Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR

Abstract

Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM--its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels--suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature.

Figure 1. PCP induction of PA3720-armR.

qRT-PCR results showing impact of PCP and/or a nalC mutation on PA3720 expression (as a measure of PA3720-armR expression). Expression of PA3720 is reported relative to the rpsL internal control for wild type P. aeruginosa K767 and its nalC derivative, K1454 exposed or not to PCP (0.75 mM; 1.5 hr). Results shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.

Figure 2. PCP modulation of NalC repressor binding to the PA3720-armR upstream region.

A) Mobility shift assay in which 50 ng of a 209-bp DNA fragment encompassing the nalC-PA3720 intergenic region was incubated without (lane 1) or with 200 (lane 2), 300 (lane 3), 400 (lane 4), 500 (lane 5), 800 (lane 6), 1000 (lane 7), 2000 (lane 8) or 3000 (lane 9) ng of purified NalC-His. B) Mobility shift assay in which purified NalC-His (800 ng) was incubated with 50 ng of a 209-bp DNA fragment encompassing the nalC-PA3720 intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.

Figure 3. Identifying the NalC binding site upstream of PA3720-armR.

A–B) Mobility shift assay in which 50 ng of A) the 107-bp PA3720-distal DNA fragment (sequence delineated by endpoints I in Fig. 4A) or B) the 102-bp PA3720-proximal DNA fragment (sequence delineated by endpoints II in Fig. 4A) were incubated without NalC (lane 1) or with 300 (lane 2), 500 (lane 3), 800 (lane 4) or 1000 (lane 5) ng of purified NalC-His. C–E) Mobility shift assay in which 0 (lane 1), 300 (lane 2), 500 (lane 3), 800 (lane 4) and 1000 (lanes 5) and 2000 (lanes 6) ng of purified NalC-his was incubated with 40 ng of C) the annealed oligonucleotide NalC-1 and its reverse complement (corresponds to sequence delineated by endpoints III in Fig. 4A), D) the annealed oligonucleotide NalC-2 and its reverse complement (corresponds to sequence delineated by endpoints IV in Fig. 4A), and E) annealed oligonucleotide NalC-3 and its reverse complement, in which the AGAACTGT sequence of NalC-1 corresponding to the first half of the inverted repeat highlighted in Fig. 4(A) (shaded sequence overlapping the putative −10 regions for nalC and PA3720-armR) is changed to TCTTGACA.

Figure 4. Mapping the PA3720-armR transcriptional start site.

The nalC/PA3720-armR intergenic region highlighting the RACE-determined transcription start site for PA3720-armR (bolded and italicized), the predicted transcriptional initiation site (marked with an asterisk; assessed using neural network promoter prediction software provided by M. G. Reese at http://www.fruitfly.org/seq_tools/promoter.html), the nalC and PA3720 start codons (arrows), and putative −10/−35 sites for nalC (overlined) and PA3720-armR (underlined) promoters. The end-points of PCR-generated PA3720-distal (I) and -proximal (II) fragments and oligonucleotides (III and IV) used in EMSAs with purified NalC (see Fig. 2B) are identified by arrowheads above the sequence. The shaded sequence corresponds to an inverted repeat and possible NalC-binding site.

Figure 5. NalC and MexR dependence of PCP induction of mexAB-oprM.

Expression of mexA (as a measure of mexAB-oprM) was assessed in P. aeruginosa strains K767 (wild type; WT), K1454 (nalC), K2276 (nalC ΔarmR) and K2568 (ΔmexR) exposed or not to PCP (0.75 mM; 1.5 hr) using qRT-PCR. Expression was normalized to rpsL controls and is reported relative (fold change) to untreated P. aeruginosa K767. Results shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.

Figure 6. ArmR-/PA3720-independence of PCP induction of mexAB-oprM.

Expression of mexA (as a measure of mexAB-oprM) was assessed in P. aeruginosa strains K767 (wild type; WT), K3145 (ΔarmR), K3146 (ΔPA3720) and K3130 (ΔPA3720-armR) exposed or not to PCP (0.75 mM; 1.5 hr) using qRT-PCR. Expression was normalized to rpsL controls and is reported relative (fold change) to untreated P. aeruginosa K767. Results shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.

Figure 7. Lack of impact of PCP on MexR repressor binding to the mexR-mexA intergenic region.

Mobility shift assay in which purified MexR-His (500 ng) was incubated with 40 ng of a 351-bp DNA fragment encompassing the mexR-mexA intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.