Regulation of the Pseudomonas aeruginosa Quorum-Sensing Regulator VqsR

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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-C 12 -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.