RhlR expression in Pseudomonas aeruginosa is modulated by the Pseudomonas quinolone signal via PhoB-dependent and -independent pathways

Abstract

The expression of virulence determinants in Pseudomonas aeruginosa is coordinately regulated in response to both the social environment--commonly referred to as quorum sensing--and to environmental cues. In this study we have dissected the various independent regulation levels for pyocyanin production, which is influenced by the homoserine lactone- and Pseudomonas quinolone signal (PQS)-mediated quorum-sensing systems as well as by iron and phosphate availability. We demonstrate that the phosphate regulon is involved in the transcriptional activation of rhlR and the augmentation of PQS and pyocyanin production under phosphate limitation. However, we also observed an enhancement of rhlR transcription under low-iron medium conditions and after the addition of PQS that was independent of the phosphate regulon. These results highlight the complexity of secondary metabolite production in P. aeruginosa via environmental cues and the quorum-sensing system.

FIG. 1.

rhlR promoter activity at three different time points in SCV 20265 and the phoB mutant cultivated in low-phosphate (0.8 mM) (A) and high-phosphate (4 mM) (B) medium supplemented with 50 μM and 2.5 μM ferric citrate, respectively. This graph is representative of at least three independent experiments.

FIG. 2.

PhoB sequence logo derived from genome-wide matches with the preferred spacer length of 4 bp.

FIG. 3.

Thin-layer chromatography of dichloromethane extracts of the SCV 20265 (A) and the isogenic phoB mutant (B) grown to stationary phase. Bacterial cultures were extracted after cultivation in low-phosphate (0.8 mM) and high-phosphate (4 mM) medium supplemented with 500 μM, 150 μM, 50 μM, and 2.5 μM ferric citrate. Synthetic PQS and HHQ served as controls. This TLC is representative of at least three independent experiments.

FIG. 4.

rhlR promoter activity in SCV 20265 and the isogenic phoB mutant grown in high-phosphate (4 mM) medium supplemented with 50 μM ferric citrate with or without the addition of 40 μM PQS. This graph is representative of at least three independent experiments.

FIG. 5.

Pyocyanin production of SCV 20265 grown for 96 h under high- and low-phosphate medium conditions and supplemented with 50 μM ferric citrate with or without the addition of 40 μM PQS. This graph is representative of at least three independent experiments.

FIG. 6.

Model of the complex regulatory circuit of secondary metabolite production in P. aeruginosa.