1 Interactions Plantes et Micro-organismes de la Rhizosphère, Institut des Sciences du Végétal, CNRS, Bâtiment 23, Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX, France
2 Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK
3 Laboratoire des Sciences de la Terre, École Normale Supérieure de Lyon, 43 Allée D'Italie, 69364 Lyon CEDEX 07, France
Correspondence
Yves Dessaux
dessaux{at}isv.cnrs-gif.fr
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ABSTRACT |
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INTRODUCTION |
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AHL-dependent QS is conserved in a broad range of micro-organisms living in various environments, and regulates very diverse physiological functions (Fuqua et al., 2001; Swift et al., 2001
; Whitehead et al., 2001
). Amongst these, several pathogenicity-related functions are controlled in a population-density-dependent fashion in bacterial species pathogenic for plants and animals (e.g. Jones et al., 1993
; Milton et al., 1997
; Zhang et al., 1993
; reviewed by Von Bodman et al., 2003
). QS also controls functions responsible for the interaction of the microbe with both its physical and biological environments, including swimming, swarming, biofilm maturation and symbiosis. With respect to the interaction between bacteria and higher organisms, QS is likely to confer a selective advantage which enables the bacterium to express groups of genes with a highly relevant biological impact, considering that (i) the expression is co-ordinated and (ii) the relevant microbial cell population needs to attain a high density before QS-controlled genes are induced (Fuqua et al., 2001
; Swift et al., 2001
; Whitehead et al., 2001
; Winans & Bassler, 2002
; Winzer et al., 2002
). Interestingly, AHLs are also perceived by higher organisms. A striking example is the attraction of zoospores from the green alga Enteromorpha by AHLs (Joint et al., 2002
). These zoospores exhibit chemotaxis for AHLs, leading to their enhanced settlement on AHL-producing biofilms. In addition, the legume plant Medicago truncatula responds to AHLs, as shown by proteomic analysis as well as activation of tissue-specific reporter gene fusions (Mathesius et al., 2003
). Furthermore, long-chain AHLs such as those produced by the opportunistic pathogen Pseudomonas aeruginosa have been shown to exert immunomodulatory (Chhabra et al., 2003
; Telford et al., 1998
) and cardiovascular effects (Gardiner et al., 2001
) in mammalian hosts
Evidence is beginning to accumulate indicating that inhibition of QS may be a strategy adopted by eukaryotic organisms to combat potentially pathogenic bacteria. The production of AHL antagonists has been demonstrated for the marine red alga Delisea pulchra (Givskov et al., 1996), higher plants (Teplistki et al., 2000
) and the bryozoan Flustra foliacea (Peters et al., 2003
). Under alkaline growth conditions, AHLs are rapidly inactivated by pH-dependent lactonolysis (i.e. opening of the HSL ring) since the corresponding N-acylhomoserine cannot activate LuxR-type response-regulator proteins (Byers et al., 2002
; Yates et al., 2002
). The ability to inactivate AHLs enzymically has also been demonstrated for a range of bacterial genera belonging to the
-Proteobacteria (Agrobacterium, Zhang et al., 2002
), the
-Proteobacteria (Variovorax, Leadbetter & Greenberg, 2000
; Ralstonia, Lin et al., 2003
; and Comamonas, Uroz et al., 2003
), the
-Proteobacteria (Pseudomonas, Huang et al., 2003
; Uroz et al., 2003
; Acinetobacter, Kang et al., 2004
), the low-G+C Gram-positive bacteria (Bacillus, Dong et al., 2000
, 2002
; Fray, 2002
; Lee et al., 2002
) and the high-G+C Gram-positive bacteria (Rhodococcus, Uroz et al., 2003
). AHL-inactivating activity has also been reported in plants (Delalande et al., 2005
) and mammalian cells (Chun et al., 2004
). The AHL-inactivating enzymes described to date belong to two families: the AHL lactone hydrolases (e.g. AiiA, AttM, AiiB, Carlier et al., 2003
; Dong et al., 2000
; Fray, 2002
; Lee et al., 2002
; Zhang et al., 2002
) and the AHL acylases (AiiD; Lin et al., 2003
). However, the physiological function(s) of these AHL-inactivating enzymes and whether AHLs are their primary substrates have not yet been entirely clarified, although an involvement of AttM from Agrobacterium, in
-butyrolactone degradation, has been proposed recently (Carlier et al., 2004
).
Amongst the bacteria exhibiting AHL catabolic activity, Rhodococcus erythropolis strain W2 was of special interest because analysis of its degradative properties revealed that it exhibits a broad AHL-degradation spectrum and rapid AHL inactivation kinetics (Uroz et al., 2003). In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers, indicating its potential as a biocontrol agent. These traits motivated a more detailed examination of the catabolic activities and pathways involved in AHL inactivation. The results reported in this paper demonstrate that strain W2 grows on various AHLs as the sole carbon and energy source, with a preference for short-chain compounds. Two enzyme activities involved in AHL inactivation were identified: an oxidoreductase which converts 3-oxo-AHLs to their corresponding 3-hydroxy derivatives, and an amidolytic acivity which cleaves the amide bond linking the acyl chain to the HSL residue.
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METHODS |
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Synthesis of AHLs and related compounds.
The AHLs investigated in this study, as well as the analogue 3O,6Ph,C6-HSL (Fig. 1) were synthesized as described previously (Chhabra et al., 1993
, 2003
). The compound 3O,C12-NH2 (Fig. 1
) was synthesized as described for AHL synthesis. HSL, homoserine and dansyl chloride were obtained from Sigma.
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Preparation of resting R. erythropolis cells and crude cell extracts.
R. erythropolis strain W2 cells were harvested after 2 days of culture in rich LBm medium, at approximately 109 c.f.u. ml1. A 1 l culture was centrifuged for 10 min at 4 °C, 10 000 g. The cells were resuspended in 100 ml PBS and washed twice in PBS. The resulting concentrated cell suspension was used directly as a source of resting cells for in vivo AHL inactivation assays or disrupted using a cell disrupter (Constant Systems) under 15 kPa pressure. The lysate obtained was recycled five times through the cell disrupter. Cell debris was removed by centrifugation (120 min, 4 °C, 10 000 g). The resulting supernatant was filtered through a 0·22 µm membrane filter and stored at 4 °C. This crude cell extract (CCE) was the source of enzyme used for the in vitro AHL inactivation assays. The protein concentration of the CCE was determined according to the manufacturer's instructions (Sigma protein detection kit), using the Bradford method with bovine serum albumin as the standard.
Separation and analysis of AHLs.
AHLs were detected using the lux-based biosensors E. coli[pSB401] and E. coli[pSB1075], for short-chain and long-chain compounds respectively (Winson et al., 1998). Bioassays were performed with the above sensors using a microtitre plate bioassay as described by Reimmann et al. (2002)
. Wells containing AHLs were visualized as bright wells in a dark background when viewed with a Luminograph LB980 photon video camera (Berthold). Reverse-phase HPLC analysis of AHLs was performed on a Kromasil C8 5µ column, 2·1x250 mm (Jones Chromatography), using a Waters 625 HPLC system coupled with a Waters 996 PDA photodiode array detector, and eluted with acetonitrile/water isocratic or gradient combinations as described before (Swift et al., 1996
; Yates et al., 2002
).
AHL inactivation assays.
For the whole-cell assays, aliquots of AHLs in ethyl acetate were dispensed into sterile tubes and the solvent evaporated to dryness under a stream of sterile nitrogen. These tubes were filled with 1 ml of a resting R. erythropolis cell suspension obtained as indicated above, rehydrating the AHLs and providing a final AHL concentration of 100 µM. The resting cell suspensions were incubated at 25 °C for up to 360 min. The reactions were stopped at regular intervals by the addition of ethyl acetate (1 ml), which also served to extract any remaining AHLs. For AHL inactivation assays in vitro using CCEs, the reaction mixture contained 0·5 mg ml1 of bacterial protein and 100 µM AHLs in a final volume of 500 µl. Assays were incubated at 25 °C or 37 °C for up to 360 min and stopped at regular intervals by addition of 3 vols (1·5 ml) ethyl acetate. For both whole-cell and CCE assays, ethyl acetate containing the residual AHL was removed and evaporated to dryness. The solution was reconstituted in acetonitrile (100 µl) and residual AHL concentrations determined using the AHL biosensors and HPLC (see above). The percentage of AHLs inactivated and the specific activity were determined by estimating the amount of AHL (by comparison of the reduction in peak areas for a given retention time) with respect to AHL solutions of known concentration. For both whole-cell and CCE assays, control experiments involving un-inoculated medium or extraction buffer incubated with AHLs, and cells or extracts incubated without AHLs, were always performed. Heat-processed cells and cell crude extracts were also used as negative controls.
Identification of AHL degradation products.
To identify the breakdown products generated following the incubation of R. erythropolis CCE with AHLs, we used HPLC in conjunction with LC-MS/MS (Waters Micromass Quattro Ultima). Synthetic standards for each assayed molecule were always used as control for HPLC analysis. Free amines released by the action of AHL-inactivating enzymes were chemically trapped by dansylation with dansyl chloride as described by Jiang et al. (1998). The formation of the ring-opened AHLs, i.e. the formation of the corresponding N-acylhomoserine, was detected using the method described by Yates et al. (2002)
. This is based on the acidification of the reaction mixture with 10 mM HCl to induce lactone recyclization.
In vitro determination of the temperature and pH sensitivity of the amidolytic activity of CCEs.
R. erythropolis CCEs were incubated for 10, 20 and 30 min at a range of temperatures (25, 50, 75 and 100 °C) in the absence of AHLs. The resulting samples were stored at 20 °C. To evaluate the effect of temperature on AHL-inactivating activity, the CCEs were incubated with AHLs as described above. To examine the influence of pH, the pHs of CCEs in PBS were adjusted from 6·5 to 1·0 with HCl prior to incubation. CCE activity was subsequently determined by incubation with 3O,C10-HSL (100 µM) for 120 min at 25 °C with shaking. The extent of 3O,C10-HSL degradation was monitored by HPLC as described above.
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RESULTS |
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No oxidoreductase activity was observed with disrupted W2 cells, implying a co-factor requirement. The addition of NADH or NADPH to CCE did not lead to the formation of NAD+ or NADP+ upon incubation with a 3-oxo-AHL as determined by spectrophotometric analysis (data not shown), suggesting either that alternative cofactors are required, or that the enzyme is sensitive to cellular disruption.
Evidence for pH- and temperature- dependent AHL-inactivating activity in cell extracts of R. erythropolis W2
Prolonged incubation of 3-oxo- and 3-hydroxy-substituted and unsubstituted AHLs resulted in the complete removal of these QS signal molecules from the incubation medium. These data suggested the presence of additional AHL-degrading activities. To investigate this possibility, 3O,C10-HSL was incubated with W2 CCE. Under these conditions, the peak corresponding to 3O,C10-HSL in the HPLC spectrum was reduced over time (Fig. 5) but no new peak corresponding to 3OH,C10-HSL was apparent. This degradative activity was investigated in more detail at 25 °C and 37 °C using 3O,C10-HSL as a substrate. After a 120 min incubation period, the apparent activities [expressed as nmol AHL degraded min1 (mg protein)1] were 1·17 at 25 °C and 1·18 at 37 °C, with a 5 % experimental error. After 180 min incubation, the activities were reduced to 1·0 and 0·8 nmol AHL degraded min1 (mg protein)1 at 25 °C and 37 °C respectively.
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AHL degradation by cell extracts of R. erythropolis W2 proceeds via an amidolytic activity
The AHL-inactivating activity of W2 CCEs is likely to be due to either a lactonase or an amidohydolase. Lactonases convert AHLs to the corresponding N-acylhomoserine compound, which can be converted back to the AHL by incubation at acidic pHs (<2·0; Yates et al., 2002). To evaluate this hypothesis, 3O,C10-HSL was incubated with CCE and the reaction mixture acidified prior to extraction with ethyl acetate. The recyclization of the lactone ring was observed for control experiments performed using N-acylhomoserine compounds incubated in the absence of CCE and acidified. However, 3O,C10-HSL could not be recovered by acidification after incubation with CCE (data not shown). These data indicated that the AHL-inactivating activity of W2 CCEs was not due to lactonolysis.
To determine whether W2 CCEs contained enzyme(s) capable of cleaving the amide bond linking the acyl side chain to the HSL ring, dansyl chloride was used to chemically trap any HSL released upon incubation of CCE with 3O,C10-HSL. Fig. 5 shows that under the experimental conditions chosen, about 70 % of the 3O,C10-HSL incubated with the CCE was degraded after 120 min and approximately 90 % after 180 min, with an apparent activity of about 1 nmol min1 (mg protein)1. Following the addition of dansyl chloride, the presence of dansylated HSL was detected after incubation for 120 and 180 min. After 300 min, the concentration of HSL decreased, and was comparable to that observed at the 120 min time point. These results clearly indicate that HSL is released from 3O,C10-HSL during incubation with R. erythropolis W2 CCEs, indicating the presence of an amidolytic activity. They also suggest that the HSL generated via AHL degradation is further degraded by the W2 CCEs. A similar production of HSL was observed using 3O,C6-HSL and the hydroxy-AHL 3OH,C10-HSL as substrates, demonstrating that the W2 CCE contained an amidolytic activity capable of cleaving the amide bond of both short- and long-chain AHLs.
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DISCUSSION |
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In PBS buffer, R. erythropolis W2 whole cells metabolized a variety of AHLs with a carbon chain ranging from C6- to C14. Remarkably, 3-oxo compounds were much more efficiently turned over than their OH-subsituted or unsubstituted counterparts. Furthermore, complete degradation of C6-HSL in PBS required the addition of an energy source (glucose) even though C6-HSL could sustain the growth of W2 when supplied as the sole carbon and nitrogen source in the medium described by Leadbetter & Greenberg (2000). The reason(s) for this apparent discrepancy are not clear but it is likely that the lack of a nutrient absent from PBS may be overcome by the provision of glucose. These data did however suggest that R. erythropolis possesses multiple mechanisms for metabolizing AHLs.
HPLC analysis and LC-MS/MS revealed that whole W2 cells but not CCEs exhibited a novel oxidoreductase activity which converted 3-oxo-substituted AHLs to the corresponding 3-hydroxy derivatives (Fig. 6), provided that the AHL contained an acyl chain of at least eight carbons (from 3O,C8- to 3O,C14-HSL). W2 cells also reduced 3O,6Ph,C6-HSL and 3O,C12-NH2 to the corresponding hydroxy compounds, indicating that the oxidoreductase activity observed is not specific for naturally occurring AHLs and does not require the presence of the HSL ring. Furthermore, the reaction is not stereospecific, since the D-isomer of 3O,C12-HSL was converted to 3OH,C12-HSL by resting W2 cells. The loss of oxidoreductase activity on heat treatment of the W2 cells indicates that the reduction of the 3-oxo-AHLs is enzymic. Although we have so far been unable either to purify the enzyme involved or to clone the corresponding gene, a carbonyl reductase has been purified from R. erythropolis (Zelinski & Kula, 1994
; Zelinski et al., 1994
). This enzyme accepts a broad range of aliphatic and aromatic ketones as substrates and for example reduces methyl 3-oxobutanoate and ethyl 4-chloro-3-oxobutanoate to the corresponding hydroxy compounds. Whether this enzyme can reduce 3-oxo-AHLs is not known.
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While CCEs prepared from W2 lacked the oxidoreductase activity associated with whole cells, CCEs retained the capacity to inactivate AHLs. The temperature and pH dependency of this activity suggested the involvement of one or more enzymes. Since several different bacteria produce lactonases (Dong et al., 2000; Lee et al., 2002
; Park et al., 2003
; Zhang et al., 2002
), R. erythropolis W2 CCEs were examined for such an activity. Since no lactonolysis was observed, an alternative mechanism for metabolizing AHLs is the cleavage of the amide bond linking the acyl side chain to the HSL ring. This mechanism of AHL inactivation has previously been observed only in Gram-negative bacteria (Huang et al., 2003
; Leadbetter & Greenberg, 2000
; Lin et al., 2003
). For example, both Variovorax and Ralstonia species cleave the AHL amide bond during utilization of QS signal molecules as growth nutrients; HSL is released as a product of these reactions and the acyl moiety is further metabolized as an energy source.
To determine whether R. erythropolis CCEs inactivated AHLs by cleaving their amide bond, dansyl chloride (Jiang et al., 1998) was used to trap chemically any HSL released. When 3O,C6-, 3OH,C10- or 3O,C10-HSL were incubated with W2 CCEs, these compounds yielded dansylated HSL, indicating that W2 possesses an amidohydrolase activity (Fig. 6
). This activity exhibits the characteristics of an enzyme since it is thermosensitive and pH dependent, and HSL is released in a time-dependent manner. In a strain of Ralstonia, an amidohydrolase, AiiD, has been characterized and shown to be capable of cleaving AHLs with either long or short acyl side chains (Lin et al., 2003
). An AiiD homologue, termed PvdQ because it is located among a cluster of genes required for the synthesis of the siderophore pyoverdin, is present in P. aeruginosa (Huang et al., 2003
; Lamont & Martin, 2003
). Although PvdQ specifically cleaves long-chain AHLs, e.g. 3O,C12-HSL, pvdQ mutants still grow on 3O,C12-HSL as the sole energy source, but produce wild-type levels of 3O,C12-HSL (Huang et al., 2003
). The presence of a putative aiiD-homologous sequence was investigated in strain W2 using temperature-gradient PCR. However, no homologous sequence was obtained and therefore further work is required to identify the R. erythropolis amidohydrolase gene.
The ability to degrade AHLs is a recently identified characteristic of the genus Rhodococcus (Uroz et al., 2003), which is well known for its remarkable ability to degrade diverse complex organic compounds (see for example Bock et al., 1996
; Chauvaux et al., 2001
; Haroune et al., 2002
; Sakai et al., 2003
; Van der Werf & Boot, 2000
; and for a review Warhurst & Fewson, 1994
). Two enzymic activities conferring the ability to modify and degrade AHLs upon strain W2 were identified: an AHL oxidoreductase, and an AHL amidolytic activity (Fig. 6
). The capacity to reduce 3-oxo AHLs to the corresponding 3-hydroxy compounds has not previously been observed in any bacterium exhibiting AHL-degrading activity. Furthermore, while amidolytic activity towards AHLs has been observed in Gram-negative bacteria including Ralstonia (Lin et al., 2003
) and Pseudomonas (Huang et al., 2003
), and postulated for Variovorax paradoxus (Leadbetter & Greenberg, 2000
), it has not to our knowledge been detected in a Gram-positive bacterium prior to this study.
The AHL inactivation mechanism(s), or at least the mechanism(s) that interfere(s) with cellcell communication via AHLs amongst microbial communities, appear(s) to be multiple and widespread within diverse bacterial genera. Whether AHLs are the primary substrates for the relevant enzymic activities in strain W2 as in other AHL-degraders is still unclear, such that the true physiological role of these enzyme activities remains to be elucidated. Recent data obtained in Agrobacterium, however, indicate that AHL-inactivating lactonases may play another and possibly a major role in the intracellular metabolism of lactone compounds such as -butyrolactone (Carlier et al., 2004
). Clearly, investigation of the substrate specificity and contribution to endogenous metabolic pathways, especially in AHL-nonproducing organisms, will be needed to address the question of the biological function(s) of these AHL-inactivating enzymes. The oxidoreductase activity described in this report appears not to be specific for AHLs, but additional work is required to evaluate this question at the purified protein level. As suggested from earlier work (Uroz et al., 2003
), it could, however, be of potential interest to target 3-oxo AHLs within the development of novel biocontrol agents (Fray, 2002
) or therapeutic strategies (Cámara et al., 2002
) directed at infection control in both plants and animals.
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ACKNOWLEDGEMENTS |
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Received 11 February 2005;
revised 20 June 2005;
accepted 30 June 2005.
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