Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C 8 -HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C 8 -HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.Quorum sensing (QS) is a cell density-dependent system of gene regulation in prokaryotes. Through the accumulation of bacterially produced signal molecules, autoinducers (AIs), the bacterial population is able to sense increases in cell density and then alters gene expression accordingly (50). This enables the coordinated expression of genes at the population level which are most effective at higher cell densities, such as those coding for pathogenicity and production of extracellular proteins (18, 45). Many QS mechanisms involve N-acyl-homoserine lactones (AHLs) in gram-negative bacteria and modified oligopeptides in gram-positive bacteria (50). Bacteria are also able to detect and respond to the number of unrelated species through an interspecies QS system involving furanosyl diesters that at least in some cases contain boron (autoinducer 2 [AI-2]). AI-2 activity has been detected in many different bacteria, and luxS, the gene involved in AI-2 synthesis, has been identified in more than 50 different bacteria (13).As QS regulates expression of pathogenicity-related phenotypes, such as biofilm formation and virulence in many bacteria, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, this is a potential target for antipathogen treatment (6, 32). There are many signal interference mechanisms called anti-QS mechanisms. They are distinguished as "quorum sensing inhibition" (QSI) for those that interfere with the QS gene regulation system and "quorum quenching" for those that degrade the AI molecules (33, 53). As the many levels of complexity of QS regulation are elucidated, possible new targets for anti-QS are revealed.Many of the original investigations of QS relied on pure culture analysis for determination of the QS signals (11,30,31). In contrast, the search for novel quorum-quenching strategies...

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

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