The Pseudomonas aeruginosa Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS

Abstract

In Pseudomonas aeruginosa , many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators, N -(3-oxododecanoyl)- l -homoserine lactone (3O-C12-HSL) and N -butanoyl- l -homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in a lasR mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene, lecA , was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the lecA translational start codon. A lux box-type element together with RpoS (ς S ) consensus sequences was identified upstream of the putative promoter region. In Escherichia coli , expression of a lecA :: lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in P. aeruginosa PAO1, the expression of a chromosomal lecA :: lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of rpoS abolished lectin synthesis in P. aeruginosa , demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the lecA :: lux reporter in E. coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P. aeruginosa . This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of lecA expression.