Catabolic Pathway of Gamma-caprolactone in the Biocontrol Agent

Nov 16, 2011 - 55 rue Saint-Germain, 27000 Evreux, France. ‡. SPPN - Comitй Nord, Station de Recherche et de Crйation Variйtale, 76110 Brettevill...
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Catabolic Pathway of Gamma-caprolactone in the Biocontrol Agent Rhodococcus erythropolis Corinne Barbey,† Alexandre Crepin,†,‡ Amelie Cirou,‡,§ Aurelie Budin-Verneuil,|| Nicole Orange,† Marc Feuilloley,† Denis Faure,§ Yves Dessaux,§ Jean-Franc-ois Burini,† and Xavier Latour*,† †

)

Laboratoire de Microbiologie Signaux et Microenvironnement, Normandie Universite, EA 4312 Universite de Rouen, IUT Evreux 55 rue Saint-Germain, 27000 Evreux, France ‡ SPPN - Comite Nord, Station de Recherche et de Creation Varietale, 76110 Bretteville du Grand Caux, France § Institut des Sciences du Vegetal, CNRS UPR2355, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France Laboratoire de Microbiologie de l’Environnement, Normandie Universite, USC INRA 2017 EA 956 IFR ICORE 146 Universite de Caen, 14032 Caen cedex, France ABSTRACT: Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by twodimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by β- and ω-oxidation coupled to the Krebs cycle and β-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones. KEYWORDS: protein identification, MALDI, Rhodococcus erythropolis, γ-caprolactone, lactonase, quorum quenching, biostimulation

’ INTRODUCTION 5-Ethyl-dihydro-2(3H)-furanone or more simply γ-caprolactone (GCL, CAS number: 695-06-7) is a cyclic monoester with a five-member ring belonging to the butyrolactone family. In nature, this compound is produced by plants, as a component of aromas flower flagrances and aromas of fruits and vegetables.1 It is also a pheromone for the damaging grain beetle Trogoderma granarium.2 GCL is best known for its usage as a food additive, in products intended for human consumption. It is a colorless oily liquid with a nutty and malty odor. It is prepared industrially by oxidation of phenyl or cyclohexanone groups and commonly used in beverages, ice cream, candy and tobacco.13 GCL is also a chemical promoting the growth of biocontrol bacteria aimed at soft-rot pathogens.4 Added to the plant growth substrate in hydroponic or soil systems, GCL promotes the growth of bacteria capable of degrading both this substrate but also N-acyl-homoserine lactones (NAHSLs).5,6 NAHSLs are synthesized by several Proteobacteria as small diffusible signaling molecules involved in cell-to-cell communication, named quorum sensing (QS) systems.7 The key role of NAHSLs as signals that control the production of macerating enzymes (and sometimes that of harpin toxins) in soft-rot plant pathogens Pectobacterium spp. has been extensively r 2011 American Chemical Society

investigated.8,9 The resulting diseases affect several important crops such as the world’s fourth most produced food commodity, the potato Solanum tuberosum L.10,11 Indeed, an insertion in the bacterial gene that codes the enzyme responsible for NAHSL synthesis or the intracellular degradation of signaling molecules before their release in the microenvironment suffice to suppress all maceration symptoms on potato tuber and defense reaction mechanisms (hypersensitive response) on nonhost plants.12,13 As a result of these observations, Pectobacterium and its NAHSLQS system have become the target for novel biocontrol strategies using NAHSL-degrading bacteria stimulated by GCL amendment.14,15 To investigate the biostimulating properties of GCL, this compound was applied to cultures of S. tuberosum grown under hydroponic conditions.5,6 A significant increase of the ratio of NAHSL-degrading bacteria among total cultivable bacteria was observed. Most of these bacteria, the growth of which was stimulated by GCL amendment, were also able to use GCL as a sole carbon Special Issue: Microbial and Plant Proteomics Received: September 15, 2011 Published: November 16, 2011 206

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Table 1. Bacterial Strains, Plasmids and qsdA Gene Occurrence characteristicsb

strain or plasmid

presence of qsdAa

source or reference

Rhodococcus erythropolis strains tested for presence of the qsdA gene W2

NAHSL degrading strain isolated from soil

+

Uroz et al.46

DCL14

Limonene degrading strain isolated from a ditch sediment sample in The Netherlands

+

De Carvalho and da Fonseca35

R54

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R74

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R78

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R107

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R108 R130

NAHSL degrading isolate recovered from hydroponic culture of potato plants NAHSL degrading isolate recovered from hydroponic culture of potato plants

+ +

Cirou et al.5 Cirou et al.5

R132

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R133

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R134

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R135

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R136

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R138

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

R139 R140

NAHSL degrading isolate recovered from hydroponic culture of potato plants NAHSL degrading isolate recovered from hydroponic culture of potato plants

+ +

Cirou et al.5 Cirou et al.5

R141

NAHSL degrading isolate recovered from hydroponic culture of potato plants

+

Cirou et al.5

DH5α

supE44 ΔlacU169 (Φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1



Lab collection

BL21(DE3)

FompT hsdSB(rB‑mB‑)gal dcm



Novagen

BL21(DE3)

BL21(DE3) strain with the empty vector pET22b+



This study

BL21(DE3) strain expressing the qsdA gene

+

This study

Escherichia coli strains

[pET22] BL21(DE3) [pET22-qsdA]

Plasmids pET22b+ pET22-qsdA a

Novagen qsdA ORF amplified and cloned into pET22b+

This study

The presence (+) or absence (-) of a qsdA homologue was determined by PCR. b NAHSL, N-acyl homoserine lactone.

source. They mainly belong to the Rhodococcus erythropolis species, an Actinobacterium able to degrade numerous recalcitrant molecules (for review see de Carvalho and da Fonseca16). Interestingly, only R. erythropolis strains isolated from the hydroponic cultures supplemented with GCL showed biocontrol activity against Pectobacterium in tuber assays, suggesting that GCL stimulates the growth of effective NAHSL-degrading species.5,6 Altogether, these results designate GCL as both a potential biostimulator and biocontrol compound, probably because GCL exhibits a γ-butyrolactone ring coupled with an aliphatic chain, two traits common to NAHSL signals. To our knowledge, no catabolic pathway of GCL is known in prokaryote or eukaryote cells. The aim of the present study was to characterize the metabolic pathway involved in the degradation of GCL by R. erythropolis, using a proteomic approach that included two-dimensional gel electrophoresis coupled to matrixassisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. To achieve this goal, the proteins synthesized by a model strain of R. erythropolis grown in the presence of GCL or an aliphatic structural analogue were compared. The role of a particular lactonase involved in the first step of GCL degradation was demonstrated and verified by expression in a heterologous host (Escherichia coli). The function of this enzyme

in Rhodococcus metabolism and its occurrence in other strains of R. erythropolis are discussed.

’ MATERIALS AND METHODS Bacterial Strains, Growth Media and Culture Conditions

Bacterial strains and plasmids are listed in Table 1. E. coli strains were grown in LuriaBertani medium (AES Chemunex, Bruz, France). For R. erythropolis strains, the nonselective growth medium was TY which is made up of 0.5% tryptone (Difco, Le Pont de Claix, France), 0.2% yeast extract (Difco) supplemented with 5% (v/v) of a phosphate source (6% K2HPO4, 4% NaH2PO4, Merck, Fontenay-sous-Bois, France). For growth assessment and protein analysis of R. erythropolis R138, experiments were performed using the minimal medium described by Leadbetter and Greenberg,17 with the following modification: a vitamin solution was replaced by yeast nitrogen base without amino acids (Difco). The autoclaved medium was supplemented with one of the following carbon sources purchased from Sigma-Aldrich (St. Quentin Fallavier, France): hexanoate sodium salt (6 mM), γ-butyrolactone (GBL, 9 mM), γ-valerolactone (GVL, 7.2 mM), GCL (6 mM), γ-heptalactone (GHL, 5.14 mM), γ-octalactone (GOL, 4.5 mM). When necessary, growth media were supplemented with ampicillin (100 mg/L, Sigma-Aldrich, France) and 207

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Bio-Rad Laboratories). Three replicate gels were first analyzed individually in the software. The software detects spots on every single gel image separately. The gel image with the best spot quality was then selected as template (Master) and all spots of the remaining gel images were matched onto it to create the MatchSet Master. For each MatchSet, the mean and standard deviation (SD) of spot intensity were calculated by comparing spot intensities of the three replicate gels in the two culture conditions. Individual protein quantities were expressed as parts per million (ppm). Unique spots to each tested culture condition and common spots with intensity superior or equal to 10 ppm were selected for further analysis by trypsin digestion and MALDI-MS analysis.

solidified with agar (15 g/L, AES Chemunex). R. erythropolis strains were grown at 25 °C and Escherichia coli at 37 °C on a rotary shaker (180 rpm). Growth was monitored spectrophotometrically at 580 nm. All cultures were inoculated at an initial Optical Density (OD) of 0.05. Protein Extraction

R. erythropolis R138 protein extracts were prepared from cells obtained from 250 mL cultures grown in the presence of sodium hexanoate or GCL as a sole carbon source. When an OD580 of 0.5 was reached, the cells were harvested at 10000 g for 15 min at 4 °C. They were then washed three times in an alkaline buffer, 50 mM Tris (Sigma-Aldrich) pH 8 by centrifugation at 10000 g for 15 min at 4 °C. After pellet resuspension in the above alkaline buffer supplemented with a protease inhibitor cocktail (SigmaAldrich), the cells were disrupted by sonication (six times, 1 min bursts with 2 min breaks) on ice. Unbroken cells were removed by centrifugation at 10000 g for 15 min at 4 °C. Proteins were precipitated with 10% (w/v) trichloroacetic acid (TCA, SigmaAldrich) on ice for 1 h. The precipitated proteins were harvested by centrifugation at 13000 g for 20 min at 4 °C, washed three times in cold acetone (Fluka, quality upgraded, St. Quentin Fallavier, France), dried and dissolved in rehydration solution containing 7 M urea (Fluka), 2 M thiourea (Sigma-Aldrich), 2% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, Bio-Rad Laboratories, Marnes la coquette, France), 2% (w/v) 3-(decyldimethylammonio)-propane-sulfonate inner salt (SB310, Sigma-Aldrich), 1% (v/v) ampholytes pH 310 (Bio-Rad Laboratories), 70 mM dithiothreitol (DTT, Fluka) and 5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Sigma-Aldrich). Protein concentration was determined according to the Bio-Rad protein assay (Bio-Rad Laboratories) using bovine serum albumin as the standard.

In-gel Trypsin Digestion

A procedure slightly modified from the one developed by Schevchenko et al.18 was used for in-gel digestion. Briefly, spots of interest were manually excised from 2-DE gels and washed in 100 mM ammonium bicarbonate (Sigma-Aldrich)/acetonitrile (Merck) (1:1, v/v) until completely destained. After drying, gel fragments were placed on ice in 20 μL of trypsin solution (Trypsin Gold Mass Spectrometry grade, Promega, Charbonnieres, France, at 13 ng/μL in 10 mM ammonium bicarbonate containing 10% (v/v) acetonitrile) for 30 min. Then, digestion was performed at 37 °C for 16 h. Peptide extraction was performed twice for 1 h with 20 μL 50% acetonitrile/5% formic acid (Sigma-Aldrich). Trypsin digests were then concentrated in a Speed Vac (Thermo Fisher Scientific, Courtaboeuf, France) to about 10 μL. Some samples were desalted using Zip-Tips (C18 resin; P10, Millipore Corporation, Molsheim, France) according to the protocol described by Dauly et al.19 Mass Spectrometry Analysis

In-gel trypsin digestion products were analyzed by a Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometer (MALDI-TOF/TOF LIFT, AutoFlexIII, Bruker Daltonics, Wissembourg, France) in positive/reflector mode controlled by FlexControl software Version 3.3. Instrument calibration was achieved by using peptide calibration standard II (Bruker Daltonics) as a reference. Samples were spotted to MTP 384 ground steel targets (Bruker Daltonics) using standard protocol for the dried droplet method: 1 μL of peptide mixture was spotted onto the target plate, and 1 μL of the freshly prepared α-cyano-4hydroxycinnamic acid matrix solution (10 mg/mL in a solution of 0.1% trifluoroacetic acid and 50% acetonitrile) was added onto each sample. Each spectrum was established over an average of 5001000 laser shots. The FlexAnalysis software Version 3.3 generated an MS peak list which was submitted for a peptide mass fingerprinting (PMF) search and used as a “survey scan” to determine peptide precursors for MS/MS acquisition. All data (MS and MS/MS), through the integrated software Biotools Version 3.2, were used to search the NCBI no redundant database by using MASCOT software available online (http://www.matrixscience. com/cgi/nph-mascot.exe). Search parameters were set as follows: taxonomy was set on Bacteria; trypsin was selected as the enzyme; the number of missed cleavage sites was set to one; carbamidomethylation and methionine oxidation were selected as fixed and variable modifications, respectively; and mass values were set to monoisotopic. Searches were performed setting a peptide mass tolerance of 50 ppm and a fragment ion mass tolerance of 0.5 Da. The statistical analyses of the sequences were determined by the probability-based Mowse score offered by MASCOT software. A p-value of less than 0.05 was considered significant and used to

Two-Dimensional Gel Electrophoresis (2-DE)

Immobilized linear pH gradients (IPG) strips with a 310 pI range (17 cm, Bio-Rad Laboratories) were first used for the first dimension. But all proteins were present in the pH 47 range. In consequence, the isoelectric focusing (IEF) step in the present study was performed using pH 47 IPG strips (17 cm). A total of 300 μg of protein was applied to each IPG strip for active rehydration at 50 V, 25 °C during 15 h. The IEF step was carried out at 25 °C on the Protean IEF System (Bio-Rad Laboratories) at 10000 V until a total of 48000 V was reached. After IEF, the IPG Strips were reduced and alkylated in an equilibration buffer made of 6 M urea, 0.375 M Tris-HCl (Sigma-Aldrich, pH 8.8), 2% (w/v) SDS (Fluka), 20% (v/v) Glycerol (Sigma-Aldrich), and 2% (w/v) DTT for reduction or 2.5% (w/v) iodoacetamide (Bio-Rad Laboratories) for alkylation. The second dimension SDS-PAGE was achieved on 10% polyacrylamide (37.5:1 acrylamide/bisacrylamide, Bio-Rad Laboratories) resolving gels at 60 mA for 3 h. The Bio-Rad low molecular weight standard marker was used as a protein molecular weight marker. The gels were stained with Coomassie Brillant Blue R-250 (Bio-Rad Laboratories). Image Analysis and Quantification

Gel images were captured using a GS-800 densitometer (BioRad Laboratories). For each condition, three protein extractions were performed with three independent cultures and then analyzed in six separate gels given that each protein sample was loaded onto two gels. Image analyses were conducted using the PDQuest quantitative image analysis software (Version 7.4.0, 208

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Bioinformatic Tools to Analyze Amino Acid Sequences

Figure 1. Growth curves of Rhodococcus erythropolis R138 cultivated in minimal medium with sodium hexanoate (white triangles), γ-butyrolactone (GBL, black diamonds), γ-valerolactone (GVL, white diamonds), γ-caprolactone (GCL, black squares), γ-heptalactone (GHL, white squares), or γ-octalactone (GOL, black triangles) as a sole carbon source. Each point is the average of at least three values.

Conserved protein domains were identified by using CDD software based on NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). This software uses Reverse PositionSpecific BLAST (RPS-BLAST) to quickly scan a set of precalculated position-specific scoring matrices (PSSMs) with a protein query. The results of CD-Search are presented as an annotation of protein domains on the user query sequence, and can be visualized as domain multiple sequence alignments with embedded user queries. High confidence associations between a query sequence and conserved domains are shown as specific hits. Proteins with undefined function were submitted to this software by entering the accession number with the following parameters: Search against database, CDD-40526 PSS Ms; Expert Value threshold, 0.01; Maximum number of hits, 500; Result mode, consive.

’ RESULTS AND DISCUSSION Assimilation of γ-Butyrolactone Derivative Molecules by R. erythropolis

The γ-butyrolactone family contains a wide variety of molecules in which the lactone ring can be either nonsubstituted, such as GBL, or substituted with aliphatic chains of various lengths, such as GVL, GCL, GHL and GOL (Figure 1). To determine the capability of R. erythropolis to assimilate these γ-butyrolactone derivative molecules, these were tested as growth substrates in minimal medium containing one of them as the sole carbon source and energy. The chemistry of the lactone ring suggests that theses molecules may be hydrolyzed under alkaline conditions to their cognate open-chain forms.21 As a result, the pH of the culture medium was carefully controlled and kept at about 6.0 during growth thanks to the buffering agent 2-(N-morpholino)-ethanesulfonic acid (MES). Comparison of growth on GBL, GVL, GCL, GHL, GOL, or on hexanoate, was performed with the same number of moles of carbon for each tested substrate. As shown in Figure 1, growth was similar in the presence of GVL, GCL, GHL and GOL with a mean doubling time of 4 h. On the other hand, growth was significantly slower in the presence of sodium hexanoate with a mean doubling time of 6 h. No growth was observed in the presence of GBL. Thus, R. erythropolis was able to assimilate GCL (as already observed by Cirou et al.5), GVL, GHL and GOL but unable to assimilate GBL as a sole carbon source. The assimilation of γ-butyrolactone molecules by R. erythropolis therefore requires the presence of an aliphatic chain branched on the lactone ring. This metabolic behavior differs from that observed in Agrobacterium tumefaciens, another NAHSL-degrading bacteria that is able to assimilate GBL.22 In this plant pathogen, GBL assimilation interferes with NAHSL signaling, but probably using pathways that differ from those involved in GCL assimilation in R. erythropolis.

generate the results. The criteria used to accept protein identification based on PMF data included the score probability greater than the score threshold of 82 (p < 0.05) for the present study, the extent of sequence coverage (minimum of 30%) and the numbers of matched peptides (minimum of nine). Protein identification only based on tandem MS was accepted if at least three matched peptides had ions scores greater than the score threshold of 50 (p < 0.05) for this study. The false discovery rate (FDR) was calculated from the combination of six to ten MS/MS spectra by an automatic decoy database search. MS/MS spectra were manually checked to verify the validity of the Mascot results. Sequences corresponding to irrelevant identifications were discarded. PCR Amplification of the qsdA Gene

Bacteria from a single colony of each studied R. erythropolis strain were suspended in 50 μL of sterile water and the mixture was boiled for 10 min. After centrifugation at 10000 g for 5 min, 5 μL of the supernatant were subjected to PCR amplification using standard conditions with the Extensor Hi Fidelity polymerase (Thermo scientific, Courtaboeuf, France). PCR primers for the detection of qsdA gene (Forward primer: 50 AGTTCAGTACAAACCGTTCGTG 30 ; reverse primer: 50 CAGCTCTCGAAGTACCGACG 30 ) and for cloning the qsdA gene into pET22b(+) expression vector (Novagen, Darmstadt, Germany; Forward primer (with NdeI site italicized): 50 GACAGTTCATATGAGTTCAGTACAAACCGT 30 ; reverse primer (with EcoRI site italicized): 50 AGACCAGAATTCCTCTCGAAGTACCGACG 30 ) were designed on the available R. erythropolis PR4 genome sequence (GenBank accession number NC_012490).

Identification of the Assimilative Pathway of GCL in R. erythropolis

Heterologous Expression of qsdA in E. coli and Colorimetric Quantification of GCL

To identify the metabolic pathway involved in GCL assimilation in R. erythropolis, we used a proteomic approach based on the comparative proteome analysis of R. erythropolis grown in a minimal medium containing GCL or hexanoate as a sole carbon source. This later compound was chosen because (i) it is one of the most closely related molecules to the open ring form of GCL and (ii) it is not able to form a ring in contrast to some hydroxylated hexanoate (e.g., 4-hydroxy hexanoate). Quantitative image gel analysis of replicate 2-DE gels using PDQuest software

The PCR-amplified qsdA gene was cloned into pET22b(+) and the recombinant pET-qsdA vector was transformed into E. coli BL21(DE3) following the recommendations of the supplier (Novagen). The appropriate insert was confirmed by sequencing. The qsdA expressing E. coli strain was cultivated in LB medium supplemented with 2 g/L GCL. The quantification of GCL in the medium was performed using a colorimetric assay developed by Yang et al.20 209

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Figure 2. Proteins of Rhodococcus erythropolis R138 grown in minimal medium containing sodium hexanoate or γ-caprolactone (GCL) as a sole carbon source. (A) 2D gel images representative of three independent protein extractions. Unique proteins to the hexanoate and GCL-culture condition are indicated in white and black arrows, respectively. These were numbered according to Table 2. (B) Segments of 2D gel map showing the spot variations between the two tested conditions. (C) Histogram with mean and standard deviation (SD) of spot intensities for unique proteins to the hexanoate (white bars) and GCL- (black bars) culture condition, obtained by comparing the three replicate gels using the PDQuest quantitative image analysis software.

allowed the detection of approximately 70 spots and revealed the presence of four and five unique spots to the hexanoate and GCL-culture condition, respectively (Figure 2). In order to confirm the involvement of enzymes in the degradation of fatty acids, commons spots between the two culture conditions were considered and consequently used as a control to validate our differential proteome analysis. Thirty-seven of them were retained on the basis of their intensity superior or equal to 10 ppm. All of these 46 spots were excised, subjected to in-gel trypsin digestion and analyzed by MALDI-MS, as described in the Materials

and Methods. Forty-three of them were successfully identified using PMF searches with protein scores greater than the score threshold of 82 (p < 0.05), with a minimum sequence coverage of 30% and a minimum number of matched peptides of nine (Table 2). The three remaining spots were identified only by tandem-MS searches with the following individual ions scores markedly greater than the score threshold of 50 (p < 0.05) in the present study and a FDR value of 0.00%: β-keto-acyl-[acylcarrier-protein] reductase (three matched peptides with ions scores of 86, 112 and 132), acyl-CoA dehydrogenase (four matched 210

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211

YP_002768932

YP_002767532

8

9

YP_002768642

YP_002766458

YP_705039

metK

gap

YP_002768704

7

YP_002766476

YP_002768930

6

qsdA

etfB

YP_002768947

5

atpH

YP_002765820

YP_002767355

4

etfA

YP_002768606

3

map

YP_002768257 YP_002765821

YP_002763675

2

aceA

YP_002768607

1

gene

name

YP_002765021

accession nb

spot nba

NCBI

protein

Mascot score

94

dehydrogenase

β-hydroxyacyl-CoA

93

160

108

108 152

157

245

culture conditions

Common proteins to the two

134

172

90

binding protein

113

Quinone oxidoreductase

91

106

134

249

106

for PMF searchesc

(QOR1) Rossmann fold NAD(P)+

(GCL) culture condition

Unique proteins to γ-caprolactone

Uncharacterized conserved domain

dehydrogenase/reductase

Classical short chain

Unique proteins to hexanoate culture condition

domainsb

S-adenosylmethionine synthetase

Acyl-CoA dehydrogenase

dehydrogenase

Glyceraldehyde-3-phosphate

beta subunit

Electron transfer flavoprotein

alpha subunit

Thiosulfate sulfurtransferase Electron transfer flavoprotein

Isocitrate lyase

Acyl-CoA dehydrogenase

Acyl-CoA dehydrogenase

Oxidoreductase

Oxidoreductase

lactonase

N-acyl-homoserine

delta chain

ATP synthase

Hypothetical protein RER_51590

peptidase

Methionine amino

Oxidoreductase

functions

putative

Table 2. MALDI MS Identification of Rhodococcus erythropolis R138 Proteins

14

21

16

14

9

14 12

21

15

24

14

12

15

9

12

19

13

peptides

nb of matched

48

73

42

56

52

68 67

60

61

61

50

51

57

55

40

93

58

(%)

coverage

MW

26 413/5.22

43 294/5.06

41 237/5.14

35 975/5.18

28 011/4.58

31 058/4.77 31 950/4.64

46 875/5.06

39 671/4.87

43 042/5.09

26 391/4.86

34 327/5.12

35 551/4.69

28 245/5.57

40 813/5.17

26 189/4.79

24 701/4.43

(Da)/pId

69.5

76.55

93.9

97.15

105.1

119.45 114.1

158.25

15.5

63

72.5

73

94

7.35

32

83.95

696.25

mean

2.4

5.55

0.4

3.75

10.4

6.85 1.2

11.25

0.5

7

7.5

7

6

0.25

2.1

1.75

58.75

SD

spot intensitye

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spot nba

gene

putative

protein

212

Isocitrate lyase Fumarate hydratase class II Cysteine synthase

aceA

fumC

cysK

YP_002765021

YP_002767687

YP_002764538

acn

YP_002766515

Glyceraldehyde-3-phosphate

β-keto-acyl-[acyl-

68 129

β-keto-acyl-[acyl-carrierprotein] reductasef Acyl-CoA dehydrogenase

fabG

fadE

YP_002768573

147

290

Siderophore-interacting protein

Aconitase

carrier-protein] reductase

117

147

Phage shock protein A (PspA)

aminotransferase Hypothetical protein

RER_27420

166

141

127

138

135

87

127

86

106

114

198

146

149

117

Acetyl-ornithine

Enoyl-CoA hydratase

dehydrogenase

(cell division initiation protein)

hydrolase superfamily DivIVA domain

Adenine nucleotide α-

acetaldehyde dehydrogenase II-like

116

YP_002768933

YP_002766151

fabG

argD

YP_002768933

YP_002766189

YP_002766728

YP_002765811

gap

Transaldolase

tal

YP_002766491

YP_002766476

Fumarate hydratase class II

fumC

YP_002767687

Hypothetical protein RER_51590

60 kDa chaperonin

groEL

YP_002768606

Phosphoglycerate kinase

pgk

YP_002764973

RER_44690 Hypothetical protein RER_35440

Hypothetical protein

delta chain

ATP synthase

YP_002766477

YP_002766991

YP_002767916

atpH

Aldehyde dehydrogenase

YP_002767715

YP_002765257

YP_002767355

147

alpha subunit β-ketoadipyl-CoA thiolase NAD+-dependent

183

Succinyl-CoA synthetase

sucD

YP_002767875

200

Enolase

92

eno

Ralstonia eutrophus

Mascot score for PMF searchesc

YP_002767730

domainsb

Elongation factor Ts

functions

tsf

name

YP_002765990

accession nb

NCBI

Table 2. Continued

14

6

12

41

13

22

14

18

13

13

20

10

11

10

10

15

19

16

11

10

12

16

16

18

13

peptides

nb of matched

54

32

53

56

65

51

63

77

54

54

52

33

48

37

34

40

65

74

59

60

34

61

68

60

57

(%)

coverage

MW

42 161/4.93

25 895/5.33

30 821/4.75

101 072/4.73

25 895/5.33

29 028/6.01

41 309/4.87

26 472/4.87

35 975/5.18

40 323/4.45

49 207/5.16

40 528/5.17

32 703/4.99

49 207/5.16

46 875/5.06

56 296/4.78

42 110/4.68

29 728/5.02

31 188/4.97

28 245/5.57

55 074/5.08

42 445/4.80

30 656/5.04

44 958/4.53

29 346/5.04

(Da)/pId

11.85

12

12.35

12.55

15.2

17.45

18.75

19.75

20.3

21.9

22.45

22.6

24.25

28

31.3

33.3

34.6

38.4

36.75

38.8

41.15

43.6

46.8

61.05

67.45

mean

0.55

0.2

1.35

0.55

2.1

2.65

0.45

0.45

0

0.5

2.15

0.3

2.95

2.9

4.8

1.4

3.5

1.2

1.25

3.3

3.05

3.3

1

4.25

2.35

SD

spot intensitye

Journal of Proteome Research ARTICLE

dx.doi.org/10.1021/pr200936q |J. Proteome Res. 2012, 11, 206–216

cyclohydrolasef

Figure 3. Degradation kinetic of γ-caprolactone by qsdA-expressed Escherichia coli.

peptides with ions scores of 86, 90, 101 and 136) and methylenetetrahydrofolate dehydrogenase (four matched peptides with ions scores of 69, 80, 96 and 101). The 46 spots encode 40 different proteins (Table 2). Six proteins were distributed over two spots, demonstrating the presence of isoforms. For hypothetical proteins and undefined oxidoreductase proteins, a potential function could however be assigned in relation with the presence of conserved domains detected by the NCBI CDD algorithm. The comparison between hexanoate and GCL culture conditions led to the identification of unique proteins for each tested condition. Concerning hexanoate culture condition, four spots were detected, corresponding to two proteins, an oxidoreductase and a methionine aminopeptidase, and two isoforms of proteins were also identified in the GCL-culture condition. According to a conserved domain search, the oxidoreductase specifically detected under the hexanoate culture condition belongs to classical short chain dehydrogenase/reductase and could be involved in NAD(P)+-dependent dehydrogenation during oxidation of hexanoate or corresponding catabolites.23 The methionine aminopeptidase was described as catalyzing the removal of the amino-terminal (initiator) methionine from nascent proteins.24 Concerning the GCL-culture condition, five spots corresponding to five different proteins were specifically identified. They were identified as an oxidoreductase with a quinolone oxidoreductase domain probably involved in a respiratory chain, another oxidoreductase with an NAD(P)+ binding protein domain, two acyl-CoA dehydrogenases implicated in β-oxidation and the lactonase QsdA probably involved in ring-opening. The proteins found in the two culture conditions belonged to several functional categories, which could be grouped into two types of pathways: catabolism and anabolism. For the degradation of both the fatty acid hexanoate and GCL, different enzymes involved in the β-oxidation process were identified. They included different acyl-CoA dehydrogenases, enoyl-CoA hydratase and β-hydroxy acyl-CoA dehydrogenase. The identification of an aldehyde dehydrogenase suggests the involvement of a ω-oxidation process, as proposed by Kunz and Weimer.25 These authors demonstrated that Pseudomonas sp. was able to catalyze the ω-oxidation of hexanoate into 6-hydroxy hexanoate and the further oxidation into adipic acid. Our statements are supported by the nonproduction of these enzymes when the strain R138 was grown on a minimal medium with succinate as a sole carbon source, a short organic acid not catabolized by β- or ω-oxidation

a

folD YP_002765445

YP_002767533

gene NCBI

Table 2. Continued

spot nba

b

Methylene-tetrahydrofolate

dehydrogenase/methenyltetrahydrofolate

Acetate-CoA ligase acs YP_002763943

Acyl-CoA dehydrogenasef

Chaperone protein DnaK dnaK YP_002764798

Numbering is according to Figure 2. Protein domain as deduced from sequence analysis with the NCBI conserved domain algorithm. c Protein scores for PMF searches with a sore threshold of 82 (p < 0.05). d Theoretical values. e The mean and standard deviation (SD) of spot intensity were calculated by PDQuest quantitative image analysis software (version 7.4.0). Individual protein quantities were expressed as parts per million (ppm). f Proteins only identified by tandem-MS searches with ions scores for 34 matched peptides markedly greater than the score threshold of 50 (p < 0.05) and a FDR of 0.00%, as described in the Results and Discussion section.

3.5 30 405/5.57 58

6

27

10.1

3.5

4 45 289/4.78 19 62

12

69 923/5.00

10.2

0.5

ARTICLE

Acyl protein synthetase

98

13

30

10.5

SD

35 13

mean

66 106/4.68

(%)

94

MW

(Da)/pId

coverage

peptides

Mascot score protein

domainsb functions name accession nb

putative

for PMF searchesc

nb of matched

10.8

spot intensitye

Journal of Proteome Research

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ARTICLE

Figure 4. Proposed catabolic pathway for the γ-caprolactone degradation in Rhodococcus erythropolis R138 deduced from proteome analysis. Enzymes identified in the present study are indicated in gray.

pathways (data not shown). The identification of the aconitase, succinyl-CoA synthetase and fumarate hydratase enzymes revealed the implication of Krebs cycle enzymes, possibly at the “final” metabolic stages. Finally, the intervention of the β-ketoadipate pathway connecting ω-oxidation to the Krebs cycle could be revealed by β-ketoadipyl-CoA thiolase. This enzyme catalyzes the conversion of β-ketoadipyl-CoA to succinyl- and acetyl-CoA, two intermediates of Krebs cycle.26 Several enzymes identified in this study are involved in anabolism. The identification of an isocitrate lyase indicates that in Rhodococcus, under the experimental conditions, the Krebs cycle was coupled to the glyoxylate cycle, a feature that leads to a bypass of the two decarboxylation steps for the synthesis of succinate and oxaloacetate, two precursors of carbohydrates synthesis. Some proteins are also related to amino acid biosynthesis pathways, (e.g., cysteine synthase) or fatty acid biosynthesis (e.g., β-ketoacyl-ACP reductase). Other identified spots correspond to enzymes involved in general cell processes.27 For example, the acetate-CoA ligase is crucial for the maintenance of optimal levels of acetyl-CoA, a key intermediate in many important biosynthetic and catabolic processes. Proteins involved in protein biosynthesis were also detected: they include the elongation factor EFTs, and the chaperonins DnaK and GroEL,

essential for assisting folding of newly translated proteins, disaggregation of protein aggregates, and proteolysis of unstable proteins.28 Other proteins were identified as being involved in iron metabolism, in sulfate metabolism and cell division.29 The last functional category common to the two culture conditions concerned metabolic pathways involved in energy and reduced coenzymes production: glycolysis (glyceraldehyde-3phosphate dehydrogenase, phosphoglycerate kinase, enolase) and pentose phosphate pathway (transaldolase) also for production of pentoses. We also identified an ATP synthase and the two subunits of the electron transfer protein implicated in the reoxidation of the reduced form of acyl-CoA dehydrogenase. Lactonase QsdA

Special attention was given to the lactonase QsdA because this enzyme is involved in NAHSL degradation via lactone ringopening,30,31 and therefore appeared as an interesting candidate protein to catalyze the first step of the GCL degradation pathway. To investigate whether QsdA is capable of hydrolyzing GCL, qsdA was PCR amplified from Rhodococcus and cloned into a vector allowing its heterologous expression in E. coli, a bacterium unable to degrade this lactone. After 24 h of incubation, the qsdAexpressing E. coli strain BL21 (DE3) is able to degrade 30% of the 214

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Journal of Proteome Research input GCL (Figure 3), a feature that confirms the involvement of QsdA in GCL degradation. QsdA (formerly AhlA) is described in literature as phosphotriesterase-like lactonase. It is more accurately a true lactonase degrading various compounds with a preference for relatively hydrophobic lactones including the plant aromatic dihydrocoumarin.32 As stated above, QsdA also degrades a wide range of NAHSLs with an acyl chain ranging from C6 to C14 with or without substitution at carbon 3.31 As expected by the results obtained in stimulated quorum quenching approaches,5,6 it appears that the hydrolysis of the biostimulant (GCL) and that of signaling molecules (NAHSL) share common steps. In addition, QsdA exhibits a much lower phosphotriesterase activity. Because phosphotriesters are not natural compounds, this promiscuous activity may have appeared recently in evolution with the use of compounds such as paraoxon insecticide.32,33 To determine the occurrence of qsdA genes in rhizospheric isolates of R. erythropolis, PCR with primers specific to qsdA was performed on the 17 strains described in Table 1. A PCR product with expected size was obtained for strain W2 used as a control,31 DCL14 a strain known for its versatile metabolism including hydrophobic compounds,3436 and for all potato isolates (that can be used as biocontrol agent). These results are consistent with previous data that reported the detection of qsdA in five other R. erythropolis strains, including the species type strain, some aromatic compound-degrading strains and a mycorrhizosphere isolate.31 Ubiquity of the QsdA enzyme argues in favor of a biocontrol strategy based on the stimulation of R. erythropolis indigenous populations found in cultivated areas.

ARTICLE

the detoxification of putative antibiotic activity exerted by ketoNAHSL and their byproduct.44 (iii) Finally a third role can be attributed to QsdA among anabolic pathways of R. erythropolis. This enzyme could be involved not in the degradation of lactones but in their synthesis, such as during the transformation of alkanediols into flavoring γ-lactones.45 All this confirms that the metabolic potential of R. erythropolis remains immense and of great interest for diverse biotechnical applications like bioremediation, biocontrol and food bioengineering.

’ AUTHOR INFORMATION Corresponding Author

*Xavier Latour, Laboratory of Microbiology Signals and Microenvironment, Normandy University, EA 4312 University of Rouen, IUT Evreux, 55 rue Saint-Germain, 27000 Evreux, France. Phone: +33.232.291.549. Fax: +33.232.291.550. E-mail: xavier.latour@ univ-rouen.fr.

’ ACKNOWLEDGMENT This research was supported by grants from the Region HauteNormandie & Ministere delegue a l’Enseignement Superieur et a la Recherche, GRR VATA & FEDER (European Union). The authors also thank Dr Carla de Carvalho for kindly providing the R. erythropolis DCL14 strain, Melanie Hillion for technical assistance, and Christine Farmer for linguistic support. ’ REFERENCES (1) Maga, J. A. Lactones in foods. CRC Crit. Rev. Food Sci. Nutr. 1976, 8 (1), 1–56. (2) Nu~ nez, M. T.; Martin, V. S. Efficient oxidation of phenyl group to carboxylic acids with ruthenium tetraoxide. A simple synthesis of (R)-γcaprolactone, the pheromone of Trogoderma granarium. J. Org. Chem. 1990, 55, 1928–1932. (3) Murib, J. H.; Kahn, J. H. Process for preparing gamma-caprolactone by isomerization of epsilon-caprolactone. U.S. Patent 4,611,069, September 9, 1986. (4) Faure, D.; Cirou, A.; Dessaux, Y. Chemicals promoting the growth of N-acylhomoserine lactone-degrading bacteria. U.S. Patent 20100050719 A1, March 4, 2010. (5) Cirou, A.; Diallo, S.; Kurt, C.; Latour, X.; Faure, D. Growth promotion of quorum-quenching bacteria in the rhizosphere of Solanum tuberosum. Environ. Microbiol. 2007, 9 (6), 1511–1522. (6) Cirou, A.; Raffoux, A.; Diallo, S.; Latour, X.; Dessaux, Y.; Faure, D. Gamma-caprolactone stimulates growth of quorum-quenching Rhodococcus populations in a large-scale hydroponic system for culturing Solanum tuberosum. Res. Microbiol. 2011, 162 (9), 945–950. (7) Waters, C. M.; Bassler, B. L. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 2005, 21, 319–346. (8) Barnard, A. M.; Salmond, G. P. Quorum sensing in Erwinia species. Anal. Bioanal. Chem. 2007, 387 (2), 415–423. (9) Liu, H.; Coulthurst, S. J.; Pritchard, L.; Hedley, P. E.; Ravensdale, M.; Humphris, S.; Burr, T.; Takle, G.; Brurberg, M. B.; Birch, P. R.; Salmond, G. P.; Toth, I. K. Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum. PLoS Pathog. 2008, 4 (6), e1000093. (10) Latour, X.; Faure, D.; Diallo, S.; Cirou, A.; Smadja, B.; Dessaux, Y.; Orange, N. Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora). Cahiers Agric. 2008, 17 (4), 355–359. (11) Diallo, S.; Crepin, A.; Barbey, C.; Orange, N.; Burini, J. F.; Latour, X. Mechanisms and recent advances in biological control mediated through the potato rhizosphere. FEMS Microbiol. Ecol. 2011, 75 (3), 351–364.

’ CONCLUSIONS On the basis of previous knowledge of the Rhodococcus metabolism3739 and proteome analysis of strain R138, we investigated the metabolism of GCL by R. erythropolis. Our results suggest that GCL is both completely dissimilated (CO2 production during Krebs cycle) and assimilated (glyoxylate cycle, lipids biosynthesis) during bacterial growth without the production of toxic compounds. We propose a catabolic pathway starting with hydrolysis of GCL by the lactonase QsdA then followed by β- and ω-oxidation of 4-hydroxy hexanoate coupled to the Krebs cycle and β-ketoadipate pathway (Figure 4). The environmental role of QsdA in the Rhodococcus metabolism remains to be clarified. To this end, at least three hypotheses can be proposed. (i) R. erythropolis is a bacterium known for its ability to degrade a wide range of recalcitrant molecules including molecules derived from lignin, pesticides or petroleum.16,34,37 The degradation steps of these compounds are often associated to an intermediate lactonase activity (i.e., enol-lactone-hydrolyzing activities) responsible for linearization of the molecule before joining β-ketoadipate or β-oxidation pathways.38,4042 QsdA activity could also have this role. Indeed, analyzing neighboring sequences downstream QsdA reveals the presence of a contiguous sequence encoding for the long-chain fatty acid-CoA ligase FadD (data not shown). This enzyme is associated with the plasma membrane and is strongly suspected to extract fatty acids from the outer membrane with activation by CoA thioester.43 This step is followed by a β-oxidation of the acyl-CoA or phospholipids incorporation that is an argument in favor of cyclic molecule assimilation. (ii) Another role assigned by Uroz and co-workers31 to QsdA is that of an NAHSL lactonase involved both in the quenching of Gram-negative bacterial communication and in 215

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large-spectrum quorum-quenching lactonases. Appl. Environ. Microbiol. 2008, 74 (5), 1357–1366. (32) Afriat, L.; Roodveldt, C.; Manco, G.; Tawfik, D. S. The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase. Biochemistry 2006, 45 (46), 13677–13686. (33) Singh, B. K. Organophosphorus-degrading bacteria: ecology and industrial applications. Nat. Rev. Microbiol. 2009, 7 (2), 156–164. (34) van der Werf, M. J.; Boot, A. M. Metabolism of carveol and dihydrocarveol in Rhodococcus erythropolis DCL14. Microbiology 2000, 146 (Pt 5), 1129–1141. (35) de Carvalho, C. C.; da Fonseca, M. M. Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14. FEMS Microbiol. Ecol. 2005, 51 (3), 389–399. (36) de Carvalho, C. C.; Wick, L. Y.; Heipieper, H. J. Cell wall adaptations of planktonic and biofilm Rhodococcus erythropolis cells to growth on C5 to C16 n-alkane hydrocarbons. Appl. Microbiol. Biotechnol. 2009, 82 (2), 311–320. (37) Lofgren, J.; Haddad, S.; Kendall, K. Metabolism of alkanes by Rhodococcus erythropolis. In Emerging technologies in hazardous waste management V; Tedder, W., Pohland, F. G., Eds; ACS symposium series Vol. 607; ACS: Washington, D.C., 1995; pp 252263. (38) Cha, C. J.; Cain, R. B.; Bruce, N. C. The modified betaketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism. J. Bacteriol. 1998, 180 (24), 6668– 6673. (39) Alvarez, H. M. Relationship between β-oxidation pathway and the hydrocarbon-degrading profile in actinomycetes bacteria. Int. Biodeterior. Biodegrad. 2003, 52 (1), 35–42. (40) Whyte, L. G.; Hawari, J.; Zhou, E.; Bourbonniere, L.; Inniss, W. E.; Greer, C. W. Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp. Appl. Environ. Microbiol. 1998, 64 (7), 2578–2584. (41) van der Vlugt-Bergmans, C. J.; van der Werf, M. J. Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14. Appl. Environ. Microbiol. 2001, 67 (2), 733–741. (42) Eulberg, D.; Lakner, S.; Golovleva, L. A.; Schlomann, M. Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity. J. Bacteriol. 1998, 180 (5), 1072–1081. (43) Weimar, J. D.; Ribusso, C. C.; Delio, R. Functional role of fatty acyl-coenzyme A synthase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport. J. Biol. Chem. 2002, 277, 29369–29376. (44) Kaufmann, G. F.; Sartorio, R.; Lee, S. H.; Rogers, C. J.; Meijler, M. M.; Moss, J. A.; Clapham, B.; Brogan, A. P.; Dickerson, T. J.; Janda, K. D. Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (2), 309–314. (45) Moreno-Horn, M.; Martinez-Rojas, E.; G€orisch, H.; Tressl, R.; Alexander Garbe, L. Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst. J. Mol. Catal., B: Enzymatic 2007, 49, 24–27. (46) Uroz, S.; D’Angelo-Picard, C.; Carlier, A.; Elasri, M.; Sicot, C.; Petit, A.; Oger, P.; Faure, D.; Dessaux, Y. Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorumsensing-regulated functions of plant-pathogenic bacteria. Microbiology 2003, 149 (8), 1981–1989.

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