Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease

Abstract

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

FIG. 1.

Effect of lon disruption on C₄-HSL production by P. aeruginosa. The extracts from the supernatants of cultures of bacterial strains PAO1 (lon⁺) and CS9008 (Δlon) were applied to C₁₈ reversed-phase thin-layer plates and then developed with methanol/water (60:40 [vol/vol]). The spots were visualized with the C. violaceum reporter strain. C₄-HSL (0.125 μmol) and C₆-HSL (0.0125 μmol) were applied as HSL standards.

FIG. 2.

Expression of rhlI (A) and rhlR (B) in wild-type and lon-disrupted cells. Total RNAs were prepared from strains PAO1 (lon⁺), CS9008 (Δlon), and CS9053 (Δlon plon⁺) grown in L broth for 24 h 37°C. The levels of rhlI and rhlR transcripts were measured by quantitative, real-time RT-PCR and then normalized to rplU expression. The values represent the means and standard deviations of changes in comparison with the transcription level in PAO1.

FIG. 3.

Cellular levels and in vivo stabilities of RhlR. (A) Whole-cell extracts were prepared from strains PAO1 (lon⁺ rhlR⁺), CS9008 (Δlon rhlR⁺), CS9011 (lon⁺ ΔrhlR), and CS9053 (Δlon plon⁺) grown in L broth for 24 h at 37°C and then separated on 15% SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes and then immunostained with anti-RhlR serum. (B) Cells of strains PAO1 (lon⁺) and CS9008 (Δlon) were grown to exponential phase. They were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine for 1 min at 37°C and chased with unlabeled methionine and cysteine. Samples were taken at the times indicated, followed by immunoprecipitation of RhlR. (C) Quantification of the precipitated RhlR protein relative to the value at 1 min. Mean values and standard deviations of at least three independent experiments are given.

FIG. 4.

Cellular levels of LasI (A) and relative levels of lasI expression (B). (A) Whole-cell extracts were prepared from strains PAO1 (lon⁺ lasR⁺), CS9008 (Δlon lasR⁺), CS9013 (lon⁺ ΔlasR), CS9027 (Δlon ΔlasR), and CS9038 (lon⁺ ΔlasI) grown in L broth for 24 h at 37°C and then separated on 15% SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes and then immunostained with anti-LasI serum. (B) Total RNAs were prepared from strains PAO1 (lon⁺ lasR⁺), CS9008 (Δlon lasR⁺), CS9013 (lon⁺ ΔlasR), and CS9027 (Δlon ΔlasR) grown in L broth to exponential phase at 37°C. The levels of lasI transcript were measured by quantitative, real-time RT-PCR and then normalized to rplU expression. The values represent the means and standard deviations of changes in comparison with the transcription level in PAO1.

FIG. 5.

In vivo stabilities of LasI. (A) Cells of strains PAO1 (lon⁺) and CS9008 (Δlon) were grown to exponential phase and pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine for 1 min at 37°C and then chased with unlabeled methionine and cysteine. Samples were taken at the times indicated, followed by immunoprecipitation of LasI. (B) Quantification of the precipitated LasI protein relative to the value at 1 min. Mean values and standard deviations of at least three independent experiments are given.

FIG. 6.

Cellular levels of RhlR (A) and relative levels of rhlR expression (B) in the absence of a lasR and/or rhlI gene in cells. (A) Whole-cell extracts were prepared from strains PAO1 (lon⁺ lasR⁺ rhlI⁺), CS9008 (Δlon lasR⁺ rhlI⁺), CS9013 (lon⁺ ΔlasR rhlI⁺), CS9027 (Δlon ΔlasR rhlI⁺), CS9051 (lon⁺ ΔlasR ΔrhlI), and CS9062 (Δlon ΔlasR ΔrhlI) grown in L broth for 24 h at 37°C and then separated on 15% SDS-polyacrylamide gel. The separated proteins were transferred to polyvinylidene difluoride membranes and then immunostained with anti-RhlR serum. (B) Total RNAs were prepared from strains PAO1 (lon⁺ lasR⁺ rhlI⁺), CS9008 (Δlon lasR⁺ rhlI⁺), CS9013 (lon⁺ ΔlasR rhlI⁺), CS9027 (Δlon ΔlasR rhlI⁺), CS9051 (lon⁺ ΔlasR ΔrhlI), and CS9062 (Δlon ΔlasR ΔrhlI) grown in L broth to exponential phase at 37°C. The levels of lasI transcript were measured by quantitative real-time RT-PCR and then normalized to rplU expression. The values represent the means and standard deviations of changes in comparison with the transcription level in PAO1.