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.

Genetic and phenotypic analysis of the GacS/GacA system in the moderate halophile Halomonas anticariensis

“…The neighbour-joining phylogenetic trees resulting from the predicted GacS and GacA protein sequences of H. anticariensis both show that the sequences from the halophilic strains cluster together in a separate group from the Pseudomonadaceae and Enterobacteriaceae (Fig. S1), which concurs to a great extent with the topology of the GacA-protein-based tree found by de Souza et al (Bertani & Venturi, 2004) and Serratia plymuthica (Ovadis et al, 2004). Nevertheless, AHL production was not fully restored when the gacS gene was complemented in trans in H. anticariensis FP35 T .…”

“…It has been suggested that the three systems, GacS/GacA, QS and RpoS, interact in a complex regulatory network, which is probably modulated according to environmental conditions. In some Pseudomonas species the QS and RpoS systems regulate each other whilst GacA positively regulates the production of AHLs (Bertani & Venturi, 2004). A third pathway begins with the GacS/GacA TCS triggering the transcription of one or several genes that encode small untranslated RNAs (sRNAs) (Bejerano-Sagie & Xavier, 2007;Lapouge et al, 2008).…”

Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391

“…A role of rpoS in antibiotic production has been reported (Sarniguet et al, 1995;Suh et al, 1999), but not for regulating PCN production. The inhibitory effect of RpoS on quorum sensing and pyocyanin in P. aeruginosa (Whiteley et al, 2000) or of PsrA and RpoS on quorum sensing in P. putida WCS358 (Bertani & Venturi, 2004) is the opposite of what we observed for strain PCL1391. Surprisingly, RpoS was previously shown not to be involved in homoserine lactone production by P. putida WCS358 (Kojic et al, 1999).…”

“…Surprisingly, RpoS was previously shown not to be involved in homoserine lactone production by P. putida WCS358 (Kojic et al, 1999). This is interesting for our study, because in the latter case P. putida was grown in minimal medium (Kojic et al, 1999), whereas in the most recent study P. putida was grown in the complex LB medium (Bertani & Venturi, 2004). In P. aeruginosa, the effect of quorum sensing on rpoS transcription in P. aeruginosa is mild (Schuster et al, 2004).…”

“…GacS and GacA are involved in the regulation of a substantial set of genes and of multiple traits, such as production of metabolites like HCN and 2,4-diacetylphloroglucinol (Phl) in Pseudomonas fluorescens CHAO (Laville et al, 1992), of enzymes like exoprotease and phospholipase C in P. fluorescens CHAO (Sacherer et al, 1994) and of various phenazines in P. aureofaciens 30-84 and Pseudomonas aeruginosa PAO1 (Chancey et al, 1999;Reimmann et al, 1997). GacS and GacA exert their effect on secondary metabolism by modulating the expression of various regulators (Chatterjee et al, 2003;Haas & Defago, 2005), including quorum sensing (Bertani & Venturi, 2004;Chancey et al, 1999;Reimmann et al, 1997) and s s (Schmidt-Eisenlohr et al, 2003;Whistler et al, 1998). In P. chlororaphis strain PCL1391, a mutation in gacS results in a severe decrease of PCN production to undetectable levels, while the Nacylhomoserine lactone (N-AHL) production is also much lower than in the wild-type (Chin-A-Woeng et al, 2005).…”