4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.

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Acyl homoserine lactone (AHL) lactonase has been proved to be the AHL-degrading enzyme with the highest substrate specificity for AHL molecules and has shown a considerable potential as low-cost and efficient quorum quenching (QQ) technique. However, few studies focused on its inhibitory effect on biofilm formation which is also a quorum sensing (QS)-regulated phenomenon. In this study, QQ activity of six isolates from biofouled reverse osmosis membranes was studied using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 as biosensors under various conditions. All of the isolates belonged to the genus Bacillus and showed QQ activity regardless of the acyl chain length or substitution of AHL molecule. The isolates were capable of significantly inhibiting biofilm formation (46.7-58.3%) by Pseudomonas aeruginosa PAO1 and produced heat-sensitive extracellular QQ substances. The LC-MS analysis of the QQ activity of a selected isolate, RO1S-5, revealed the degradation of N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL) and the production of corresponding acyl homoserine (3-oxo-C12-HS), which indicated the activity of AHL lactonase. The broad AHL substrate range and high substrate specificity suggested that the isolate would be useful for the control of biofilm-related pathogenesis and biofouling in industrial processes. Keywords:Pseudomonas aeruginosa, AHL lactonase, biofilm, quorum quenching, quorum sensing *For correspondence. E-mail: ysoh@mju.ac.kr; Tel.: Quorum sensing (QS) allows bacteria to perceive the density of the surrounding bacterial populations using signal molecules such as N-acyl homoserine lactone (AHL) and to coordinately respond to this information by regulating expression of various genes (Smith and Iglewski, 2003a). QS has been known to play significant roles in many biological processes such as virulence factor production, bioluminescence, antibiotic production, sporulation, and biofilm formation (Novick and Geisinger, 2008;Ng and Bassler, 2009; Williams and Cámara, 2009).Among these, biofilm formation is one of the major contributors to the pathogenesis of many clinically important bacteria, and the loss of efficiency in medical, industrial, and environmental processes such as medically-implanted devices, oil drilling, paper production, food processing, fish farming, and membrane separation processes (Donlan, 2001; Defoirdt et al., 2004; Adonizio et al., 2008; Ammor et al., 2008; Wevers et al., 2009; Van Houdt and Michiels, 2010). Biofilm is not only ubiquitous and costly but also endows the cells with resistance to many antimicrobial agents, which has made it difficult to suggest prevalent techniques to control biofilm development (Davies et al., 1998;Stewart and Costerton, 2001;Hoffman et al., 2005). Therefore, there has been a high demand for effective strategies to control biofilm formation, and targeting the QS regulatory mechanism has been a highly considerable candidate due to the critical role of QS in biofilm formation (Davies et al., 1998;Smith and Iglewski,...