A Novel Quorum-Quenching N-Acylhomoserine Lactone Acylase from Acidovorax sp. Strain MR-S7 Mediates Antibiotic Resistance

ABSTRACT N-Acylhomoserine lactone acylase (AHL acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here, we isolated and characterized a novel and unique AHL acylase (designated MacQ) from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs, ranging from C6 to C14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum, was dramatically attenuated by coculture with MacQ-overexpressing Escherichia coli, whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of β-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identity to any of the known acylases (<39%) and has among the broadest substrate range. Our findings provide the possibility that AHL acylase genes can be an alternative source of antibiotic resistance genes posing a threat to human health if they migrate and transfer to pathogenic bacteria. IMPORTANCE N-Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum sensing), and AHL acylase, which is able to degrade AHLs, has been recognized as a major target for quorum-sensing interference (quorum quenching) in pathogens. In this work, we succeeded in isolating a novel AHL acylase (MacQ) from a multidrug-resistant bacterium and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple β-lactam antibiotics by deacylation activities. Although quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme and further providing the possibility that a hitherto-overlooked antibiotic resistance mechanism mediated by the quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

A diverse array of microorganisms communicate with each other in a cell densitydependent manner through the exchange of diffusible signal molecules called autoinducers, e.g., N-acylhomoserine lactones (AHLs), in some Gram-negative bacteria and in small peptides in Gram-positive bacteria, respectively (1). This intercellular communication system, termed quorum sensing (QS), is responsible for the regulation of gene expression involved in pathogenicity (2), biofilm formation (3), secondary metabolite production (4), motility (5), and respiration (6). In particular, since a number of different pathogens utilize AHLs to control the production of virulence factors and biofilm formation, which are serious problems in nosocomial infectious diseases (7,8), it is expected that disruption of the QS system (i.e., quorum quenching) could inhibit bacterial infectious diseases (9).
Quorum quenching is mainly achieved through enzymatic degradation of AHLs by two different families of enzymes, AHL lactonases and AHL acylases (10). AHL acylases are well-known members of the Ntn hydrolase family proteins (11), and they inactivate AHLs by cleaving the acyl side chains from the homoserine lactone (Fig. 1). AHL acylase activity has been identified in various bacteria, including Gram-negative and Grampositive bacteria (12)(13)(14)(15)(16)(17)(18), but it differs in substrate specificity based on the different acyl chain substitutions of AHLs.
Based on the fact that AHLs and ␤-lactam antibiotics are structurally similar and both have an acyl side chain, we postulate that there should be bifunctional AHL acylases that mediate ␤-lactam antibiotic resistance and disrupt cell-cell communications ( Fig. 1); if so, hitherto-unrecognized antibiotic-resistant candidates may be present in nature. However, very little is known about bifunctional acylases and their biological functions. So far, it has been reported that only two experimentally characterized AHL acylases (AhlM from Streptomyces sp. strain M664 and KcPGA from Kluyvera citrophila DMSZ 2660) catalyze penicillin G as just one of the substrates (19,20), and their in vivo contributions to antibiotic resistance have never been discussed. Furthermore, the phylogenetic diversity of bifunctional acylases remains largely unclear, because the bifunctionality is rarely found.
In the present study, we found a new candidate gene encoding bifunctional acylase from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7, which is capable of inactivating AHLs (21), by cloning and expressing the gene from the bacterium and characterizing its catalytic functions and phylogenetic relatedness with previously identified acylases.

RESULTS AND DISCUSSION
Cloning of a putative bifunctional acylase gene and heterologous expression in E. coli. In the present study, we identified one putative acylase gene (macQ) in the strain MR-S7 genome based on homology and domain searches. In an amino acid sequence comparison, MacQ is related to Ntn hydrolase family proteins and shows the highest identity to AHL acylase (QqaR) (39%) from Deinococcus radiodurans R1 (16) among the verified acylases. Interestingly, MacQ also shares high similarity to putative ␤-lactam acylase (78%) from Acidovorax ebreus TPSY. Since the result implicates the potential bifunctionality of MacQ, we purified the enzyme from the recombinant Escherichia coli strain Origami 2(DE3) using a Ni affinity column. SDS-PAGE analysis showed that MacQ consisted of two subunits (␣-subunit, 20 kDa; ␤-subunit, 62 kDa) (Fig. 2) corresponding to the predicted molecular mass (84 kDa) from the amino acid sequence (806 amino acids).
Enzymatic properties of MacQ. To demonstrate whether MacQ can degrade both AHLs and ␤-lactams, gas chromatography-mass spectroscopy (GC-MS) analyses were performed to detect the presence of decanoic acid and phenylacetic acid generated by deacylation of C 10 -HSL and penicillin G, respectively. After incubation of purified MacQ with C 10 -HSL or penicillin G, we observed that each product emerged, with GC retention times of 10.42 min (Fig. 3A) and 8.86 min (Fig. 3B), respectively. MS analyses of the 10.42-and 8.86-min GC fractions showed the [M-H] ions at m/z 173 (Fig. 3C) and m/z 136 (Fig. 3D), corresponding to the molecular weights of decanoic acid and phenylacetic acid, respectively. These results obviously showed that MacQ has the bifunctional capacity to degrade both AHL and ␤-lactam antibiotics by deacylation activity.
We further characterized the AHL degradation capacity of purified MacQ using green fluorescent protein (GFP)-based and virulence-based AHL-detectable biosensor strains, as described in Materials and Methods. MacQ was able to inactivate all tested AHLs ranging from C 6 to C 14 chains with or without 3-oxo substitutions (Table 1). Besides, we observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum, was dramatically attenuated by coculture with MacQoverexpressing Escherichia coli Origami 2(DE3), whereas E. coli with an empty vector was unable to quench the pathogenicity (Fig. 4). Although whether or not the production of plant cell wall-degrading enzymes was inhibited by MacQ is unclear, it is quite possible that MacQ acts as a quorum-quenching enzyme and disrupts the quorum-sensing system in the pathogen.
Sequence analysis of MacQ. The first 24 residues of MacQ are predicted to be a signal sequence by SignalP (24), implying that MacQ is an extracellular enzyme, as well as the known Ntn hydrolase proteins (19,25). Indeed, amino acid residues (Ser-234 and a E. coli MT102 harboring pJBA132 was used for detecting C 6 -HSL, C 8 -HSL, C 10 -HSL, C 12 -HSL, OC 6 -HSL, OC 8 -HSL, OC 10 -HSL, and OC 12 -HSL, and P. putida F117 harboring pKR-C12 was used for detecting OC 14 -HSL. The plus and minus signs indicate degradation and nondegradation activities, respectively. ND, not determined.  (14,26,27) were highly conserved in MacQ and other Ntn hydrolase proteins. This accords with the result of SDS-PAGE analysis of MacQ, implicating that MacQ might be synthesized as a precursor polypeptide and modified into two enzymatically active subunits by posttranslational modification (Fig. 2), as other known acylases (19,25). Intriguingly, three residues (Ile-282 and Ser-289 in AHL acylase [AiiD] and Trp-443 in KcPGA) associated with the substrate specificity (28,29) were not fully conserved in MacQ, and we identified alternative residues (i.e., Phe-283, Gln-290, and Trp-401 in MacQ) with the same alignment positions (Fig. 5). We infer that the lack of amino acid sequence conservation may contribute to the broad substrate specificity and bifunctionality of MacQ, although further investigation with crystal structure analysis is required to verify this. Phylogenetic analysis of bifunctional acylases. Based on phylogenetic analysis using amino acid sequences, we found that the Ntn hydrolase family proteins were   divided into three groups (i.e., one ␤-lactam acylase group and AHL acylase groups A and B), and that MacQ was classified into AHL acylase group A (Fig. 6). Two AHL acylases, AhlM and KcPGA, known to degrade penicillin G were categorized into AHL acylase group A and a ␤-lactam acylase group, respectively (Fig. 6). Thus, the bifunctional acylases degrading both AHLs and ␤-lactams might be broadly distributed among the phylogeny, which implies that the bifunctionality may be conserved in other acylases of a wide variety of microorganisms, perhaps including pathogens. In fact, Ntn hydrolase proteins in this phylogram are derived from phylogenetically diverse bacteria: members of the phyla Actinobacteria, Cyanobacteria, Deinococcus-Thermus, and Proteobacteria, some of which are clinically and agriculturally important pathogens, such as Acinetobacter baumannii ATCC 19606, Mycobacterium tuberculosis ATCC 25618, Pseudomonas aeruginosa PAO1, and Pseudomonas syringae B72a. Further biochemical characterization is necessary for known and putative acylases to verify our assumption. Conclusions. We isolated and characterized a new AHL acylase (MacQ) with the bifunctional capability to degrade not only a variety of AHLs but also multiple ␤-lactam antibiotics. It has been recognized that antibiotic resistance and quorum quenching are distinct biological functions in bacteria. This study has, however, linked ␤-lactam degradation with quorum quenching in an explicit way by discovering a bifunctional enzyme. Our findings further provide the possibility that quorum-quenching genes (i.e., AHL acylase genes) would be an alternative source of antibiotic resistance genes.

MATERIALS AND METHODS
Bacterial strains, culture media, and growth conditions. A multidrug-resistant bacterium, Acidovorax sp. strain MR-S7, was previously isolated from activated sludge in a penicillin G production wastewater treatment system (21). Escherichia coli strain DH5␣ (TaKaRa, Tokyo, Japan) and strain Origami 2(DE3) (Novagen, Madison, WI) were used as the host strains for DNA manipulation and expression of the cloned gene, respectively. E. coli strain MT102 and Pseudomonas putida strain F117 with the gfp reporter plasmids pJBA132 and pKR-C12, respectively, were used as biosensors for AHL degradation activity in the bioassay (30,31). Strain MR-S7, P. putida, and E. coli strains were cultured on Luria-Bertani (LB) agar or in LB broth at 30°C, 30°C, and 37°C, respectively. When appropriate, antibiotics were added at the following concentrations: kanamycin, 50 g/ml; tetracycline, 20 g/ml; and gentamicin, 25 g/ml.
Cloning and heterologous expression of a putative AHL acylase gene. To explore the genes encoding AHL acylase or ␤-lactam acylase, a homology search and protein domain search were conducted using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/) and the InterProScan sequence search program (http://www.ebi.ac.uk/interpro/search/sequence-search), respectively, against the draft genome sequence data of strain MR-S7 obtained in our previous study (21). Signal sequence prediction analysis was conducted using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/). A macQ gene coding region was amplified with PrimeSTAR HS DNA polymerase (TaKaRa) using the following primers: For_mac, 5=-GGAATTCCATATGGCCTGCGGAGGCAGCGGC-3= (NdeI site underlined); and Rev_mac, 5=-CCGCTCGAGCTCCTTCACGGTGGCCGTGCG-3= (XhoI site underlined). PCR amplification was performed with initial denaturation at 98°C for 5 min, followed by 40 cycles at 98°C for 10 s, and then 68°C for 2.5 min. The PCR product was then gel purified using a QIAquick gel extraction kit (Qiagen, Valencia, CA), digested by each restriction enzyme, and subcloned into expression vector pET-28b (Novagen). The resulting plasmid, pMQ28, was transformed into E. coli strain Origami 2(DE3). To induce recombinant protein overexpression, 0.1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) was added to the E. coli cultures. The cells were harvested by centrifugation at 5,800 ϫ g for 10 min after 18 h of cultivation at 18°C, washed with suspension buffer (50 mM Na 2 PO 4 , 300 mM NaCl, 10% glycerol [pH 7.5]), resuspended in the same buffer, and disrupted using an ultrasonic disintegrator (Sonifier 250; Branson Ultrasonics). The cell debris was removed by centrifugation at 5,800 ϫ g for 10 min. The supernatant was applied to HIS-Select nickel affinity gel (Sigma-Aldrich, St. Louis, MO), and the His-tagged recombinant protein was purified according to the manufacturer's instructions. The purified protein was treated with marker dye (1% SDS, 1% 2-mercaptoethanol, 10 mM Tris-HCl [pH 6.8], 20% glycerin, and 1 mg/ml bromophenol blue) and Bootstrap values greater than 50% and 90% estimated using neighbor-joining (NJ) and maximum likelihood (ML) methods (1,000 replications) are shown by circle and square symbols at branching points, respectively. heated for 5 min at 98°C. The sample was subjected to a 10% PAGE gel under 30 mA for 80 min. The protein band was stained with Bio-Safe Coomassie brilliant blue (Bio-Rad, Hercules, CA).
Antibiotic susceptibility testing. MICs were determined as the lowest concentration of antibiotics preventing visible growth of E. coli strains on the agar. E. coli Origami 2(DE3) harboring plasmid pMQ28 or pET-28b was cultivated in LB broth with kanamycin. After incubation with IPTG, each culture of recombinant E. coli cells was equated and inoculated on LB agar plate with a selected ␤-lactam antibiotic. All plates were incubated at 37°C for 18 h under ambient atmosphere. The tested antibiotics were penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil, and the final concentrations were 8,16,32,64,128,256, and 512 g/ml.
Metabolite analyses of C 10 -HSL and penicillin G degradation. A purified MacQ protein (100 g) was mixed with 3 mM C 10 -HSL or 2 mM penicillin G solution and incubated at 30°C for 3 h or 12 h, respectively. After incubation, the digestion mixture was extracted with equal volumes of ethyl acetate three times; thereafter, the combined organic phase was evaporated to dryness in a vacuum. The redissolved samples in methanol were introduced onto a Hitachi M7200A GC/3DQMS system equipped with a DB-5ms capillary column (30 mm by 0.25 mm; J&W Scientific, Folsom, CA) coated with (5%phenyl)-methylpolysiloxane (250 nm thickness). Helium was used as carrier gas at a flow rate of 1.5 ml/min. The column temperature profile was initially 60°C for 1 min, was increased to 320°C at a rate of 10°C/min, and was finally held at 320°C for 5 min.
Inhibition of virulence factor production. In a plant-pathogenic bacterium, Pectobacterium carotovorum subsp. carotovorum, two different AHLs (OC 6 -HSL and OC 8 -HSL) regulate the development of soft rot symptoms on host plants (32,33). The assay was carried out on potato tubers (each n ϭ 5), as described previously (34). Briefly, potatoes were washed with running tap water, sterilized with 70% ethanol, and finally dried under sterile conditions. Escherichia coli expressing MacQ protein was used as a quorum quencher, whereas E. coli harboring an empty vector was used as a nonquenching control. P. carotovorum strain NBRC 3830 was cultivated in growth medium (1% polypeptone, 0.2% yeast extract, 0.1% MgSO 4 [pH 7.0]) at 30°C for 15 h and mixed with the culture of recombinant E. coli for equal volumes. The reaction mixture was incubated at 30°C for 2 h with gentle shaking and introduced directly into the surface of sliced potato tubers. The tubers were incubated at 28°C under 70% humidity for 2 days. The results were estimated by visual inspection of the infected area.
Conserved amino acid sequences and phylogenetic analysis. Multiple-amino-acid-sequence alignment analysis was performed using CLUSTAL W2 and the GENETYX software (Genetyx, Tokyo, Japan). The amino acid sequence of MacQ was aligned with four amino acid sequences of Ntn hydrolase family proteins, including a known ␤-lactam acylase (EcPGA from Escherichia coli ATCC 11105), AHL acylase (AiiD from Ralstonia sp. strain XJ12B), and two AHL acylases known to degrade penicillin G (AhlM and KcPGA). The phylogenetic trees were constructed with MEGA6 by using the neighbor-joining method based on the JTT matrix-based model (35). Amino acid sequences of experimentally identified Ntn hydrolase proteins were used. In addition, homologous sequences of bifunctional acylases were retrieved from the UniProt database (http://www.uniprot.org/) and clustered using the CD-HIT clustering program (36). Bootstrap resampling analysis using neighbor-joining and maximum likelihood methods (each 1,000 replications) was performed.
Accession number(s). The complete nucleotide sequence of the macQ gene was deposited in the NCBI GenBank database with accession no. AB702957.