Abstract
Quorum-sensing (QS) signals of the N-acylhomoserine lactone (NAHL) class are cleaved by quorum-quenching enzymes, collectively named NAHLases. Here, functional metagenomics allowed the discovery of a novel bacterial NAHLase in a rhizosphere that was treated with γ-caprolactone. As revealed by rrs-DGGE and rrs-pyrosequencing, this treatment increased the percentage of the NAHL-degrading bacteria and strongly biased the structure of the bacterial community, among which Azospirillum dominated. Among the 29 760 fosmids of the metagenomic library, a single one was detected that expressed the qsdB gene conferring NAHL-degradation upon E. coli and decreased QS-regulated virulence in Pectobacterium. Phylogenetic analysis of the 34 orfs of the fosmid suggested that it would belong to an unknown Proteobacterium - probably a γ-proteobacterium. qPCR quantification of the NAHLase-encoding genes attM, qsdA, and qsdB revealed their higher abundance in the γ-caprolactone-treated rhizosphere as compared to an untreated control. The purified QsdB enzyme exhibited amidase activity. QsdB is the first amidase signature (AS) family member exhibiting NAHLase-activity. Point mutations in the AS-family catalytic triad K-S-S abolished the NAHLase activity of QsdB. This study extends the diversity of NAHLases and highlights a common phylogenic origin of AS-family enzymes involved in the degradation of natural compounds, such as NAHLs, and xenobiotics, such as nylon and linuron.
Highlights
N-acylhomoserine lactones (NAHLs) are diffusible signals used by many Proteobacteria to correlate gene expression to cell density via a regulatory pathway called quorum-sensing (QS) [1,2]
While GCL was introduced at 0.8 g/L (260.4 g/L), it was detected at 3.2 1024 g/L in the nutritive solution and at 4.9 1024 mg/g of fresh weight in plant tissues recovered from treated batch at 42-day
The novel metagenomic NAHL-amidase QsdB identified in this study is distantly related to the previously identified NAHLases and belongs to the amidase signature (AS) family that comprises most than 200 proteins from diverse origins including Bacteria, Archaea and Eukarya [44]
Summary
N-acylhomoserine lactones (NAHLs) are diffusible signals used by many Proteobacteria to correlate gene expression to cell density via a regulatory pathway called quorum-sensing (QS) [1,2]. Aside from the enzymes implicated in biosynthesis of NAHLs, others are able to cleave or modify NAHLs, to disrupt QS-signaling [3] They have been identified in bacteria and eukaryotes, and are collectively called NAHLases or quorum-quenching enzymes [4]. The biological role(s) of NAHLases generally remains unclear, except in some firmicutes where they may contribute to the degradation of toxic NAHL-derivatives [20], or in the plant pathogen Agrobacterium tumefaciens in which they slightly modulate QS-functions [21,22] In spite of this lack of information on the biological roles of NAHLases, these enzymes have been used successfully to quench QS-regulated functions. Virulence in the plant pathogen Pectobacterium could be reduced or abolished either via the production of transgenic plants expressing NAHLase-encoding gene [23] or by the selection of bacterial isolates or populations exhibiting NAHLase activities [24,25,26,27]
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