LasI/R and RhlI/R quorum sensing in a strain of Pseudomonas aeruginosa beneficial to plants

Abstract

Pseudomonas aeruginosa possesses three quorum-sensing (QS) systems which are key in the expression of a large number of genes, including many virulence factors. Most studies of QS in P. aeruginosa have been performed in clinical isolates and have therefore focused on its role in pathogenicity. P. aeruginosa, however, is regarded as a ubiquitous organism capable of colonizing many different environments and also of establishing beneficial associations with plants. In this study we examined the role of the two N-acyl homoserine lactone systems known as RhlI/R and LasI/R in the environmental rice rhizosphere isolate P. aeruginosa PUPa3. Both the Rhl and Las systems are involved in the regulation of plant growth-promoting traits. The environmental P. aeruginosa PUPa3 is pathogenic in two nonmammalian infection models, and only the double las rhl mutants are attenuated for virulence. In fact it was established that the two QS systems are not hierarchically organized and that they are both important for the colonization of the rice rhizosphere. This is an in-depth genetic and molecular study of QS in an environmental P. aeruginosa strain and highlights several differences with QS regulation in the clinical isolate PAO1.

FIG. 1.

TLC analysis of AHLs produced by wild type and QS mutants. (A and B) Ethyl acetate extracts of P. aeruginosa strain PUPa3 wild type, PAO1 wild type, lasI mutants, rhlI mutants, and rsaL mutants of both PUPa3 and PAO1 strains. Standards (STDs) were synthetic C6-HSL (0.15 nmol) and C4-HSL (0.2 nmol) (for panels A and C) or 3-oxo-C12-HSL (4 nmol) and 3-oxo-C10-HSL (4 nmol) (for panels B and D). (C and D) Ethyl acetate extracts of P. aeruginosa strain PUPa3 WT and the rsaL mutant. For panels A and C TLCs were overlaid with the bacterial biosensor C. violaceum CV026; for panels B and D, TLCs were overlaid with the bacterial biosensor E. coli(pSB1075). In all the samples the equivalent of an extraction of 10⁹ cells was run on the TLC, while for the rsaL mutant (only in panel D), the equivalent of 10⁷ cells was run, since the rsaL mutant produces approximately 100 times more 3-oxo-C12-HSL and 3-oxo-C10-HSL.

FIG. 2.

Root colonization assays of rice rhizosphere P. aeruginosa strain PUPa3 and QS mutant derivatives in four independent experiments (A to D). PUPa3, P. aeruginosa PUPa3 wild-type strain; LASI, lasI mutant; LASR, lasR mutant; RHLI, rhlI mutant; DMI, lasI rhlI double mutant; DMR, lasR rhlR double mutant; RSAL, rsaL mutant. The colonization of the wild-type strain was significantly higher than that of all QS mutant strains.

FIG. 3.

Killing of G. mellonella larvae by P. aeruginosa PUPa3, the rhlI mutant RHLI, the lasI mutant LASI, the DMI (lasI rhlI) and DMR (lasR rhlR) double mutants, and the rsaL mutant RSAL. Larvae were infected with approximately 30 cells of the various P. aeruginosa strains and incubated in the dark at 30°C. Dead larvae were determined after 24 h. Data represent means and standard errors of three independent trials. The treatments that showed significant differences (using ANOVA; see text for details) from the wild-type P. aeruginosa PUPa3 strain were the two double mutants DMI and DMR and are indicated in the figure by an asterisk.

FIG. 4.

Killing of C. elegans by P. aeruginosa PUPa3, the rhlI mutant RHLI, the lasI mutant LASI, the DMI and DMR double mutants, and the rsaL mutant RSAL. P. aeruginosa strains were grown on NGM overnight, and 20 to 40 nematodes were then placed onto the plates. Surviving worms were counted after 72 h of incubation at 20°C. Data represent means and standard errors of three independent trials. The treatments that showed significant differences (using ANOVA; see text for details) from the wild-type P. aeruginosa PUPa3 strain were the two double mutants DMI and DMR and the rsaL mutant and are indicated in the figure by an asterisk.

FIG. 5.

Working model for the role of QS in P. aeruginosa PUPa3. Both QS systems positively regulate swimming, swarming (with the Rhl system being more important than the Las system), lipase, root colonization, and antifungal activity; the LasI/R system positively regulates protease activity, while RhlI/R does not regulate this activity. The two systems acting independently of each other and are not hierarchically organized. The LasI/R system produces 3-oxo-C12-HSL and undergoes positive autoregulation. The RhlI/R system produces C4-HSL and C6-HSL (C4 is the cognate AHL), and it also undergoes positive autoregulation. Both systems together are necessary for the infection of C. elegans and G. mellonella, whereas both independently are important for rhizosphere colonization. RsaL is a negative regulator of the LasI/R system and it is important for nematocidal killing, for antifungal activity, and for root colonization.