Metagenome-Derived Clones Encoding Two Novel Lactonase Family Proteins Involved in Biofilm Inhibition in Pseudomonas aeruginosa

Abstract

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...

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Metagenomic analyses faciliated the discovery of QlcA, BipB01, BipB04, BipB05, and BipB07 (68,74,75), and screens for AHL-degrading bacteria led to the identification of QsdA of Rhodococcus erythropolis strain W2 (70), AiiM of Microbacterium testaceum (71), AidH of Ochrobactrium sp. strain T63 (72), and QsdH of Pseudoalteromonas byunsanensis strain 1A01261 (73).…”

AiiM, a Novel Class of N -Acylhomoserine Lactonase from the Leaf-Associated Bacterium Microbacterium testaceum

“…240B1 (4), AttM (9.9% identity) from A. tumefaciens C58 (34), AhlD (11.7% identity) from Arthrobacter sp. IBN110 (23), QsdA (12.5% identity) from R. erythropolis W2 (30), and QlcA (14.9% identity), BpiB01 (12.9% identity), BpiB04 (13.3% identity), and BpiB07 (12.4% identity) from the soil metagenome (24,27). A conserved HXHXDH sequence is a zinc-binding motif that is found in the metallo-␤-lactamase superfamily and is common to many AHL lactonase enzymes (5).…”

“…Human and murine paraoxonase degrades AHL by hydrolyzing its lactone ring (21). Novel AHL lactonase genes have been isolated from a metagenomic library which was constructed from environmental soil samples (24,27). AHL-degrading genes have also been utilized in the biocontrol of plant diseases.…”

Degradation of N-Acylhomoserine Lactone Quorum Sensing Signaling Molecules by Potato Root Surface-Associated Chryseobacterium Strains

“…Genes encoding AHL-acylase, which hydrolyzes the amide bond of AHL, have been cloned from Ralstonia spp., Anabaena spp., Streptomyces spp., Shewanella spp., and Pseudomonas aeruginosa. Novel AHLdegrading genes have been isolated from metagenomic libraries constructed from soil samples (19,22). Recently, it was reported that some members of the α/β hydrolase family have AHL-lactonase activity (11,30).…”