Abstract

Bacteria communicate with each other to regulate cell density-dependent gene expression via a quorum-sensing (QS) cascade. In Pseudomonas aeruginosa, two known QS systems, las and rhl, control the expression of many factors that relate to virulence, pathogenicity, and biofilm development. Microarray studies of the las and rhl regulons led to our hypothesis that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. Here, we examined a QS-regulated gene, vqsR, which encodes a probable transcriptional regulator with a putative 20-bp operator sequence (las box) upstream. The transcriptional start site for vqsR was determined. The vqsR promoter was identified by examining a series of vqsR promoter-lacZ fusions. In addition, an Escherichia coli system where either LasR or RhlR protein was expressed from a plasmid indicated that the las system was the dominant regulator for vqsR. Electrophoretic mobility shift assays (EMSA) demonstrate that purified LasR protein binds directly to the vqsR promoter in the presence of 3O-C12-HSL. Point mutational analysis of the vqsR las box suggests that positions 3 and 18 in the las box are important for vqsR transcription, as assayed with a series of vqsRp-lacZ fusions. EMSA also shows that positions 3 and 18 are important for binding between the vqsR promoter and LasR. Our results demonstrate that the las system directly regulates vqsR, and certain nucleotides in the las box are crucial for LasR binding and activation of the vqsR promoter.