Quorum-sensing regulation of a copper toxicity system in Pseudomonas aeruginosa

Abstract

The LasR/LasI quorum-sensing system in Pseudomonas aeruginosa influences global gene expression and mediates pathogenesis. In this study, we show that the quorum-sensing system activates, via the transcriptional regulator PA4778, a copper resistance system composed of 11 genes. The quorum-sensing global regulator LasR was recently shown to directly activate transcription of PA4778, a cueR homolog and a MerR-type transcriptional regulator. Using molecular genetic methods and bioinformatics, we verify the interaction of LasR with the PA4778 promoter and further demonstrate the LasR binding site. We also identify a putative PA4778 binding motif and show that the protein directly binds to and activates five promoters controlling the expression of 11 genes--PA3519 to -15, PA3520, mexPQ-opmE, PA3574.1, and cueA, a virulence factor in a murine model. Using gene disruptions, we show that PA4778, along with 7 of 11 gene targets of PA4778, increases the sensitivity of P. aeruginosa to elevated copper concentrations. This work identifies a cellular function for PA4778 and four other previously unannotated genes (PA3515, PA3516, PA3517, and PA3518) and suggests a potential role for copper in the quorum response. We propose to name PA4778 cueR.

FIG. 1.

cueR expression changes in response to acyl-HSL signals. (A) Strain PAO1-MW1 was grown in the presence of 3OC₁₂-HSL, C₄-HSL, or both, and RNA was sampled in mid-log phase. Microarray expression profiles were then generated, and transcript expression levels were normalized to the acyl-HSL-free control cultures to give the fold changes. The experiment was done in both LB and modified FAB media. (B) Plasmid pJTT-PA4778 was integrated into the genome of strain PAO1 lasI rhlI, and cueR expression was assayed in the presence and absence of exogenous acyl-HSL signals.

FIG. 2.

Direct activation of promoters by LasR and RhlR in a heterologous host. E. coli DH5α received two plasmids, a reporter vector containing a promoter-lacZ transcriptional fusion and a regulator vector containing either lasR (pJTT201), rhlR (pJTT202), or a negative control (pMMB67). The acyl-homoserine lactone molecules 3OC₁₂-HSL and C₄-HSL were added as indicated. The rsaL promoter directly binds LasR and is shown here as a positive control. The upstream region of mvfR that was tested here has not been shown to bind LasR and was a negative control.

FIG. 3.

Electrophoretic mobility shift analysis of the cueR promoter. Purified LasR was added to 100 fmol of the labeled promoter DNA. Where indicated, nonlabeled specific or nonspecific competitor DNA was present in the reaction mixture. LasR had previously been shown to bind the rsaL-lasI bidirectional promoter region, and this interaction is verified here and shown for reference. The PA2588 region tested here has never been shown to bind LasR and was a negative control.

FIG. 4.

A LasR binding motif upstream of cueR. (A) The weblogo shown here is a LasR binding motif from Schuster et al. (47), and the sequence below it is the cueR upstream sequence from −77 to −57 relative to the translational start codon. The nucleotides that perfectly match the binding motif are highlighted in black. (B) Activation of wild-type and mutant cueR promoters in PAO1 lasI. The extent of the cueR upstream region in each construct is indicated relative the cueR translational start codon. β-Galactosidase activity is given in Miller units, along with standard deviations. pJTT-PA4778-mut1 through pJTT-PA4778-mut15 each contain a single-base-pair mutation, indicated by an arrow, in the LasR binding motif. The strains were grown both in the presence and absence of 3OC₁₂-HSL, as shown.

FIG. 5.

The MerR family of transcriptional regulators. Residues that are either known or predicted to bind metal are highlighted in yellow and marked with arrows. Identical residues in each subgroup are shown in blue, and conserved residues are shown in red. The figure is modified from reference with permission of the publisher. P putida, Pseudomonas putida; Y pestis, Yersinia pestis; V cholerae, Vibrio cholerae; S typhi, Salmonella enterica serovar Typhi; S meliloti, Sinorhizobium meliloti; R leguminosarum, Rhizobium leguminosarum; S typhimurium, Salmonella enterica serovar Typhimurium; S oneidensis, Shewanella oneidensis; R metallidurans, Ralstonia metallidurans; S marcesens, Serratia marcesens.

FIG. 6.

Copper sensitivity of PAO1 and the isogenic cueR mutant. The pJTT200 vector contains the cueR gene under the control of an IPTG-inducible promoter, and pMMB67 was the negative-control vector. Twenty microliters of overnight culture was spread onto an LB-gentamicin agar plate. A paper disk containing 10 μl of 1 M copper sulfate was placed on each plate, and the cells were incubated overnight at 37°C. Similar experiments were performed with silver, nickel, cadmium, iron, mercury, zinc, cobalt, and manganese, though no change in metal sensitivity was observed in the cueR mutant (data not shown).

FIG. 7.

CueR binding sites in E. coli (A) and P. aeruginosa (B). The weblogos in both panels were generated from the respective E. coli CueR and P. aeruginosa CueR binding motifs. Putative −35 and −10 sequence elements are shaded, regions of dyad symmetry are indicated by arrows, and the positions of the bases relative to the start codons are displayed above each binding motif.

FIG. 8.

Promoter activities of CueR-regulated genes in response to copper. Promoter-lacZ transcriptional fusions were integrated into the genomes of PAO1 and PAO1 cueR with the site-specific integration vector mini-ctx-lacZ. Cultures were grown in LB to log phase and then divided in half, with one half receiving copper sulfate to 1 mM and the other half receiving the equivalent volume of water. After 1 h of incubation at 37°C with shaking, β-galactosidase activity was assayed and is shown here in Miller units with standard deviations.

FIG. 9.

Gel shift analysis of CueR and its target promoters. Purified CueR was added to 100 fmol of the labeled promoter DNA. Where indicated, nonlabeled specific or nonspecific competitor DNA was present in the reaction mixture.