Regulation of the N-acyl homoserine lactone-dependent quorum-sensing system in rhizosphere Pseudomonas putida WCS358 and cross-talk with the stationary-phase RpoS sigma factor and the global regulator GacA

Abstract

Quorum sensing is a cell population-density dependent regulatory system which in gram-negative bacteria often involves the production and detection of N-acyl homoserine lactones (AHLs). Some Pseudomonas putida strains have been reported to produce AHLs, and one quorum-sensing locus has been identified. However, it appears that the majority of strains do not produce AHLs. In this study we report the identification and regulation of the AHL-dependent system of rhizosphere P. putida WCS358. This system is identical to the recently identified system of P. putida strain IsoF and very similar to the las system of Pseudomonas aeruginosa. It is composed of three genes, the luxI family member ppuI, the putative repressor rsaL, and the luxR family member ppuR. A genomic ppuR::Tn5 mutant of strain WCS358 was identified by its inability to produce AHLs when it was cross-streaked in close proximity to an AHL biosensor, whereas an rsaL::Tn5 genomic mutant was identified by its ability to overproduce AHL molecules. Using transcriptional promoter fusions, we studied expression profiles of the rsaL, ppuI, and ppuR promoters in various genetic backgrounds. At the onset of the stationary phase, the autoinducer synthase ppuI gene expression is under positive regulation by PpuR-AHL and under negative regulation by RsaL, indicating that the molecules could be in competition for binding at the ppuI promoter. In genomic rsaL::Tn5 mutants ppuI expression and production of AHL levels increased dramatically; however, both processes were still under growth phase regulation, indicating that RsaL is not involved in repressing AHL production at low cell densities. The roles of the global response regulator GacA and the stationary-phase sigma factor RpoS in the regulation of the AHL system at the onset of the stationary phase were also investigated. The P. putida WCS358 gacA gene was cloned and inactivated in the genome. It was determined that the three global regulatory systems are closely linked, with quorum sensing and RpoS regulating each other and GacA positively regulating ppuI expression. Studies of the regulation of AHL quorum-sensing systems have lagged behind other studies and are important for understanding how these systems are integrated into the overall growth phase and metabolic status of the cells.

FIG. 1.

(A) Gene map of the 2.456-kb KpnI fragment isolated in this study, which contained the gacA response regulatory gene. The ORFs spanning this region are shown, as is the position of the Tn5 insertion which was isolated in this study in the cloned gene and transferred by homologous recombination into the genome of P. putida WCS 358, creating IBE1 (see text for details). Tn5 was at position 1507 of this fragment, and gacA spanned from position 1177 to position 1707. The ORFs adjacent to gacA displayed high identity to pp4101 and pp4100 of P. putida KT2440, which encoded an acetyl transferase belonging to the GNAT family and a transcriptional regulator belonging to the Cro/Ci family, respectively. (B) Gene map of the 2.683-kb SalI-KpnI fragment isolated in this study, which contained the AHL-dependent system of P. putida WCS358. The locations within this fragment of the ppuI gene (spanning positions 1383 to 790), the rsaL gene (spanning positions 1523 to 1753), and the ppuR gene (spanning positions 2473 to 1757) are shown. Also shown are the positions of the Tn5 insertions in the different Tn5 genomic mutants of P. putida WCS358 isolated in this study. The positions of the genomic Tn5 insertions into the rsaL gene are labeled 3C (Tn5 inserted at position 1640) and 2B (Tn5 inserted at position 1672). The positions of the genomic Tn5 insertions into the ppuR gene are labeled 49 (Tn5 inserted at position 1793), 15 (Tn5 inserted at position 2073), 133 (Tn5 inserted at position 2133), and 50 (Tn5 inserted at position 2292). The work described here was performed by using Tn5 mutants 2B (IBE3) and 50 (IBE2); the sequences of these loci have been deposited in data banks under accession numbers AJ629219 and AJ629218, respectively.

FIG. 2.

TLC analysis of AHLs produced by parent strain P. putida WCS358 and mutant derivatives. In panels A, B, and C the E. coli(pSB401) AHL biosensor was used for detection of the spots, whereas in panel D, E. coli(pSB1075) was used. In all experiments a volume corresponding to 5 × 10⁸ CFU was loaded. Synthetic AHL compounds were used as reference compounds.

FIG. 3.

ppuI, rsaL, and ppuR promoter activities in parent strain P. putida WCS358 and mutant derivatives. All experiments were performed in triplicate, and the mean values are indicated; the standard deviations are not shown and were all within 10% of the reported values. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started with an inoculum of 1.6 × 10⁸ CFU. The growth curves of all mutants were comparable to the curve obtained for the parent strain, as indicated in each graph. (A) ppuI promoter activities obtained by using plasmid pPUI220. Also shown are the promoter activities in P. putida IBE3(pPUI220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (B) rsaL promoter activities obtained by using plasmid pRSA220. Also shown are the promoter activities in P. putida IBE3(pRSA220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (C) ppuR promoter activities obtained by using pPUR220 in P. putida WCS358. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 3.

ppuI, rsaL, and ppuR promoter activities in parent strain P. putida WCS358 and mutant derivatives. All experiments were performed in triplicate, and the mean values are indicated; the standard deviations are not shown and were all within 10% of the reported values. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started with an inoculum of 1.6 × 10⁸ CFU. The growth curves of all mutants were comparable to the curve obtained for the parent strain, as indicated in each graph. (A) ppuI promoter activities obtained by using plasmid pPUI220. Also shown are the promoter activities in P. putida IBE3(pPUI220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (B) rsaL promoter activities obtained by using plasmid pRSA220. Also shown are the promoter activities in P. putida IBE3(pRSA220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (C) ppuR promoter activities obtained by using pPUR220 in P. putida WCS358. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 3.

ppuI, rsaL, and ppuR promoter activities in parent strain P. putida WCS358 and mutant derivatives. All experiments were performed in triplicate, and the mean values are indicated; the standard deviations are not shown and were all within 10% of the reported values. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started with an inoculum of 1.6 × 10⁸ CFU. The growth curves of all mutants were comparable to the curve obtained for the parent strain, as indicated in each graph. (A) ppuI promoter activities obtained by using plasmid pPUI220. Also shown are the promoter activities in P. putida IBE3(pPUI220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (B) rsaL promoter activities obtained by using plasmid pRSA220. Also shown are the promoter activities in P. putida IBE3(pRSA220) in the presence of pMQS1, which carried the intact rsaL gene; the values for these activities are indicated by black bars. (C) ppuR promoter activities obtained by using pPUR220 in P. putida WCS358. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 4.

rpoS promoter activities in parent strain P. putida WCS358 and mutant derivatives obtained by using plasmid pRPO220B. All experiments were performed in triplicate, and the means are shown; the standard deviations are not shown and were all within 5% of the values reported. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started an inoculum of 1.6 × 10⁸ CFU. The growth curves of all mutants were comparable to the curve obtained for the parent strain, as indicated in each graph. The values for P. putida mutants MKO1 and M17 are shown in the same graph; the darker bars indicate the values for mutant M17. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 4.

rpoS promoter activities in parent strain P. putida WCS358 and mutant derivatives obtained by using plasmid pRPO220B. All experiments were performed in triplicate, and the means are shown; the standard deviations are not shown and were all within 5% of the values reported. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started an inoculum of 1.6 × 10⁸ CFU. The growth curves of all mutants were comparable to the curve obtained for the parent strain, as indicated in each graph. The values for P. putida mutants MKO1 and M17 are shown in the same graph; the darker bars indicate the values for mutant M17. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 5.

gacA promoter activities in parent strain P. putida WCS358 obtained by using plasmid pGAC220. All experiments were performed in triplicate, and the means are shown; the standard deviations are not shown and were all within 5% of the values reported. β-Galactosidase activities were determined at various times after a 20-ml LB medium culture was started an inoculum of 1.6 × 10⁸ CFU. The growth curve was comparable to the curve obtained for the parent strain, as indicated in the graph. See text for details. β-gal, β-galactosidase; OD600, optical density at 600 nm.

FIG. 6.

Working model for regulation of the AHL-dependent quorum-sensing system of P. putida WCS358 at the onset of the stationary phase when organisms are grown in LB media. ppuI is negatively regulated by the RsaL repressor and positively regulated by PpuR-AHL. RsaL is negatively autoregulated and is negatively regulated by RpoS. GacA has a positive effect either directly or indirectly on ppuI expression, while RpoS has a negative effect. PpuR and RsaL have a positive effect on rpoS expression; it is not known whether this effect is direct. All effects of RpoS observed are also observed in the psrA null mutant, as this is an important positive transcriptional regulator directly regulating rpoS transcription. See text for details.