The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation

Abstract

Background: In Pseudomonas putida and Pseduomonas aeruginosa, the similar PpuR/RsaL/PpuI and LasR/RsaL/LasI acyl homoserine lactones (AHLs) quorum sensing (QS) systems have been shown to be under considerable regulation by other global regulators. A major regulator is the RsaL protein which strongly directly represses the transcription of the P. putida ppuI and P. aeruginosa lasI AHL synthases. In this study we screened a transposon mutant bank of P. putida in order to identify if any other regulators were involved in negative regulation of AHL QS.

Results: In our screen we identified three Tn5 mutants which displayed overproduction of AHLs in P. putida strain WCS358. Two of the mutants had a Tn5 located in the rsaL gene, whereas in one mutant the transposon was located in the lon protease gene. Lon proteases play important roles in protein quality control via degradation of misfolded proteins. It was determined that in the P. putida lon mutant, AHL levels, PpuR levels and ppuI promoter activity all increased significantly; we therefore postulated that PpuR is a target for Lon. The Lon protease had no effect on AHL production in P. aeruginosa.

Conclusion: The Lon protease is a negative regulator of AHL production in P. putida WCS358. The Lon protease has also been shown by others to influence AHL QS in Vibrio fischeri and Agrobacterium tumefaciens and can thus become an important regulator of AHL QS timing and regulation in bacteria.

Figure 1

Physical map of the 4.4 kb EcoRI DNA fragment containing the lon protease cloned from the chromosome of P. putida WCS358. Position of the first and last nucleotide of the lon gene are shown together with the position of the Tn5 insertion. The ORF or part of ORFs downstream the lon gene display 88% and 92% similarity to the pp_2303 and pp_2304 ORFs of P. putida KT2440. In the upstream region of the lon gene, part of clpX ORF is localized.

Figure 2

C12-3-oxo-AHL measurement produced by P. putida WCS358, by lon protease mutant derivative P. putida IBE4 and lon mutant containing a plasmid expressing the Lon protease, P. putida IBE4 (pBBRlon). C12-3-oxo-AHL was extracted from spent supernatants, AHL levels were measured with P. putida SM17 (prsaL220) with a volume of extract corresponding to an amount of 5 × 10⁸ cfu as described in the Methods section. C12-3-oxo-AHL levels are proportional to β-galactosidase activity (Miller Units). Standard deviation bars are given on the mean value of three independent cultures. Statistical analysis was performed with Anova resulting in a significant main effect of the mutation with F(2,12) = 20.45 and p < .001. The same measurement was also performed using 1 μM of synthetic C12-3-oxo-AHL (obtained from P. Williams, University of Nottingham, UK).

Figure 3

Gene promoter activities of ppuI and rsaL in P. putida WCS358 and lon mutant derivatives. A. Promoter activities of the ppuI C12-3-oxo-AHL synthase in P. putida WCS358 (pPPUI220), in the lon protease mutant derivative P. putida IBE4 (pPPUI220) and lon mutant containing a plasmid expressing the Lon protease, P. putida IBE4 (pPPUI220)(pBBRlon). The ppuI promoter activities are expressed in β-galactosidase activity (Miller Units) as the plasmid construct pPPUI220 contains the ppuI promoter transcriptionally fused to a promoterless lacZ gene (see text for all details). Standard deviation bars are given on the mean value of three independent experiments. Logarithmic phase (Log.) corresponds to 8 hours of growth, stationary phase (Stat.) to 16 hrs and late stationary (Late stat.) to 48 hours of growth. Statistical analysis was performed with Anova resulting in a significant main effect of the mutation with F(1,14) = 18.90 and p < .001. Statistical analysis including the age of the cultures results in a significant difference between the wild type and the IBE4 mutant in the Logarithmic phase of growth [F(1,14) = 28.70 p < .001]. B. Promoter activities of the rsaL in P. putida WCS358 (pRSA220), in the lon protease mutant derivative P. putida IBE4 (pRSA220) and lon mutant containing a plasmid expressing the Lon protease, P. putida IBE4 (pRSA220)(pBBRlon). The ppuI promoter activities are expressed in β-galactosidase activity (Miller Units) as the plasmid construct pRSA220 contains the rsaL promoter transcriptionally fused to a promoterless lacZ gene. Standard deviation bars are given on the mean value of three independent experiments. Logarithmic phase (Log.) corresponds to 8 hours of growth, stationary phase (Stat.) to 16 hrs and late stationary (Late stat.) to 48 hours of growth. Statistical analysis was performed with Anova resulting in a significant main effect of the mutation with F(1,18) = 126.12 and p < .000001. Statistical analysis including the age of the cultures results in a significant difference between the wild type and the IBE4 mutant in the late stationary phase of growth [F(1,18) = 18.79 p < .001].

Figure 4

TLC analysis of C12-3-oxo-AHL produced by parent strain P. putida WCS358 (lane 1), by lon mutant derivative P. putida IBE4 (lane 2) and lon mutant derivative carrying a plasmid expressing Lon P. putida IBE4 (pBBRlon) (lane 3). Lane 4 contains the standards C10-3-oxo-AHL and C12-3-oxo-AHL. The AHLs were visualized with AHL-sensor strain E. coli (pSB1075); a volume corresponding to 5 × 10⁸ cfu was loaded on the TLC assay.

Figure 5

Western hybridization performed using anti-PpuR antibody on total cellular protein extracts. Total protein extracts were performed using different protein amounts of either parent strain P. putida WCS358 or lon mutant derivative P. putida IBE4 each carrying a plasmid expressing PpuR (pBBRppuR). Lane 1 corresponds to 10 μg of purified PpuR-6His; lane 2 and lane 3 correspond to total proteins from 2 × 10⁷ cfu of WCS358(pBBRppuR) and IBE4(pBBRppuR) respectively; lanes 4 and 5 correspond to total proteins from 10⁷ cfu. For lanes 4 and 5 the images were scanned using a Versadoc (Biorad) and the QuantityOne software; results of this quantification are displayed as a histogram. See text for all details.

Figure 6

C12-3-oxo-AHL measurement produced by of P. aeruginosa PAO1 and by lon protease mutant derivative P. aeruginosa 18577. C12-3-oxo-AHL was extracted from spent supernatants, AHL levels were measured with P. putida SM17 (prsaL220) with a volume of extract corresponding to an amount of 5 × 10⁸ cfu as described in the Methods section. C12-3-oxo-AHL levels is proportional to β-galactosidase activity (Miller Units). Standard deviation bars are given on the mean value of three independent cultures. Differences between PAO1 and PAO1 ID18577, analysed using T-test for independent groups were not significant (t = 2.2; p = .06; df = 6;t(6) = 2.2). The same measurement was also performed using 1 μM of synthetic C12-3-oxo-AHL (obtained from P. Williams, University of Nottingham, UK).