A Rhodococcus qsdA-Encoded Enzyme Defines a Novel Class of Large-Spectrum Quorum-Quenching Lactonases

A gene involved in N-acyl homoserine lactone (N-AHSL) degradationwas identified by screening a genomic library of Rhodococcuserythropolis strain W2. This gene, named qsdA (for quorum-sensingsignal degradation), encodes an N-AHSL lactonase unrelated tothe two previously characterized N-AHSL-degrading enzymes, i.e.,the lactonase AiiA and the amidohydrolase AiiD. QsdA is relatedto phosphotriesterases and constitutes the reference of a novelclass of N-AHSL degradation enzymes. It confers the abilityto inactivate N-AHSLs with an acyl chain ranging from C₆ toC₁₄, with or without substitution at carbon 3. Screening ofa collection of 15 Rhodococcus strains and strains closely relatedto this genus clearly highlighted the relationship between theability to degrade N-AHSLs and the presence of the qsdA genein Rhodococcus. Bacteria harboring the qsdA gene interfere veryefficiently with quorum-sensing-regulated functions, demonstratingthat qsdA is a valuable tool for developing quorum-quenchingprocedures.

INTRODUCTION

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INTRODUCTION

Gram-negative bacteria couple gene expression to populationdensity by a regulatory mechanism named quorum sensing (QS).QS relies upon the production and the perception of one or moresignal molecules by the bacterial population. An important classof these signals is the N-acyl homoserine lactone (N-AHSL) class(9). Molecules belonging to this class exhibit a conserved structure,with a backbone composed of a homoserine lactone (HSL) N-linkedto an acyl chain via an amide bond. Variation in N-acyl chainlength and the oxidation status of N-AHSLs provide for specificityof the signal. Of particular interest is the finding that QSregulates pathogenicity, or pathogenicity-related functions,in bacteria of medical or environmental importance (15, 32,50).

If QS and N-AHSLs are important components of the strategy ofadaptation by bacteria to their biotic environment, especiallya plant surface, one might suspect that the eukaryotic hostsand competing bacteria might have developed strategies to interferewith this communication system. Indeed, QS inhibition was reportedthrough the production of antagonists (10) or the productionof N-AHSL degradation enzymes by plants (5), animals (2, 34),and a wide range of bacterial genera (14, 16, 18-20, 30, 31,44, 49). In spite of the large diversity of N-AHSL-degradingbacteria, only two families of N-AHSL-inactivating enzymes (N-AHSLases)have been described to date: the AiiA-like N-AHSL lactonases(6, 19, 49) and the AiiD-like N-AHSL amidohydrolases (14, 20,30). Whatever their physiological role, N-AHSLases have beenused efficiently to interfere with the expression of QS-regulatedfunctions in bacteria (6, 19, 20, 35, 42). This strategy hasbeen termed quorum quenching (QQ). It proved to be a valuabletrail toward definition of novel biocontrol agents such as naturalisolates degrading N-AHSLs (8, 23, 44). QQ occurs in naturalenvironments as indicated by the coexistence of N-AHSL-producingand -degrading strains in biofilms (14) or in the rhizosphere(4). Among the species harboring an N-AHSLase activity, Rhodococcuserythropolis is remarkable because it is the only bacteriumin which three enzymatic activities directed at N-AHSLs havebeen characterized: an oxidoreductase activity, which reduces3-oxo-N-AHSLs to their hydroxylated equivalents (43); an amidohydrolase(43); and a lactonase (29). The marked R. erythropolis QQ capabilitiessuggest that it might be used in biocontrol protocols, especiallysince it is a natural inhabitant of soils worldwide (44).

In this study we report the isolation of one of the three genesencoding N-AHSLase activities from R. erythropolis strain W2.We show that this gene encodes a phosphotriesterase (PTE)-likebroad-spectrum N-AHSL lactonase, which is found only in theRhodococcus genus and solely in strains capable of degradingN-AHSLs.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains, media, growth conditions, and chemicals.
Bacterial strains and plasmids are listed in Table 1. All strainswere grown at 25°C, with the exception of the Chromobacteriumviolaceum biosensor and Escherichia coli, which were grown at30°C and 37°C, respectively. Unless otherwise stated,LBm medium was buffered at pH 6.5 to prevent spontaneous lactonolysisof N-AHSLs and used as the rich medium (44). All media weresolidified with 16 g/liter of agar. Antibiotics were used atthe following final concentrations (in µg/ml; see Table1 for specific strain requirements): ampicillin, 100; gentamicin,25; tetracycline, 10; rifampin, 100; and streptomycin, 250.X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)and IPTG (isopropyl-β-D-thiogalactopyranoside) were includedin media at 40 µg/ml and 100 µg/ml, respectively.Hexanoyl-HSL (C₆-HSL) and its 3-oxo derivative were purchasedfrom Sigma. The other N-AHSLs were generous gifts from S. R.Chhabra and P. Williams (University of Nottingham, United Kingdom).

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TABLE 1. Bacterial strains and plasmids

Detection of N-AHSLs.
N-AHSLs were separated and visualized on thin-layer chromatography(TLC) plates as described previously (37). Saturated short-chainN-AHSLs were detected by TLC using the Chromobacterium violaceumbiosensor CV026 (22). Oxo and hydroxy derivatives and long-chainN-AHSLs were detected using the Agrobacterium biosensor NTL4(pZLR4)(21).

Detection of N-AHSL degradation products.
The N-AHSL degradation assays were performed as described earlier(45), using actively growing DH5 ${alpha}$ cells expressing or not theqsdA gene in pH 6.5 buffered LB medium. The incubation timewas set to 24 h, because this allowed work at low concentrationand thus reduction of the interference of the medium with thedata collected. Under these conditions, a fraction of ca. 25%of the initial amount of C₆-HSL is spontaneously converted intoC₆-HS by chemical lactonolysis in the presence or absence ofE. coli cells. The enzymatic degradation of N-AHSLs can proceedthrough two different routes (Fig. 1), which lead to the formationof either N-AHS (lactonolysis) (Fig. 1, top) or HSL and an alkylchain (amidolysis) (Fig. 1, bottom). The presence of HSL inthe incubation medium was determined after trapping of the freeamine with dansyl chloride as described earlier (48). The formationof the ring-opened derivative of N-AHSLs following lactone hydrolysiswas investigated using high-pressure liquid chromatography (HPLC)on a Waters chromatograph equipped with a Waters separationmodule 2659 and an Atlantis reverse-phase column (4.6 by 150mm; 5 µm) coupled to an electrospray ionization-mass spectrometrydetector (Waters Micromass ZQ 200). Retention times and massspectra were determined for individual molecules in solutionas standards. Thirty microliters of the degradation assay samplewas injected and eluted with water-0.1% formic acid (solventA) and acetonitrile-0.1% formic acid (solvent B) with the followingelution sequence: 100% solvent A for 5 min, a linear gradientto reach 20% solvent B in 5 min, and 80% solvent A and 20% solventB for 10 min. Between samples, the column was rinsed by applying100% B solvent (3 min). The column was then reequilibrated with100% solvent A for 7 min. The specific fragments expected toappear in the mass spectra of C₆-HSL and C₆-HS are all present;fragment 200 is characteristic for C₆-HSL, while fragment 218is characteristic for C₆-HS. Fragment 102 corresponds to HSLand therefore appears in the C₆-HSL spectrum, while fragment120 is characteristic for HS and appears in the C₆-HS spectrum(28).

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FIG. 1. Scheme for identification of N-AHSL-degradation products. N-AHSL inactivation in bacteria proceeds through two known pathways: lactonolysis (top) or amide hydrolysis (bottom). N-AHSL lactonolysis is a reversible reaction which yields the corresponding N-AHS, which can be separated by HPLC and identified by mass spectrometry (MS). Amide hydrolysis is an irreversible reaction which yields HSL and the corresponding acyl side chain. HSL can be detected by HPLC after trapping the free amine with dansyl chloride.

Library construction.
As strain W2 is especially difficult to lyse by conventionalmethods, genomic DNA preparation was based on the method ofEulberg et al. (7) with the following modifications. StrainW2 was grown overnight in 50 ml of LB medium buffered at pH8.0 with glycine (3%, wt/vol). Prior to DNA extraction, cellswere treated for 2 hours with ampicillin and erythromycin (100µg/ml, final concentration) to disturb cell wall synthesisand favor the subsequent lysis of the cells. A genomic libraryof strain W2 was constructed by partially digesting 100 µgof total genomic DNA with Bsp143I (Sau3AI) and ligating thefragments in BamHI-linearized cosmid vector pCP13/B as describedby Hayman and Farrand (11). The ligation products were packagedusing the Gigapack III Gold packaging kit (Stratagene, La Jolla,CA) as recommended by the manufacturer and recovered by transfectioninto E. coli VCS257. The W2 genomic library generated in thisstudy contained over 4,000 clones, with an average insert sizeof 23 kb and fewer than 1% empty clones. This corresponds toa theoretical coverage of 15 times of Rhodococcus genome (1).

Screening of the genomic library for N-AHSL degradation ability.
A total of 2,880 library clones were screened directly in theirE. coli strain VCS257 host by use of a modification of the microplateN-AHSL degradation assay described by Uroz et al. (44), usingC. violaceum CV026 as an indicator strain. Clones were firstgrown in presence of antibiotics and then subcultured in mediumdevoid of antibiotics but supplemented with 25 µM of C₆-HSL.Cosmid clones were considered to confer the ability to degradeN-AHSLs only if a total disappearance of the C₆-HSL was observedafter a 24-h period. The ability of the clones to effectivelydegrade the N-AHSL was confirmed by separating the degradationproducts by TLC and detecting the presence of N-AHSL by theappropriate biosensor. This allows the detection of false-positivedegradation due to the presence of compounds inhibiting N-AHSLdetection or the growth of the biosensor.

DNA sequence analysis.
Sequence analysis was performed with ORF FINDER. Nucleotideand amino acid sequence comparisons were made using the BLASTprotocol. Multiple alignments were performed using the Pileupsubroutine of the GCG package (version 10; GCG Inc., Madison,WI).

Subcloning of the qsdA region.
To identify the gene coding for the N-AHSL degradation activity,the 3.2-kb EcoRI fragment was subcloned as shown in Fig. 2 usingthe primers QS (5'-ATGAGTTCAGTACAAACCGTTCGTG-3'), QR (5'-TCAGCTCTCGAAGTACCGACGTGGG-3'),and IR (5'-TCACCATTTTTCAACGGCCG-3') and available restrictionsites. The qsdA gene was disrupted by insertion of a gentamicinresistance gene from pUC1318::Gm, cloned as an XmaI fragmentat the unique AgeI site of the qsdA gene. Southern hybridizationwas performed at 62°C with a qsdA probe amplified with theQS/QR primer pair and digoxigenin labeled according to the manufacturers'instructions (Roche/Boehringer Mannheim).

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FIG. 2. Identification of the qsdA gene. The genetic organization of the qsdA locus derived from the complete sequence of the 3.2-kb EcoRI fragment conferring C₆-HSL degradation upon its host is shown at the top. Broken arrows symbolize the primers used for subcloning and their orientations. Restriction sites also used for subcloning are shown. See Table 1 for a description of the plasmids. Plasmid constructions used to identify the qsdA gene were assayed for their ability (+) or inability (–) to confer N-AHSL degradation upon their host against a set of N-AHSLs, including C₆-HSL, O-C₆-HSL, C₇-HSL, C₈-HSL, O-C₈-HSL, O-C₁₀-HSL, C₁₂-HSL, O-C₁₂-HSL, and O-C₁₄-HSL. Each construct gave identical degradation results regardless of the N-AHSL present in the medium.

QQ assays.
The ability of the cloned qsdA genes to interfere with the expressionof QS-regulated functions was tested in heterologous expressionsystems. Plasmid pSU40 (qsdA_W2 cloned into pME6032) was transferredto E. coli strain DH5 ${alpha}$ , Pseudomonas fluorescens strain 1855-344,and the octopine-type conjugal transfer constitutive strainAgrobacterium tumefaciens 15955tra^c to yield DH5 ${alpha}$ (pSU40), 1855-344(pSU40),and 15955tra^c (pSU40), respectively. The ability of these strainsto interfere with QS-regulated functions was first evaluatedusing the streak assay described by Molina et al. (23), usingC. violaceum CV026 as an indicator strain. The ability to interferewith virulence in Erwinia carotovora was tested in the potatotuber assay as described by Uroz et al. (44). Additionally,the ability of the qsdA locus to interfere intracellularly withQS was tested in Agrobacterium tumefaciens. Ti plasmid conjugationassays were performed essentially as described by Oger et al.(26), using strain 15955tra^c as a transfer constitutive donorand C58C1RS as a recipient. In all experiments, strains harboringthe empty vector pME6032 were used as negative control.

Nucleotide sequence accession numbers.
The qsdA locus sequence has been deposited at GenBank underaccession number AY541692, and the qsdA allele sequences havebeen deposited under accession numbers EF218062, EF218066, EF218065,and EF589962 (R. erythropolis strains SQ1, MP50 CECT 3008, andMic1, respectively), EF218064 (R. corynebacteroides CECT 420),and EF218063 (R. rhodochrous strain CECT 3042).

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Identification of the Rhodococcus erythropolis gene involved in N-AHSL degradation.
Genes involved in QS signal degradation were isolated from agenomic library of R. erythropolis strain W2 on the basis ofthe ability of specific cosmids to confer C₆-HSL degradationcapability upon the E. coli host cells. Degradation was clearlyevidenced for 20 out of the 2,880 clones screened. Based ontheir restriction patterns, the 20 cosmids fell into four groups,which all shared single common EcoRI and BamHI fragments ofca. 3.2 and 3.5 kb, respectively. The cosmid pSU16, which harborsthe smallest insert, was chosen for further studies. DNA fragmentsresulting from BamHI or EcoRI restrictions of pSU16 DNA wereshotgun subcloned into pUC19. Only two sets of clones, containingeither a 3.5-kb BamHI (pSU16-12) (not shown) or a 3.2-kb EcoRI(pSU16-11) (Fig. 2) fragment conferred upon their E. coli hostthe ability to degrade N-AHSLs with acyl chains ranging fromC₆- to C₁₂-HSL, independently of the substitution at carbon3. HPLC analyses of the growth media of these clones confirmedthe disappearance of the N-AHSL (data not shown).

The sequence of the EcoRI fragment of pSU16-11 (3,152 bp; GenBankaccession number AY541692) contains two incomplete open readingframes (ORFs) and two complete ORFs, which were named orf1 andqsdA (for QS signal degradation) (Fig. 2). The first incompleteORF (to the right in Fig. 2) shows similarities to alleles ofthe gntR/fadR family of transcriptional regulators and is mostclosely related to that of Pseudomonas aeruginosa strain PAO1(PA1627) (identity, 40%; similarity, 58%). The first completeORF, orf1, could encode a serine-rich protein of 172 amino acids.It does not show any significant homology with peptide sequencesavailable in the databases, suggesting that it might be a pseudogene.The second complete ORF, qsdA, could encode a protein of 323amino acids related to members of the PTE superfamily, whichis found in a wide range of organisms from bacteria to eukarya.PTEs are zinc metalloenzymes which were initially identifiedas efficiently catalyzing the hydrolysis of a variety of organophosphoruscompounds (13), but a growing number of PTE homologues whichalso show amidohydrolase or lactonase activities have been characterized(36). Finally, the protein encoded by the incomplete orf3 shows48% identity with proteins annotated as acyl coenzyme A synthetases(AMP-forming)/AMP-acid ligases II of Ralstonia metalliduransand other enzymes related to the lipid metabolism and transport.Acyl coenzyme A synthetases are involved in both the synthesisand turnover of fatty acids in bacteria.

Because the ORFs qsdA and orf1 were the only uninterrupted ORFspresent in pSU16-11, they were likely to determine the C₆-HSLdegradation ability conferred by that clone. To confirm thishypothesis, various constructions were generated (Fig. 2). Asexpected, the constructions lacking one or both partial ORFs(e.g., pSU8-1 and pSUqsdA_W2) still conferred N-AHSL-degradingcapabilities upon E. coli. Conversely, constructions lackingqsdA (pSU reg1) or harboring a disrupted qsdA gene (pSU8-1::Gm)did not confer N-AHSL degradation ability upon their host. Fromthese results, it is clear that qsdA is necessary and sufficientto code for N-AHSL degradation in the original pSU16 cosmid.

qsdA codes for a PTE-like N-AHSL lactonase.
The identification of the degradation products of N-AHSLs wasperformed by a combination of HPLC and mass spectrometry analysesto detect the presence of HSL or N-AHS generated by amidolysisor lactonolysis, respectively (Fig. 1). The spontaneous degradationwas evaluated in control experiments that used uninoculatedLB medium or medium inoculated with E. coli DH5 ${alpha}$ or DH5 ${alpha}$ withthe empty cloning vector. Figure 3A presents results obtainedfor strain DH5 ${alpha}$ (pME6032). In each experiment, two peaks werevisible on the HPLC spectra after a 24-h incubation. Their positionat 15.8 and 21 min as well as mass spectra correspond to C₆-HSand C₆-HSL, respectively. The presence of C₆-HS in the controlexperiments results from the spontaneous lactonolysis of C₆-HSLin aqueous medium. Each control condition yielded the same amountof spontaneous lactonolysis (ca. 25%; data not shown), showingthat DH5 ${alpha}$ does not itself facilitate the degradation of N-AHSLs.At the end of experiments performed with E. coli DH5 ${alpha}$ (pSU40)expressing qsdA, no N-AHSL could be detected (Fig. 3B), indicatingthat a complete conversion of the initial C₆-HSL occurred. Attemptsto detect the presence of HSL, which would indicate a cleavageof the N-AHSL molecules by an amidohydrolase, failed (data notshown). At the same time, the presence of C₆-HS was clearlyvisible (Fig. 3). Thus, qsdA must code for an N-AHSL lactonaseactivity, the presence of which in Rhodococcus has been recentlyidentified by Park and colleagues (29).

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FIG. 3. QsdA lactonase activity. E. coli strains DH5 ${alpha}$ (pME6032) and DH5 ${alpha}$ (pSU40) were incubated in pH 6.5 buffered LBm medium with 50 µM C₆-HSL for 24 h. The medium was analyzed at 0 and 24 h by HPLC-mass spectrometry. Under the experimental conditions used, C₆-HS (molecular weight, 217) and C₆-HSL (molecular weight, 199) had retention times of 15.8 and 21 min, respectively, and mass spectra were composed of the following main fragments: m/z = 218, 200, and 120 for C₆-HS, and m/z = 200, 102, and 99 for C₆-HSL. (A) Spontaneous degradation of C₆-HSL in aqueous medium. (B) DH5 ${alpha}$ (pSU40) after 24 h of incubation. A single peak at a retention time of 15.8 min is visible on the HPLC spectrum, which is identified as C₆-HS. C₆-HSL has completely disappeared from the medium. The formation of C₆-HS, correlated with the absence of HSL in the medium, is indicative of a lactonase activity.

QsdA clearly belongs to the PTE family of zinc-dependent metalloproteins(Fig. 4). It is totally unrelated to the known bacterial N-AHSLlactonases, which belong to the Zn-dependent glyoxylase family,or to the known N-AHSL amidohydrolases, which belong to thecluster of β-lactam acylases. Thus, the present study extendsthe number of families of N-AHSL-degrading enzymes of bacterialorigin to three and to include the PTE family. PTEs were firstdescribed for their ability to cleave the phosphotriester bondin phosphotriesters but were later shown to be promiscuous enzymes,harboring lactonase or amidohydrolase activities (36). QsdA,however, does not confer the ability to degrade the prototypicphosphotriester methyl parathion, nor does the prototype PTEgene, opd, code for N-AHSL degradation (data not shown). AlthoughQsdA exhibits the canonical structure of PTEs, the sequenceof its metal binding site, which is central to the enzyme activityby fixing two molecules of zinc and forming the catalytic pocketin which the substrate inserts, differs from the consensus sequenceof PTEs at 3 of 12 positions (CD1 and CD2 domains in Fig. 4).In contrast, the sole PTE/QsdA homologue known from Rhodococcus,i.e., the TIG:13193 gene from strain RHA1 (Rsp_RHA1 in Fig.4), as well as the closest homologues of QsdA, i.e., the PTEalleles of several Mycobacterium strains and other actinobacteria(Mtu_CDC1551 in Fig. 4), all present the canonical signatureof the PTEs, including the 12 conserved amino acids from thezinc binding domain. Interestingly, strain RHA1 and Mycobacteriumsp. strain Tn20 are both unable to degrade N-AHSLs. In addition,when cloned and expressed in E. coli, the TIG:13193 ORF fromRHA1 does not confer N-AHSL degradation capabilities to itshost. Thus, the lack of N-AHSL degradation in strain RHA1 isdue not to a lack of expression of the protein from its nativepromoter but to the lack of N-AHSL lactonase activity of theTIG:13193 protein, which will consequently be referred to notas a QsdA but as a classical PTE homologue. QsdA is thereforethe first prokaryotic member of the PTE family with demonstratedN-AHSL lactonase activity.

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FIG. 4. Alignment of selected PTEs from bacterial and eukaryotic origins. In addition to QsdA from R. erythropolis strain W2 (Rer_W2), PTE homologues used in this alignment include a putative QsdA homologue from Rhodococcus corynebacterioides CECT 420 (Rco_420, this study); putative PTEs from Rhodococcus sp. strain RHA1 (Rsp_RHA1, accession no. TIG:13193), Agrobacterium tumefaciens strain C58 (Atu_C58, accession no. AAL43882), Pseudomonas syringae pv. Syringae strain B728a (Psy_B728a, accession no. YP_233869), Shigella flexneri strain 2457T (Sfl_2457T, accession no. AAP19319), Mycobacterium tuberculosis strain CDC1551 (Mtu_CDC1551, accession no. AAK44461), and Escherichia coli K-12 (Eco_K12, accession no. AAC76404); PTEs from Chryseobacterium balustinum strain BC9 (Cba_BC9, accession no. CAD19996); OPD from Flavobacterium sp. strain ATCC 27551 (Fsp_ATCC27551, accession no. CAD13181); and PTER from Homo sapiens sapiens (Hsa, accession no. Q96BW5) and from Mus musculus (Mmu, accession no. Q60866). The positions of the essential amino acids residues of the catalytic site (A), the substrate binding site (S), and the dimerization domains (H) are shown above the alignment. CD1 and CD2, zinc binding conserved domains 1 and 2. The 27 residues conserved among known PTE sequences are shown in reverse font. QsdA-specific amino acid substitutions which are not observed in other PTEs (including alleles not shown in the figure) are highlighted by a gray background.

QsdA is a Rhodococcus-specific PTE-like N-AHSL lactonase.
The wide occurrence of PTE homologues in species closely relatedto R. erythropolis and the specific signature and activity ofQsdA prompted us to determine the phylogenetic distributionof this gene in the actinobacterial cluster. We screened a collectionof related strains belonging to the Rhodococcus genus, i.e.,R. corynebacteroides (one strain), R. erythropolis (five strains),R. fascians (one strain), R. rhodochrous (one strain), and R.opacus (one strain), for their ability to inactivate N-AHSLsand the presence of the qsdA gene. The ability to degrade N-AHSLsappeared ubiquitous among strains of R. erythropolis, sinceall assayed isolates (5/5) degraded a range of N-AHSLs identicalto that previously reported for strain W2 (acyl chain lengthfrom C₆ to C₁₄, with or without substitution on C3 [see TableS1 in the supplemental material]). N-AHSL degradation was observedin three of the five Rhodococcus species tested, i.e., R. erythropolis,R. rhodochrous, and R. corynebacteroides, but not in R. fasciansand R. opacus, although these species are more closely relatedto R. erythropolis than are R. rhodochrous and R. corynebacteroides(Fig. 5), and not in the polychlorinated biphenyl-degradingRhodococcus sp. strain RHA1. The degradation ability was alsotested in members of genera closely related to Rhodococcus,such as Pseudonocardia autotrophica (strain CECT 3044), Corynebacteriumglutamicum (strain ATCC14752), Gordonia alkalivorans (strain98), Microbacterium sp. (strain POLB142), Mycobacterium sp.(strain Tn20), Nocardia asteroides (strain CECT 3051), Actinoplanesutahensis (strain NRRL 12052), and Flavobacterium sp. (strainATCC 27551). None of these assayed strains degraded N-AHSLs.

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FIG. 5. Phylogenetic distribution of N-AHSL degradation in Rhodococcus. The ability to degrade N-AHSLs among bacteria is highlighted on a phylogenetic tree based on the 16S rRNA gene. Strains shown in bold were tested in the present study. N-AHSL degradation data from the literature are reported for all other strains. Wherever possible the nature of the enzymatic activity is noted: A, N-AHSL amidohydrolase; L, N-AHSL lactonase; O, N-AHSL oxidoreductase; u, undetermined. The number of identified activities is also reported (e.g., A, A indicates two amidohydrolases). The presence (+) or absence (–) of a qsdA homologue was determined by hybridization with a qsdA probe. The N-AHSL-degrading strains belonging to the Rhodococcus genus are boxed. Agrobacterium tumefaciens strain C58 harbors two N-AHSL lactonases (AttM and AiiB), and R. erythropolis strain W2 harbors at least three N-AHSL modification/degradation enzymes: an amidohydrolase (A), an oxidoreductase (O), and a lactonase (L). NT, not tested.

The qsdA gene was detected by Southern hybridization, underlow-stringency conditions using the cloned qsdA gene from strainW2 as a probe, in all but one of the strains able to degradeN-AHSLs, including R. corynebacteroides, R. erythropolis, andR. rhodochrous. Southern hybridization performed on genomicDNA restricted with EcoRI, BamHI, and HindIII confirmed thatthe gene is present as a single copy in the genomes of thesestrains. The only R. erythropolis strain that did not hybridizewith the qsdA probe was strain DCL14 (46). This strain is indistinguishablefrom strain W2 in terms of 16S rRNA gene sequence and abilityto inactivate QS molecules. This result is, however, consistentwith the observation by Uroz et al. (43), who demonstrated theoccurrence of N-AHSLase activities other than lactonase in R.erythropolis strains W2 and DCL14. The qsdA gene could not bedetected in any of the Rhodococcus strains unable to degradeN-AHSLs or in the strains from the related genera.

A total of six qsdA alleles (in addition to qsdA from strainW2) were cloned into pGEM-T Easy following PCR amplificationfrom the genomic DNAs of the four R. erythropolis clones, aswell as the R. rhodochrous and R. corynebacteroides strainsshowing a positive hybridization signal in Southern analysis.All alleles conferred the ability to degrade N-AHSLs upon E.coli DH5 ${alpha}$ . The deduced QsdA peptides fell into two homology groups,which were termed A1 and A2 (Fig. 6A). Sequences (DNA and protein)were almost identical within each group and were ca. 88% identicaland 93% similar at the protein level between groups A1 and A2.The conserved zinc binding domains CD1 and CD2 of the QsdA allelesof clusters A1 and A2 diverge at one position (residue 167)(Fig. 6A). Meanwhile, domains CD1 and CD2 of allele clustersA1 and A2 also diverge from the consensus PTE domains at twoor three positions (QsdA residues 19, 167, 169), suggestingthat the six alleles might derive from the same PTE ancestor.However, the qsdA allele phylogeny is not congruent with theRhodococcus 16S rRNA gene phylogeny (Fig. 6B). Furthermore,the Rhodococcus species harboring these alleles do not appearto form a clade within the Rhodococcus genus (Fig. 5). Thissuggests that qsdA is a Rhodococcus-specific N-AHSL lactonasethat evolved in this genus and was transferred horizontallyat several points during the speciation of Rhodococcus.

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FIG. 6. Alignment of the conserved zinc biding domains of QsdA homologues. (A) OPD F.asp, canonical PTE sequence from Flavobacterium sp. strain ATCC 27551; RHA1 R.sp, deduced peptide sequence from the PTE homologue from Rhodococcus sp. strain RHA1 encoded by gene TIG:13193; PHP M.sme, PTE homologue from Mycobacterium smegmatis; consensus PTE, the 12 conserved amino acids from the PTE zinc binding domains CD1 and CD2 are indicated (#). Nonconserved positions are indicated in lowercase letters. qsdA-deduced protein sequences used in this alignment include those from Rhodococcus corynebacteroides strain CECT 420 (QsdA_420), Rhodococcus rhodochrous strain CECT 3042 (QsdA_3042), and Rhodococcus erythropolis strains SQ1 (QsdA_SQ1), CECT 3008 (QsdA_3008), W2 (QsdA_W2), Mic1 (QsdA_Mic1), and MP50 (QsdA_MP50). Divergences from the consensus PTE zinc domain sequence are underlined in the QsdA sequences. Two divergent alleles of QsdA (A1 and A2) are found in Rhodococcus. (B) Taxonomic relationship between strains harboring allele A1 (black background) and strains harboring allele A2 (white background) based on a 16S rRNA gene phylogeny.

qsdA confers QS quenching abilities in heterologous systems.
We tested the ability of all the cloned qsdA alleles, includingqsdA_W2 as well as qsdA₄₂₀, qsdA_SQ1, qsdA₃₀₀₈, qsdA_MP50, qsdA_Mic1and qsdA₃₀₄₂, to interfere with the expression of QS-regulatedfunctions, using three different quenching assays in which qsdAis expressed in heterologous systems.

All alleles conferred upon their host the ability to inactivatethe same range of N-AHSLs as the wild-type strain W2 (i.e.,N-AHSLs with or without substitution on carbon 3 and with anacyl chain ranging from 6 to 14 carbons [data not shown]), regardlessof the plasmid vector used to express the gene. They were ableto quench the synthesis of violacein by C. violaceum CV026 grownin the presence of C₆-HSL with the same efficiency as the wild-typeR. erythropolis strain W2 (data not shown).

When expressed in P. fluorescens strain 1855-344, qsdA_W2 conferredquenching capabilities closely matching that of the wild-typeR. erythropolis strain W2 (Table 2). Strains 1855-344(pSU40)and 1855-344(pME6032) were inoculated independently at variousratios with the potato soft rot pathogen P. carotovorum strainPCC797 on potato tubers. In the absence of the quencher, themaceration zones averaged 2.8 cm on the potato tubers. Conversely,in the presence of the quenching strain expressing qsdA_W2, aclear reduction of symptom severity was visible, since no macerationoccurred at quencher/pathogen ratios of 10:1 and 1:1 whateverthe concentration of pathogen used for the assay (10⁵ or 10⁶cells ml^–1).

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TABLE 2. QQ capabilities of P. fluorescens strain 1855-344 harboring qsdA

When expressed in the Agrobacterium tumefaciens conjugal transferconstitutive strain 15955tra^c, qsdA_W2 prevented the accumulationin the medium of the N-AHSLs normally produced by this strain(data not shown). In addition, the qsdA_W2 gene conferred uponthis strain the ability to degrade N-AHSLs provided exogenously(data not shown). Furthermore, the expression of the qsdA_W2gene totally abolished Ti plasmid conjugal transfer by thisstrain. Indeed, 15955tra^c(pME6032) transferred its Ti plasmidto the recipient strain C58C1RS at high frequency (1.7 10^–2transconjugants per donor cells), while 15955tra^c(pSU40) nolonger conjugated its Ti plasmid to detectable levels; i.e.,transfer frequencies were below the detection level of 5 x 10^–8transconjugants per donor cell. Therefore, the qsdA-based N-AHSL-turnoverwas sufficient to prevent the accumulation of the typical A.tumefaciens QS signal molecules in the growth medium and theQS regulation of Ti plasmid conjugal transfer.

Due to the worldwide distribution of this genus in the soiland to the marked quenching abilities of the wild-type strainsand cloned qsdA genes, Rhodococcus isolates and derivativesharboring this gene could become interesting natural biocontrolagents directed toward QS-regulated traits in plant pathogens.

Role of qsdA in Rhodococcus.
The above observations raise questions regarding the role ofqsdA in Rhodococcus. The analysis of the neighboring sequences(encoding an acyl coenzyme A synthetase and a FadR peptide analogousto a fatty acid biosynthesis regulatory protein) suggests apossible involvement in fatty acid metabolism. The observationthat even closely related clones, such as R. erythropolis strainDCL14, or species, such as R. opacus and R. fascians, do notpossess the qsdA gene or a N-AHSLase activity supports the viewthat the function encoded by qsdA may be either nonessentialor redundant. This view is also supported by the observationthat a W2 qsdA mutant (harboring a disrupted qsdA gene) hasgrowth properties and N-AHSL degradation ability similar tothose of the wild-type parent (data not shown). In such a light,the hypothesis suggested by Kaufmann et al. (17) that 3-oxo-dodecanoyl-HSLand spontaneous reorganization products could play a role asantibiotics targeting specifically gram-positive bacteria isa very tempting alternative explanation accounting for the presenceof qsdA within the Rhodococcus genus. Strain W2 and relatedN-AHSL degraders therefore appear very well equipped to resistN-AHSLs produced by gram-negative soil competitors, with a completedegradative arsenal composed of at least two N-AHSL degradationactivities, including a lactonase and an amidohydrolase, andan additional N-AHSL modification activity (29, 43). The putativetoxicity of N-AHSLs on Rhodococcus remains to be demonstrated,since it has not been observed in preliminary experiments performedwith the wild-type strains used in the present work.

ACKNOWLEDGMENTS

This work was made possible by grants from the European Union5th Framework Program EcoSafe (QLK3-2000-01759) to Y.D., fromthe French Ministère de la Recherche et de la TechnologieGEOMEX program to P.M.O., and from the French Ministèrede la Recherche et de la Technologie to S.U., all of which aregratefully acknowledged.

We thank Claudine Elmerich and Carmela Giglionne (CNRS, Gif-sur-Yvette),Annie Chaboud (IBCP-Lyon), Mohammed Bendahmane (ENS-Lyon), Robertvan der Geize (University of Groningen), and Paul Williams (Universityof Nottingham) for helpful discussions. We especially acknowledgeall the colleagues who provided the strains tested for N-AHSLdegradation in this study.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Sciences de la Terre, UMR CNRS 5570, Ecole Normale Supérieure, 46, Allée d'Italie, 69364 Lyon Cedex, France. Phone: (33) 4 72 72 87 92. Fax: (33) 4 72 72 86 77. E-mail: poger{at}ens-lyon.fr

Published ahead of print on 11 January 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Interactions Arbres Microorganismes, INRA,54280 Champenoux, France.

REFERENCES

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