Bacterial communities use ''quorum sensing'' (QS) to coordinate their population behavior through the action of extracellular signal molecules, such as the N-acyl-L-homoserine lactones (AHLs). The versatile and ubiquitous opportunistic pathogen Pseudomonas aeruginosa is a well-studied model for AHL-mediated QS. This species also produces an intercellular signal distinct from AHLs, 3,4-dihydroxy-2-heptylquinoline (PQS), which belongs to a family of poorly characterized 4-hydroxy-2-alkylquinolines (HAQs) previously identified for their antimicrobial activity. Here we use liquid chromatography (LC)͞MS, genetics, and whole-genome expression to investigate the structure, biosynthesis, regulation, and activity of HAQs. We show that the pqsA-E operon encodes enzymes that catalyze the biosynthesis of five distinct classes of HAQs, and establish the sequence of synthesis of these compounds, which include potent cytochrome inhibitors and antibiotics active against human commensal and pathogenic bacteria. We find that anthranilic acid, the product of the PhnAB synthase, is the primary precursor of HAQs and that the HAQ congener 4-hydroxy-2-heptylquinoline (HHQ) is the direct precursor of the PQS signaling molecule. Significantly, whereas phnAB and pqsA-E are positively regulated by the virulence-associated transcription factor MvfR, which is also required for the expression of several QSregulated genes, the conversion of HHQ to PQS is instead controlled by LasR. Finally, our results reveal that HHQ is itself both released from, and taken up by, bacterial cells where it is converted into PQS, suggesting that it functions as a messenger molecule in a cell-to-cell communication pathway. HAQ signaling represents a potential target for the pharmacological intervention of P. aeruginosa-mediated infections.

rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids Pseudomonas aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase. rhlA is indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P. aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties. P. aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility. INTRODUCTIONPseudomonas aeruginosa is a ubiquitous and versatile Gram-negative bacterium capable of three types of motility: flagellum-mediated swimming, type IV pilus-dependent twitching, and swarming, which was only recently reported in this species (Déziel et al., 2001; Köhler et al., 2000;Rashid & Kornberg, 2000). Swarming is a form of organized surface translocation allowing rapid colonization of surfaces that depends on extensive flagellation and cell-to-cell contact (Fraser & Hughes, 1999;Harshey, 1994). It is a multicellular phenomenon requiring cell differentiation and cooperation, two features also involved in biofilm formation and bacterial virulence (Harshey, 1994;Sharma & Anand, 2002).Swarming cells need to overcome the strong surface tension of water surrounding the colony to efficiently colonize the surface (Matsuyama & Nakagawa, 1996). This surface conditioning is often achieved by the production of surfaceactive compounds, which act as wetting agents (Lindum et al., 1998; Matsuyama et al., 1992Matsuyama et al., , 1995Mendelson & Salhi, 1996;Toguchi et al., 2000). Spreading growth of Serratia marcescens depends on the production of various extracellular lipopeptides named serrawetins (Matsuyama et al., 1986(Matsuyama et al., , 1992(Matsuyama et al., , 1995. Synthesis of serrawettin W2, a cyclic lipodepsipentapeptide required for swarming motility of Serrat...