Abstract
The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-acyl homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demonstrated that a single gene (hqiA) allowed AHL degradation. Enzymatic analysis using purified HqiA and HPLC/MS revealed that this protein has lactonase activity on a broad range of AHLs. The introduction of hqiA in the plant pathogen Pectobacterium carotovorum efficiently interfered with both the synthesis of AHLs and quorum-sensing regulated functions, such as swarming motility and the production of maceration enzymes. Bioinformatic analyses highlighted that HqiA showed no sequence homology with the known prototypic AHL lactonases or acylases, thus expanding the AHL-degrading enzymes with a new family related to the cysteine hydrolase (CHase) group. The complete sequence analysis of the fosmid showed that 31 ORFs out of the 46 identified were related to Deltaproteobacteria, whilst many intercalated ORFs presented high homology with other taxa. In this sense, hqiA appeared to be assigned to the Hyphomonas genus (Alphaproteobacteria), suggesting that horizontal gene transfer had occurred.
Highlights
Many microorganisms use a genetic regulatory mechanism to monitor their cell density and to establish a coordinated behaviour[1,2,3]
Based on acyl homoserine lactones (AHLs)-degradation assays performed with short- and long-chain AHLs (C6-HSL and C12-HSL), both thin-layer chromatography (TLC) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the activity corresponded to AHL degradation and not to an inhibition of the biosensor (Fig. 2)
The HPLC/MS analyses confirmed that E. coli S17 λ pir carrying the fosmid f10/17.1H significantly degraded AHLs compared to the control E. coli S17 λ pir (P < 0.0001)
Summary
Screening of AHL-degrading genes from a metagenomic library. Environmental DNA extracted from a hypersaline soil located in the Finca La Salina (Rambla Salada, Murcia, Spain) was used to construct a metagenomic library. The enzymatic restriction analyses revealed that the different clones recovered corresponded to a unique fosmid, which we named f10/17.1H (data not shown) This fosmid was purified and transferred into E. coli S17 λ pir to carry out additional AHL-degradation assays. A first step consisting in the cloning in pGEM-T of 5-kb fragments of the fosmid DNA insert was conducted to determine which region conferred AHL-degradation ability to E. coli. This first screening allowed the identification of a fragment containing from ORF26 to ORF31. The AHL-degradation assays performed with pGEM-T or pME6010-based constructions revealed that ORF29 (named as hqiA gene) conferred to E. coli the ability to degrade all the AHLs tested (data not shown). HqiA shows the conserved catalytic domains (D, K and C) of the CSHase family and presents a very different three-dimensional structure than the other AHL lactonases based on a Phyre[2] analysis[30] (see Supplementary Fig. S1)
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