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-C₈-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-C₈-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.
The
N-acyl-homoserine lactones (
N-AHLs) play an important role in bacterial cell–cell signaling. Up to date, however, only a few different experimentally proven classes of
N-AHL ring-cleaving ...enzymes are known. Here we report on the isolation and biochemical characterization of a novel hydrolase derived from the soil metagenome and acting on
N-AHLs. The identified protein designated BpiB05 is weakly similar to hypothetical proteins from
Bacteroides fragilis, the draft genomes of two
Burkholderia species as well as a marine metagenomic ORF but is otherwise not similar to any known protein. BpiB05 was overexpressed in
Escherichia coli as a 10× His-tagged fusion protein. The recombinant protein revealed a molecular weight of about 70
kDa and was tested for its quorum quenching (QQ) activities using a
lacZ-bioassay. Additional HPLC–MS analyses confirmed the lactonolytic activity of the purified protein in the presence of Ca
2+. Further tests suggested that BpiB05 strongly reduces motility in
Pseudomonas aeruginosa, pyocyanin synthesis and biofilm formation in this microbe. Because BpiB05 is not distantly related to any of the currently known hydrolases it forms probably a novel group within the growing number of proteins acting on
N-AHLs.
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