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<p>Abstract</p> <p>Background</p> <p>Acyl-homoserine lactone (acyl-HSL) and alkyl quinolone (AQ) based quorum-sensing (QS) systems are important for <it>Pseudomonas aeruginosa </it>virulence and biofilm formation. The effect of QS on biofilm formation is influenced by various genetic and environmental factors. Here, we used a colony biofilm assay to study the effect of the central acyl-HSL QS regulator, LasR, on biofilm formation and structure in the representative clinical <it>P</it>. <it>aeruginosa </it>isolate ZK2870.</p> <p>Results</p> <p>A <it>lasR </it>mutant exhibited wrinkled colony morphology at 37°C in contrast to the smooth colony morphology of the wild-type. Mutational analysis indicated that wrinkling of the <it>lasR </it>mutant is dependent on <it>pel</it>, encoding a biofilm matrix exopolysaccharide. Suppressor mutagenesis and complementation analysis implicated the AQ signaling pathway as the link between <it>las </it>QS and colony morphology. In this pathway, genes <it>pqsA-D </it>are involved in the synthesis of 4-hydroxyalkyl quinolines ("Series A congeners"), which are converted to 3,4-dihydroxyalkyl quinolines ("Series B congeners", including the well-characterized <it>Pseudomonas </it>Quinolone Signal, PQS) by the product of the LasR-dependent <it>pqsH </it>gene. Measurement of AQ in the wild-type, the <it>lasR pqsA</it>::Tn suppressor mutant as well as the defined <it>lasR</it>, <it>pqsH</it>, and <it>lasR pqsH </it>mutants showed a correlation between 4-hydroxyalkyl quinoline levels and the degree of colony wrinkling. Most importantly, the <it>lasR pqsH </it>double mutant displayed wrinkly morphology without producing any 3,4-dihydroxyalkyl quinolines. Constitutive expression of <it>pqsA-D </it>genes in a <it>lasR pqsR</it>::Tn<it/>mutant showed that colony wrinkling does not require the AQ receptor PqsR.</p> <p>Conclusions</p> <p>Taken together, these results indicate that the <it>las </it>QS system represses Pel and modulates colony morphology through a 4-hydroxyalkyl quinoline in a PqsR-independent manner, ascribing a novel function to an AQ other than PQS in <it>P. aeruginosa</it>.</p>