lane 2, probe and 115 pM CifR; lane 3, probe and 287.5 pM CifR; lane 4, probe and 575 pM CifR; lane 5, probe and 862.5 pM CifR; and lane 6, probe and 1.15 nM CifR. (D) EMSA EBH dose response. Lane 1, probe alone; lane 2, probe and 1.15 nM CifR; lane 3, probe, 1.15 nM CifR, and 10 μM EBH; lane 4, probe, 1.15 nM CifR, and 100 μM EBH; and lane 5, probe, 1.15 nM CifR, and 1 mM EBH.

FIG. 6.

Clinical isolates overexpressing the cif gene demonstrate decreased cifR gene expression. (A) sqRT-PCR of wild-type P. aeruginosa PA14 and four nonmucoid P. aeruginosa CF sputum isolates. Expression of the cif, cifR, and rplU genes was assayed. (B) sqRT-PCR of wild-type (WT) P. aeruginosa, the Δcif and ΔcifR mutants, and two of the clinical isolates from panel A carrying either the empty vector pMQ71 (−) or the cifR-expressing plasmid pDPM79 (+).

FIG. 7.

Model for cif gene expression. CifR represses cif gene expression by binding to the promoter region immediately adjacent to the cif-containing operon. The binding of EBH by CifR results in disassociation of the repressor from the promoter, resulting in increased cif gene expression and biosynthesis of the EH Cif, which in turn degrades EBH.