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N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa
Process Biochemistry 45 (2010) 1944–1948
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa Yang-Chun Yong a,c , Jian-Jiang Zhong a,b,∗ a
State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China c State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China b
a r t i c l e
i n f o
Article history: Received 24 January 2010 Received in revised form 2 May 2010 Accepted 4 May 2010 Keywords: Quorum sensing Homoserine lactone Aromatics Pseudomonas aeruginosa Biodegradation
a b s t r a c t N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bacteria communicate with each other by producing diffusible small signaling molecules and sensing signals in the environment, which is known as quorum sensing (QS) [1,2]. In Gram-negative bacteria, acylated homoserine lactones (AHLs) are the quorum sensing signal molecules. These AHLs are conserved at the lactone moiety and varied in their acyl-chain (including chain length and C-3 substitution) [2,3]. For example, BHL and HHL, which are the signal molecules for the rhl QS system in Pseudomonas aeruginosa, are both C-3 unsubstituted AHLs and only differ in acyl-chain length [2]. AHL-mediated QS has an important role in many essential biological processes, including animal and plant diseases [1,16].
∗ Corresponding author at: School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China. Tel.: +86 21 34206968; fax: +86 21 34204831. E-mail address: [email protected] (J.-J. Zhong). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.05.006
In environmental bioprocesses, strains such as Pseudomonas, Agrobacterium, and Aeromonas, which may produce AHLs, were isolated from activated sludges and water reclamation systems, and AHL production by these strains was confirmed in rich culture media [5–7]. Addition of AHLs changed the composition of the bacterial community in industrial activated sludges and improved the performance of these sludges through an unknown mechanism [5]. However, until now, there have been no reports of AHL production during pollutant biodegradation. In addition, the ability of these isolated AHL producers to degrade pollutants has not yet been reported. Therefore, whether any potential AHL producers could synthesize AHLs during pollutant degradation is yet to be clarified, and it is crucial to evaluate the role of AHL or AHL-mediated QS in biodegradation processes. In this study, an environmental isolate P. aeruginosa CGMCC 1.860 was selected because it could degrade a broad spectrum of aromatic compounds and produce AHLs in rich culture media. AHL production by the strain was observed during aromatic biodegradation. Additionally, the AHLs produced during aromatic biodegradation were then identified by using TLC
Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
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Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid
Description
Reference or source
Strains P. aeruginosa CGMCC 1.860 rhlIR (P. aeruginosa) rhlI (P. aeruginosa) C. violaceum CV026
Wild type; rhlI+ rhlR+ Apr Derived from CGMCC 1.860; rhlI− rhlR− Derived from CGMCC 1.860; rhlI− rhlR+ Biosensor for AHL
9, 14, CGMCCa 14 14 10, NCTCb
Plasmids pYC1000 pYC-rhlR pYC-rhlI pYC-rhlIR pME6000 pME6863
Derived from pME6000, tetAR replaced by tet from pBR322 pYC1000 with AatII insert of rhlR (PstI-KpnI of rhlIR) and partial flanking sequence, mob+ Tcr pYC1000 with AatII insert of rhlI (BamHI-BamHI of rhlIR) and partial flanking sequence, mob+ Tcr pYC1000 with AatII insert of rhlIR and partial flanking sequence, mob+ Tcr Broad-host-range cloning vector; Tcr pME6000 carrying the aiiA gene plac control; Tcr
14 14 14 14 15 15
a b
China General Microbiological Culture Collection Center (Beijing, China). National Collection of Type Cultures (London, UK).
and HPLC–APCI-MS/MS analyses. Furthermore, the AHLs were demonstrated to be involved in the phenol biodegradation process. 2. Materials and methods 2.1. Bacterial strains and culture conditions Bacterial strains and plasmids used in this study are listed in Table 1. For biodegradation, P. aeruginosa CGMCC 1.860 [9] and other strains were cultivated in M9 mineral medium [13] with an aromatic compound as the sole carbon and energy source. Chromobacterium violaceum CV026 (NCTC13278) was obtained from the National Collection of Type Culture (NCTC, London, UK) and used for the AHL plate and TLC bioassays [10]. E. coli JM109 and E. coli S17-1 were used for genetic manipulation. Bacteria were routinely grown with shaking in LB broth at 30 ◦ C. When required, antibiotics were used at the following concentrations: ampicillin (100 g/ml) or tetracycline (20 g/ml) for E. coli; and ampicillin (100 g/ml) and tetracycline (250 g/ml) for P. aeruginosa. 2.2. DNA manipulation and mutant construction The DNA fragment containing the rhlIR gene (GenBank Accession No. FJ213455) was cloned from P. aeruginosa CGMCC 1.860 as described elsewhere [14]. The mutants rhlI (246 bp StyI-StyI fragment in-frame deletion in rhlI; rhlI– rhlR+ ) and rhlIR (1003 bp KpnI-BamHI fragment in-frame deletion in rhlIR; rhlI– rhlR− ) were constructed by gene in-frame-deletion technology (Fig. S1). The rhlI or rhlIR mutants were complemented with the plasmids pYC-rhlI or pYC-rhlIR, respectively (Supplemental Materials). Plasmid pME6863, which harbored the AiiA lactonase from Bacillus, was a gift from Professor Haas [15]. Plasmid transformation for P. aeruginosa was performed by conjugation using E. coli S17-1 as the donor strain as reported [15].
separated with the developing solvent (methanol:water = 60:40, v/v). The plate was removed from the chromatography tank when the solvent front had migrated to within 1 cm of the top of the TLC plate. Then, the plate was dried in air and overlaid with a thin film (about 5 mm) of C. violaceum CV026 seeded in semi-solid LB agar (0.3%, w/v). After incubation at 30 ◦ C for 12–18 h, purple spots showed the location of AHLs. 2.5. Analysis of AHLs by HPLC–APCI-MS/MS HPLC–APCI-MS/MS was further used to identify the component of AHLs produced during aromatic biodegradation. The Agilent HPLC system (CA, USA) equipped with a Thermo C18 analytical column (5 m particle size, 250 mm × 4.6 mm i.d., Thermo, CA, USA) was used for sample separation. The mobile phase used was a solution of 60% acetonitrile and 40% ammonium acetate (4 mM, pH 4.2), and the flow rate was 1.0 ml/min. Mass spectra were recorded on Thermo Finnigan LCQ Deca xp Max mass spectrometer (Thermo, CA, USA) using an atmospheric pressure chemical ionization (APCI) source with a vaporizer temperature of 460 ◦ C, a sheath gas flow rate of 60 arbitrary units, an aux/sweep gas flow rate of 14 arbitrary units, a capillary voltage of 15 V, and a capillary temperature of 200 ◦ C. 2.6. Addition of AHL extract P. aeruginosa CGMCC 1.860 and P. aeruginosa CGMCC 1.860/pME6863 (plasmid pME6863 contained AiiA lactonase, which could degrade the AHLs) were cultivated in M9 medium supplemented with glucose. At the stationary phase, one liter of culture supernatants were collected and extracted as described in Section 2.3. Each extract was dissolved in 400 l DMSO (0.1%, v/v), and 10 l of these DMSO solutions were added into the phenol biodegradation system (50 ml) at the beginning of the cultivation (0 h). To determine the effect of AHL on phenol biodegradation, an equal volume of DMSO was added as a control.
3. Results and discussion 2.3. Homoserine lactone extraction AHL extracts were prepared as reported [10,14]. Spent supernatants were collected from stationary phase cultures of Pseudomonas grown in M9 medium complemented with glucose or aromatic compound by centrifugation at 12,000 × g for 4 min. Then, the supernatants were extracted with an equal volume of dichloromethane three times. The organic phase was pooled, dried over anhydrous magnesium sulfate, and filtrated. The dichloromethane was removed by evaporation at 30 ◦ C, and the residue was the AHL extracts. The AHL extract was stored at −70 ◦ C and dissolved with an appropriate volume of HPLC-grade acetonitrile for bioassay or HPLC–APCI-MS/MS. 2.4. Chromobacterium violaceum CV026 plate bioassay and TLC Stock solutions of N-butyryl-l-homoserine lactone (BHL) or N-hexanoyl-lhomoserine lactone (HHL) standard (Cayman Chemical Co., MI, USA) were prepared with HPLC-grade acetonitrile at 1 mg/ml and stored at −70 ◦ C. The AHL plate assay using C. violaceum CV026 was performed according to the method of McClean et al. [10]. Briefly, 50 l of an overnight LB culture of C. violaceum CV026 was mixed with 5 ml semi-solid LB agar (0.3%, w/v) and poured onto the surface of a prepared LB agar plate. After the overlaid agar solidified, wells were punched, and AHL extracts (after solvent was dried with nitrogen gas and AHL extracts were re-dissolved in LB broth) were piped into individual wells. The plates were incubated overnight at 30 ◦ C. TLC analysis was performed on RP18 TLC plates (Merck) using methanol/water (60:40, v/v) as the developing solvent [10,12]. Standard AHLs in acetonitrile or extracts of spent supernatant (dissolved in acetonitrile) were spotted (1–50 l) onto RP18 reverse-phase TLC plates and dried at room temperature. After that, samples were
3.1. Selection of an environmental isolate with AHL activity In order to understand whether AHL is present and active during pollutant biodegradation, a strain with both AHL-producing ability and pollutant-degrading ability was required. Based on our previous results [14], three out of nine environmental isolates belonging to Pseudomonas that showed phenol biodegradation ability were chosen for strain selection (Table 2). Because aromatics are the most prevalent and serious environment pollutants [17,18], they were chosen to represent typical pollutants in this work. AHL production in LB medium by these candidates was detected by the C. violaceum CV026 plate assay [10]. Two strains belonging to P. aeruginosa showed AHL-producing ability and pollutant-degrading ability simultaneously (Table 2). P. aeruginosa CGMCC 1.860, which was originally isolated from trash rubber, was eventually selected because it could degrade a broad spectrum of aromatics (including phenol, benzoate, p-hydroxy-benzoate, anthranilate, salicylate, naphthalene, and phenanthrene) and produce AHL in LB medium (Table 2). According to our previous report, P. aeruginosa CGMCC 1.860 contained an intact rhl cluster (GenBank Accession No. FJ213455) that showed about 99% identity to that of P. aerugi-
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Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
Table 2 Strain selection based on biodegradation ability and AHL production ability. No.
Strain
Degradationa d
1.860 1.926 1.37 a b c d
P. aeruginosa P. aeruginosa P. fluorescens
AHLb d
d
1
2
3
4
5
6
7
+++ + +++
+++ − NTc
+ − NT
+++ NT NT
+++ NT NT
+ NT NT
++ NT NT
++ ++ −
The aromatic compounds are 1, phenol; 2, naphthalene; 3, phenanthrene; 4, benzoate; 5, p-hydroxy-benzoate; 6, anthranilate; 7, salicylate. Detected by C. violaceum CV026 plate assay. Not tested. These results were referred with the Ref. [14].
nosa PAO1 [14]. In addition, an in-frame deletion in rhlIR (strain rhlIR) resulted in abolishing AHL activity as detected by C. violaceum CV026, but this activity could be restored by rhlI or rhlIR complementation in rhlIR (strain rhlIR/pYC-rhlI or rhlIR/pYCrhlIR) (Fig. 2). The results indicated that the environmental isolate P. aeruginosa CGMCC 1.860 could produce AHL in rich medium by RhlI, similar to P. aeruginosa PAO1 [4]. Therefore, P. aeruginosa CGMCC 1.860 was selected as a strain that possesses an AHL-producing ability and a pollutant-degrading ability. This provided us with a reasonable tool to investigate the production of and possible role of AHLs during biodegradation of pollutants. 3.2. Detection and identification of AHL during biodegradation of aromatics To study whether P. aeruginosa CGMCC 1.860 could produce AHL while using pollutants as the sole carbon and energy source, the selected strain was cultivated in M9 mineral medium complemented with an aromatic compound (phenol, benzoate, p-hydroxy-benzoate, anthranilate, salicylate, or naphthalene) as the sole carbon and energy source. In this system, biodegradation occurs together with cell growth. At the stationary growth phase, the culture supernatant was extracted by dichloromethane and analyzed by C. violaceum CV026 plate assay for the detection of AHLs. All of the tested extracts showed purple pigment development (Fig. 1), suggesting the presence of AHL during the biodegradation of the aromatics by P. aeruginosa CGMCC 1.860. Burmølle et al. [8] detected AHL activity in soil by a fluorescent biosensor and suggested that AHLs are present during litter decomposition. However, whether the AHL was really synthesized by pollutant degraders during pollutant decomposition or degradation was not clear because the litter decomposition process was not monitored. Our results, for the first time, clearly indicate that AHL is produced by the pollutant-degrading organism during biodegradation. The results also implied that AHLs might have certain roles in the biodegradation process. Further, to identify the AHLs that were produced during biodegradation of the aromatics, the AHL extracts obtained during phenol biodegradation were analyzed by TLC and HPLC–APCIMS/MS. The TLC chromatogram (Fig. 3) indicated that two different
Fig. 2. TLC analysis of AHL produced during phenol biodegradation by P. aeruginosa CGMCC 1.860 and related mutants. Plasmid pYC1000 was used as a vector control. Phenol (700 mg/L) was used as the sole carbon and energy source, and AHL extraction was performed using the stationary phase culture supernatant.
AHL molecules were present in this extract. Upon comparison to known standards, the AHLs were suggested to be BHL and HHL (Fig. 2). Furthermore, the AHLs were identified by HPLC–APCIMS/MS. In the mass spectra, positive adduct ions [M+NH4 ]+ (m/z 189.2) and [M+H]+ (m/z 172.3) were observed and suggested that the AHL was BHL, which has a molecular weight of 171 (Fig. 3(A)). By MS/MS analysis of the ion [M+H]+ (m/z 172.3), the mass fragmentation pattern of the ion [M+H]+ (Fig. 3(A), inset) was the same as that of BHL standard reported previously [11]. Meanwhile, HHL was identified as the other AHL component produced during phenol biodegradation (Fig. 3(B)). The biosensor C. violaceum CV026, which was used for AHL imaging in the TLC analysis, was much more sensitive (about 20 times) to HHL than to BHL [10]. Furthermore, BHL was proven to be the main AHL produced by rhlI in P. aeruginosa (about 15 times higher than HHL in concentration) [4]. Therefore, it could be estimated that BHL was the main component of AHLs produced during phenol biodegradation because the signal intensity in the TLC chromatogram was comparable between HHL and BHL (Fig. 3). This finding was also confirmed by HPLC–MS/MS analysis (data not shown). In addition, AHL production during phenol biodegradation could be abolished by rhlIR deletion and could be restored by complementation with wild-type rhlI or rhlIR gene (Fig. 2). As RhlI is the synthase of BHL and HHL in P. aeruginosa [4], the results confirmed that the AHLs detected during phenol biodegradation were comprised of BHL and HHL. 3.3. Involvement of AHLs in phenol biodegradation
Fig. 1. Detection of AHL production during biodegradation of various aromatic compounds by using C. violaceum CV026 plate assay.
The effect of AHLs on phenol biodegradation by P. aeruginosa CGMCC 1.860 was determined. Here, AHL extracts obtained from the culture supernatant of P. aeruginosa CGMCC 1.860 cultivated in M9 medium supplemented with glucose, conditions that produce high levels of AHLs as determined by C. violaceum CV026
Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
Fig. 3. Identification of BHL (A) and HHL (B) produced by P. aeruginosa CGMCC 1.860 during phenol biodegradation using HPLC–APCI-MS/MS. The insets show the MS/MS spectra of indicated ion peaks. The AHL extract was prepared as described in Fig. 2.
bioassay, was added into the phenol biodegradation system. As shown in Fig. 4(A), cell growth of P. aeruginosa CGMCC 1.860 was enhanced by addition of AHL extracts, and phenol biodegradation was also significantly improved. The results suggested that AHL was involved in the regulation of phenol biodegradation. In order to confirm that the improvement in phenol biodegradation could be ascribed to the addition of AHLs, the effect of extracts from P. aeruginosa CGMCC 1.860/pME6863, which was also cultivated in M9 medium supplemented with glucose but showed no AHL activity detected by C. violaceum CV026 bioassay, on phenol biodegradation was determined. The results showed that the extracts from P. aeruginosa CGMCC 1.860/pME6863 had no effect on cell growth or phenol biodegradation (Fig. 4(A)). Because the plasmid pME6863 contained AiiA lactonase, which could specifically degrade AHLs produced by P. aeruginosa [19], the main difference between the extracts added above (A, extracts from P. aeruginosa CGMCC 1.860; B, extracts from P. aeruginosa CGMCC 1.860/pME6863) was that AHLs were present under the conditions in A but not in B. Therefore, the results suggest that AHLs could improve phenol biodegradation by P. aeruginosa. Furthermore, because the mutant rhlI could not synthesize these AHLs (Fig. 2), phenol biodegradation by this strain was greatly repressed compared to the wild type strain. In addition, the repression in biodegradation was effectively reversed by
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Fig. 4. Effect of addition of AHL extracts on cell growth (dashed line) and phenol biodegradation (solid line) by P. aeruginosa CGMCC 1.860 (WT) and rhlI. A, WT type strain with addition of various AHL extracts: () control, without addition; () addition of AHL extract from P. aeruginosa CGMCC 1.860; and () addition of AHL extract from P. aeruginosa CGMCC 1.860/pME6863. B: () WT strain without addition; (䊉) rhlI without addition; and () rhlI with addition of AHL extracts from P. aeruginosa CGMCC 1.860.
addition of AHL extract in rhlI (Fig. 4(B)). These results confirm that AHLs have a positive effect on phenol biodegradation. Although Valle et al. [5] reported that addition of AHLs could affect the bacterial community in industrial activated sludge, it was unknown whether or not AHL could affect the pollutant biodegradation ability of bacteria. Our results indicated that addition of AHLs could improve the phenol biodegradation ability of P. aeruginosa CGMCC 1.860. However, the question remains of whether the endogenously synthesized AHLs during biodegradation have the same effect on phenol biodegradation as exogenously added AHLs. Because rhlI was responsible for AHL production during phenol biodegradation, AHL production was abolished in rhlIR and rhlI strains, and increased in rhlIR/pYC-rhlIR and rhlI/pYCrhlI strains (Fig. 2). The effect of endogenously produced AHLs on phenol biodegradation was analyzed among these strains. As shown in Fig. 5, phenol biodegradation was significantly repressed by rhlIR (strain rhlIR) or rhlI deletion (strain rhlI) as compared with the wild type strain. However, this repression could be reversed by complementation with the wild type rhlIR gene (strain rhlIR/pYC-rhlIR) or rhlI (strain rhlI/pYC-rhlI). Also, exogenously added BHL (the main signal molecule synthesized by rhlI) could enhance biodegradation by the wild type strain (Fig. 5). The results indicated that endogenously produced AHLs by the rhlIR QS system had a positive effect on the phenol biodegradation ability of P. aeruginosa, which is in accordance with the result obtained with
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Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
References
Fig. 5. Effect of endogenously synthesized AHLs on phenol biodegradation. Plasmid pYC1000 was used as the vector control. Experimental conditions were as follows: initial phenol concentration, 750 mg/L; ampicillin (100 g/ml) and tetracycline (250 g/ml); and BHL, 20 M. The phenol concentration shown in this figure was the residual phenol detected at 50 h of cultivation.
the addition of exogenous AHLs. In addition, rhlIR also showed stimulation of benzoate biodegradation by P. aeruginosa (Fig. S2). This work demonstrated AHL production during pollutant biodegradation and showed the involvement of AHLs in biodegradation. These findings are the first step in elucidating the role of QS during pollutant biodegradation. The mechanism for the effect of AHL on biodegradation remains to be clarified, and further work is in progress in our laboratory. Furthermore, as this work suggests, manipulation of the AHL-mediated QS system could be expected to achieve a better control of pollutant biodegradation. Acknowledgements The financial support from the National Natural Science Foundation of China (NSFC project nos. 30821005 and 20876096) and the Shanghai Leading Academic Discipline Project (#B203 and B505) is appreciated. The donation of pME6000, pME6863 and E. coli S17-1 from Professor Dieter Haas (University of Lausanne, Switzerland) and C. violaceum CV026 from Professor Paul Williams (University of Nottingham, UK) is also gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2010.05.006.
[1] Bassler BL, Losick R. Bacterially speaking. Cell 2006;125:237–46. [2] Fuqua C, Greenberg EP. Listening in on bacteria: acyl-homoserine lactone signaling. Nat Rev Mol Cell Biol 2002;3:685–95. [3] Manefield M, Whiteley AS. Acylated homoserine lactones in the environment: chameleons of bioactivity. Phil Trans R Soc B 2007;362:1235–40. [4] Parsek MR, Val DL, Hanzelka BL, Cronan JE, Greenberg EP. Acyl homoserinelactone quorum-sensing signal generation. Proc Natl Acad Sci USA 1999;96:4360–5. [5] Valle A, Bailey MJ, Whiteley AS, Manefield M. N-Acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge. Environ Microbiol 2004;6:424–33. [6] Hu JY, Fan Y, Lin YH, Zhang HB, Ong SL, Dong N, et al. Microbial diversity and prevalence of virulent pathogens in biofilms developed in water reclamation system. Res Microbiol 2003;154:623–9. [7] Morgan-Sagastume F, Boon N, Dobbelaere S, Defoirdt T, Verstraete W. Production of acylated homoserine lactones by Aeromonas and Pseudomonas strains isolated from municipal activated sludge. Can J Microbiol 2005;51: 924–33. [8] Burmølle M, Hansen LH, Sørensen SJ. Use of a whole-cell biosensor and flowcell cytometry to detect AHL production by indigenous soil community during decomposition of litter. Microbial Ecol 2005;50:221–9. [9] Sun GX, Zhou WQ, Zhong JJ. Organotin decomposition by pyochelin, secreted by Pseudomonas aeruginosa even in an iron-sufficient environment. Appl Environ Microbiol 2006;72:6411–3. [10] McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, et al. Quorum sensing and Chromobacterium violaceum: Exploitation of violacein production and inhibition for the detection of N-acyl homoserine lactones. MicrobiologySGM 1997;143:3703–11. [11] Morin D, Grasland B, Vallée-Rehel K, Dufau C, Haras D. On-line highperformance liquid chromatography–mass spectrometric detection and quantification of N-acylhomoserine lactones, quorum sensing signal molecules, in the presence of biological matrices. J Chromatogr A 2003;1002: 79–92. [12] Shaw PD, Ping G, Daly S, Cronan JE, Rinehart K, Farrand SK. Detecting and characterizing acyl-homoserine lactone signal molecules by thin layer chromatography. Proc Natl Acad Sci USA 1997;94:6036–41. [13] Tian L, Ma P, Zhong JJ. Kinetics and key enzyme activities of phenanthrene degradation by Pseudomonas mendocina. Process Biochem 2002;37:1431–7. [14] Yong YC, Zhong JJ. A genetically engineered whole-cell pigment-based bacterial biosensing system for quantification of N-butyryl homoserine lactone quorum sensing signal. Biosens Bioelectron 2009;25:41–7. [15] Reimmann C, Ginet N, Michel L, Keel C, Michaux P, Krishnapillai V, et al. Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1. Microbiology-SGM 2002;148:923–32. [16] Ishida T, Ikeda T, Takiguchi N, Kuroda A, Ohtake H, Kato J. Inhibition of quorum sensing in Pseudomonas aeruginosa by N-acyl cyclopentylamides. Appl Environ Microbiol 2007;73:3183–8. [17] Malick S, Dutta TK. Kinetics of phenanthrene degradation by Staphylococcus sp. strain PN/Y involving 2-hydroxy-1-naphthoic acid in a novel metabolic pathway. Process Biochem 2008;43:1004–8. [18] Hsieh FM, Huang C, Lin TF, Chen YM, Lin JC. Study of sodium tripolyphosphatecrosslinked chitosan beads entrapped with Pseudomonas putida for phenol degradation. Process Biochem 2008;43:83–92. [19] Wang LH, Weng LX, Dong YH, Zhang LH. Specificity and enzyme kinetics of the quorum-quenching N-acyl homoserine lactone lactonase (AHL-lactonase). J Biol Chem 2004;279:13645–51.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa Yang-Chun Yong a,c , Jian-Jiang Zhong a,b,∗ a
State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China c State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China b
a r t i c l e
i n f o
Article history: Received 24 January 2010 Received in revised form 2 May 2010 Accepted 4 May 2010 Keywords: Quorum sensing Homoserine lactone Aromatics Pseudomonas aeruginosa Biodegradation
a b s t r a c t N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bacteria communicate with each other by producing diffusible small signaling molecules and sensing signals in the environment, which is known as quorum sensing (QS) [1,2]. In Gram-negative bacteria, acylated homoserine lactones (AHLs) are the quorum sensing signal molecules. These AHLs are conserved at the lactone moiety and varied in their acyl-chain (including chain length and C-3 substitution) [2,3]. For example, BHL and HHL, which are the signal molecules for the rhl QS system in Pseudomonas aeruginosa, are both C-3 unsubstituted AHLs and only differ in acyl-chain length [2]. AHL-mediated QS has an important role in many essential biological processes, including animal and plant diseases [1,16].
∗ Corresponding author at: School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China. Tel.: +86 21 34206968; fax: +86 21 34204831. E-mail address: [email protected] (J.-J. Zhong). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.05.006
In environmental bioprocesses, strains such as Pseudomonas, Agrobacterium, and Aeromonas, which may produce AHLs, were isolated from activated sludges and water reclamation systems, and AHL production by these strains was confirmed in rich culture media [5–7]. Addition of AHLs changed the composition of the bacterial community in industrial activated sludges and improved the performance of these sludges through an unknown mechanism [5]. However, until now, there have been no reports of AHL production during pollutant biodegradation. In addition, the ability of these isolated AHL producers to degrade pollutants has not yet been reported. Therefore, whether any potential AHL producers could synthesize AHLs during pollutant degradation is yet to be clarified, and it is crucial to evaluate the role of AHL or AHL-mediated QS in biodegradation processes. In this study, an environmental isolate P. aeruginosa CGMCC 1.860 was selected because it could degrade a broad spectrum of aromatic compounds and produce AHLs in rich culture media. AHL production by the strain was observed during aromatic biodegradation. Additionally, the AHLs produced during aromatic biodegradation were then identified by using TLC
Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
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Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid
Description
Reference or source
Strains P. aeruginosa CGMCC 1.860 rhlIR (P. aeruginosa) rhlI (P. aeruginosa) C. violaceum CV026
Wild type; rhlI+ rhlR+ Apr Derived from CGMCC 1.860; rhlI− rhlR− Derived from CGMCC 1.860; rhlI− rhlR+ Biosensor for AHL
9, 14, CGMCCa 14 14 10, NCTCb
Plasmids pYC1000 pYC-rhlR pYC-rhlI pYC-rhlIR pME6000 pME6863
Derived from pME6000, tetAR replaced by tet from pBR322 pYC1000 with AatII insert of rhlR (PstI-KpnI of rhlIR) and partial flanking sequence, mob+ Tcr pYC1000 with AatII insert of rhlI (BamHI-BamHI of rhlIR) and partial flanking sequence, mob+ Tcr pYC1000 with AatII insert of rhlIR and partial flanking sequence, mob+ Tcr Broad-host-range cloning vector; Tcr pME6000 carrying the aiiA gene plac control; Tcr
14 14 14 14 15 15
a b
China General Microbiological Culture Collection Center (Beijing, China). National Collection of Type Cultures (London, UK).
and HPLC–APCI-MS/MS analyses. Furthermore, the AHLs were demonstrated to be involved in the phenol biodegradation process. 2. Materials and methods 2.1. Bacterial strains and culture conditions Bacterial strains and plasmids used in this study are listed in Table 1. For biodegradation, P. aeruginosa CGMCC 1.860 [9] and other strains were cultivated in M9 mineral medium [13] with an aromatic compound as the sole carbon and energy source. Chromobacterium violaceum CV026 (NCTC13278) was obtained from the National Collection of Type Culture (NCTC, London, UK) and used for the AHL plate and TLC bioassays [10]. E. coli JM109 and E. coli S17-1 were used for genetic manipulation. Bacteria were routinely grown with shaking in LB broth at 30 ◦ C. When required, antibiotics were used at the following concentrations: ampicillin (100 g/ml) or tetracycline (20 g/ml) for E. coli; and ampicillin (100 g/ml) and tetracycline (250 g/ml) for P. aeruginosa. 2.2. DNA manipulation and mutant construction The DNA fragment containing the rhlIR gene (GenBank Accession No. FJ213455) was cloned from P. aeruginosa CGMCC 1.860 as described elsewhere [14]. The mutants rhlI (246 bp StyI-StyI fragment in-frame deletion in rhlI; rhlI– rhlR+ ) and rhlIR (1003 bp KpnI-BamHI fragment in-frame deletion in rhlIR; rhlI– rhlR− ) were constructed by gene in-frame-deletion technology (Fig. S1). The rhlI or rhlIR mutants were complemented with the plasmids pYC-rhlI or pYC-rhlIR, respectively (Supplemental Materials). Plasmid pME6863, which harbored the AiiA lactonase from Bacillus, was a gift from Professor Haas [15]. Plasmid transformation for P. aeruginosa was performed by conjugation using E. coli S17-1 as the donor strain as reported [15].
separated with the developing solvent (methanol:water = 60:40, v/v). The plate was removed from the chromatography tank when the solvent front had migrated to within 1 cm of the top of the TLC plate. Then, the plate was dried in air and overlaid with a thin film (about 5 mm) of C. violaceum CV026 seeded in semi-solid LB agar (0.3%, w/v). After incubation at 30 ◦ C for 12–18 h, purple spots showed the location of AHLs. 2.5. Analysis of AHLs by HPLC–APCI-MS/MS HPLC–APCI-MS/MS was further used to identify the component of AHLs produced during aromatic biodegradation. The Agilent HPLC system (CA, USA) equipped with a Thermo C18 analytical column (5 m particle size, 250 mm × 4.6 mm i.d., Thermo, CA, USA) was used for sample separation. The mobile phase used was a solution of 60% acetonitrile and 40% ammonium acetate (4 mM, pH 4.2), and the flow rate was 1.0 ml/min. Mass spectra were recorded on Thermo Finnigan LCQ Deca xp Max mass spectrometer (Thermo, CA, USA) using an atmospheric pressure chemical ionization (APCI) source with a vaporizer temperature of 460 ◦ C, a sheath gas flow rate of 60 arbitrary units, an aux/sweep gas flow rate of 14 arbitrary units, a capillary voltage of 15 V, and a capillary temperature of 200 ◦ C. 2.6. Addition of AHL extract P. aeruginosa CGMCC 1.860 and P. aeruginosa CGMCC 1.860/pME6863 (plasmid pME6863 contained AiiA lactonase, which could degrade the AHLs) were cultivated in M9 medium supplemented with glucose. At the stationary phase, one liter of culture supernatants were collected and extracted as described in Section 2.3. Each extract was dissolved in 400 l DMSO (0.1%, v/v), and 10 l of these DMSO solutions were added into the phenol biodegradation system (50 ml) at the beginning of the cultivation (0 h). To determine the effect of AHL on phenol biodegradation, an equal volume of DMSO was added as a control.
3. Results and discussion 2.3. Homoserine lactone extraction AHL extracts were prepared as reported [10,14]. Spent supernatants were collected from stationary phase cultures of Pseudomonas grown in M9 medium complemented with glucose or aromatic compound by centrifugation at 12,000 × g for 4 min. Then, the supernatants were extracted with an equal volume of dichloromethane three times. The organic phase was pooled, dried over anhydrous magnesium sulfate, and filtrated. The dichloromethane was removed by evaporation at 30 ◦ C, and the residue was the AHL extracts. The AHL extract was stored at −70 ◦ C and dissolved with an appropriate volume of HPLC-grade acetonitrile for bioassay or HPLC–APCI-MS/MS. 2.4. Chromobacterium violaceum CV026 plate bioassay and TLC Stock solutions of N-butyryl-l-homoserine lactone (BHL) or N-hexanoyl-lhomoserine lactone (HHL) standard (Cayman Chemical Co., MI, USA) were prepared with HPLC-grade acetonitrile at 1 mg/ml and stored at −70 ◦ C. The AHL plate assay using C. violaceum CV026 was performed according to the method of McClean et al. [10]. Briefly, 50 l of an overnight LB culture of C. violaceum CV026 was mixed with 5 ml semi-solid LB agar (0.3%, w/v) and poured onto the surface of a prepared LB agar plate. After the overlaid agar solidified, wells were punched, and AHL extracts (after solvent was dried with nitrogen gas and AHL extracts were re-dissolved in LB broth) were piped into individual wells. The plates were incubated overnight at 30 ◦ C. TLC analysis was performed on RP18 TLC plates (Merck) using methanol/water (60:40, v/v) as the developing solvent [10,12]. Standard AHLs in acetonitrile or extracts of spent supernatant (dissolved in acetonitrile) were spotted (1–50 l) onto RP18 reverse-phase TLC plates and dried at room temperature. After that, samples were
3.1. Selection of an environmental isolate with AHL activity In order to understand whether AHL is present and active during pollutant biodegradation, a strain with both AHL-producing ability and pollutant-degrading ability was required. Based on our previous results [14], three out of nine environmental isolates belonging to Pseudomonas that showed phenol biodegradation ability were chosen for strain selection (Table 2). Because aromatics are the most prevalent and serious environment pollutants [17,18], they were chosen to represent typical pollutants in this work. AHL production in LB medium by these candidates was detected by the C. violaceum CV026 plate assay [10]. Two strains belonging to P. aeruginosa showed AHL-producing ability and pollutant-degrading ability simultaneously (Table 2). P. aeruginosa CGMCC 1.860, which was originally isolated from trash rubber, was eventually selected because it could degrade a broad spectrum of aromatics (including phenol, benzoate, p-hydroxy-benzoate, anthranilate, salicylate, naphthalene, and phenanthrene) and produce AHL in LB medium (Table 2). According to our previous report, P. aeruginosa CGMCC 1.860 contained an intact rhl cluster (GenBank Accession No. FJ213455) that showed about 99% identity to that of P. aerugi-
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Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
Table 2 Strain selection based on biodegradation ability and AHL production ability. No.
Strain
Degradationa d
1.860 1.926 1.37 a b c d
P. aeruginosa P. aeruginosa P. fluorescens
AHLb d
d
1
2
3
4
5
6
7
+++ + +++
+++ − NTc
+ − NT
+++ NT NT
+++ NT NT
+ NT NT
++ NT NT
++ ++ −
The aromatic compounds are 1, phenol; 2, naphthalene; 3, phenanthrene; 4, benzoate; 5, p-hydroxy-benzoate; 6, anthranilate; 7, salicylate. Detected by C. violaceum CV026 plate assay. Not tested. These results were referred with the Ref. [14].
nosa PAO1 [14]. In addition, an in-frame deletion in rhlIR (strain rhlIR) resulted in abolishing AHL activity as detected by C. violaceum CV026, but this activity could be restored by rhlI or rhlIR complementation in rhlIR (strain rhlIR/pYC-rhlI or rhlIR/pYCrhlIR) (Fig. 2). The results indicated that the environmental isolate P. aeruginosa CGMCC 1.860 could produce AHL in rich medium by RhlI, similar to P. aeruginosa PAO1 [4]. Therefore, P. aeruginosa CGMCC 1.860 was selected as a strain that possesses an AHL-producing ability and a pollutant-degrading ability. This provided us with a reasonable tool to investigate the production of and possible role of AHLs during biodegradation of pollutants. 3.2. Detection and identification of AHL during biodegradation of aromatics To study whether P. aeruginosa CGMCC 1.860 could produce AHL while using pollutants as the sole carbon and energy source, the selected strain was cultivated in M9 mineral medium complemented with an aromatic compound (phenol, benzoate, p-hydroxy-benzoate, anthranilate, salicylate, or naphthalene) as the sole carbon and energy source. In this system, biodegradation occurs together with cell growth. At the stationary growth phase, the culture supernatant was extracted by dichloromethane and analyzed by C. violaceum CV026 plate assay for the detection of AHLs. All of the tested extracts showed purple pigment development (Fig. 1), suggesting the presence of AHL during the biodegradation of the aromatics by P. aeruginosa CGMCC 1.860. Burmølle et al. [8] detected AHL activity in soil by a fluorescent biosensor and suggested that AHLs are present during litter decomposition. However, whether the AHL was really synthesized by pollutant degraders during pollutant decomposition or degradation was not clear because the litter decomposition process was not monitored. Our results, for the first time, clearly indicate that AHL is produced by the pollutant-degrading organism during biodegradation. The results also implied that AHLs might have certain roles in the biodegradation process. Further, to identify the AHLs that were produced during biodegradation of the aromatics, the AHL extracts obtained during phenol biodegradation were analyzed by TLC and HPLC–APCIMS/MS. The TLC chromatogram (Fig. 3) indicated that two different
Fig. 2. TLC analysis of AHL produced during phenol biodegradation by P. aeruginosa CGMCC 1.860 and related mutants. Plasmid pYC1000 was used as a vector control. Phenol (700 mg/L) was used as the sole carbon and energy source, and AHL extraction was performed using the stationary phase culture supernatant.
AHL molecules were present in this extract. Upon comparison to known standards, the AHLs were suggested to be BHL and HHL (Fig. 2). Furthermore, the AHLs were identified by HPLC–APCIMS/MS. In the mass spectra, positive adduct ions [M+NH4 ]+ (m/z 189.2) and [M+H]+ (m/z 172.3) were observed and suggested that the AHL was BHL, which has a molecular weight of 171 (Fig. 3(A)). By MS/MS analysis of the ion [M+H]+ (m/z 172.3), the mass fragmentation pattern of the ion [M+H]+ (Fig. 3(A), inset) was the same as that of BHL standard reported previously [11]. Meanwhile, HHL was identified as the other AHL component produced during phenol biodegradation (Fig. 3(B)). The biosensor C. violaceum CV026, which was used for AHL imaging in the TLC analysis, was much more sensitive (about 20 times) to HHL than to BHL [10]. Furthermore, BHL was proven to be the main AHL produced by rhlI in P. aeruginosa (about 15 times higher than HHL in concentration) [4]. Therefore, it could be estimated that BHL was the main component of AHLs produced during phenol biodegradation because the signal intensity in the TLC chromatogram was comparable between HHL and BHL (Fig. 3). This finding was also confirmed by HPLC–MS/MS analysis (data not shown). In addition, AHL production during phenol biodegradation could be abolished by rhlIR deletion and could be restored by complementation with wild-type rhlI or rhlIR gene (Fig. 2). As RhlI is the synthase of BHL and HHL in P. aeruginosa [4], the results confirmed that the AHLs detected during phenol biodegradation were comprised of BHL and HHL. 3.3. Involvement of AHLs in phenol biodegradation
Fig. 1. Detection of AHL production during biodegradation of various aromatic compounds by using C. violaceum CV026 plate assay.
The effect of AHLs on phenol biodegradation by P. aeruginosa CGMCC 1.860 was determined. Here, AHL extracts obtained from the culture supernatant of P. aeruginosa CGMCC 1.860 cultivated in M9 medium supplemented with glucose, conditions that produce high levels of AHLs as determined by C. violaceum CV026
Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
Fig. 3. Identification of BHL (A) and HHL (B) produced by P. aeruginosa CGMCC 1.860 during phenol biodegradation using HPLC–APCI-MS/MS. The insets show the MS/MS spectra of indicated ion peaks. The AHL extract was prepared as described in Fig. 2.
bioassay, was added into the phenol biodegradation system. As shown in Fig. 4(A), cell growth of P. aeruginosa CGMCC 1.860 was enhanced by addition of AHL extracts, and phenol biodegradation was also significantly improved. The results suggested that AHL was involved in the regulation of phenol biodegradation. In order to confirm that the improvement in phenol biodegradation could be ascribed to the addition of AHLs, the effect of extracts from P. aeruginosa CGMCC 1.860/pME6863, which was also cultivated in M9 medium supplemented with glucose but showed no AHL activity detected by C. violaceum CV026 bioassay, on phenol biodegradation was determined. The results showed that the extracts from P. aeruginosa CGMCC 1.860/pME6863 had no effect on cell growth or phenol biodegradation (Fig. 4(A)). Because the plasmid pME6863 contained AiiA lactonase, which could specifically degrade AHLs produced by P. aeruginosa [19], the main difference between the extracts added above (A, extracts from P. aeruginosa CGMCC 1.860; B, extracts from P. aeruginosa CGMCC 1.860/pME6863) was that AHLs were present under the conditions in A but not in B. Therefore, the results suggest that AHLs could improve phenol biodegradation by P. aeruginosa. Furthermore, because the mutant rhlI could not synthesize these AHLs (Fig. 2), phenol biodegradation by this strain was greatly repressed compared to the wild type strain. In addition, the repression in biodegradation was effectively reversed by
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Fig. 4. Effect of addition of AHL extracts on cell growth (dashed line) and phenol biodegradation (solid line) by P. aeruginosa CGMCC 1.860 (WT) and rhlI. A, WT type strain with addition of various AHL extracts: () control, without addition; () addition of AHL extract from P. aeruginosa CGMCC 1.860; and () addition of AHL extract from P. aeruginosa CGMCC 1.860/pME6863. B: () WT strain without addition; (䊉) rhlI without addition; and () rhlI with addition of AHL extracts from P. aeruginosa CGMCC 1.860.
addition of AHL extract in rhlI (Fig. 4(B)). These results confirm that AHLs have a positive effect on phenol biodegradation. Although Valle et al. [5] reported that addition of AHLs could affect the bacterial community in industrial activated sludge, it was unknown whether or not AHL could affect the pollutant biodegradation ability of bacteria. Our results indicated that addition of AHLs could improve the phenol biodegradation ability of P. aeruginosa CGMCC 1.860. However, the question remains of whether the endogenously synthesized AHLs during biodegradation have the same effect on phenol biodegradation as exogenously added AHLs. Because rhlI was responsible for AHL production during phenol biodegradation, AHL production was abolished in rhlIR and rhlI strains, and increased in rhlIR/pYC-rhlIR and rhlI/pYCrhlI strains (Fig. 2). The effect of endogenously produced AHLs on phenol biodegradation was analyzed among these strains. As shown in Fig. 5, phenol biodegradation was significantly repressed by rhlIR (strain rhlIR) or rhlI deletion (strain rhlI) as compared with the wild type strain. However, this repression could be reversed by complementation with the wild type rhlIR gene (strain rhlIR/pYC-rhlIR) or rhlI (strain rhlI/pYC-rhlI). Also, exogenously added BHL (the main signal molecule synthesized by rhlI) could enhance biodegradation by the wild type strain (Fig. 5). The results indicated that endogenously produced AHLs by the rhlIR QS system had a positive effect on the phenol biodegradation ability of P. aeruginosa, which is in accordance with the result obtained with
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Y.-C. Yong, J.-J. Zhong / Process Biochemistry 45 (2010) 1944–1948
References
Fig. 5. Effect of endogenously synthesized AHLs on phenol biodegradation. Plasmid pYC1000 was used as the vector control. Experimental conditions were as follows: initial phenol concentration, 750 mg/L; ampicillin (100 g/ml) and tetracycline (250 g/ml); and BHL, 20 M. The phenol concentration shown in this figure was the residual phenol detected at 50 h of cultivation.
the addition of exogenous AHLs. In addition, rhlIR also showed stimulation of benzoate biodegradation by P. aeruginosa (Fig. S2). This work demonstrated AHL production during pollutant biodegradation and showed the involvement of AHLs in biodegradation. These findings are the first step in elucidating the role of QS during pollutant biodegradation. The mechanism for the effect of AHL on biodegradation remains to be clarified, and further work is in progress in our laboratory. Furthermore, as this work suggests, manipulation of the AHL-mediated QS system could be expected to achieve a better control of pollutant biodegradation. Acknowledgements The financial support from the National Natural Science Foundation of China (NSFC project nos. 30821005 and 20876096) and the Shanghai Leading Academic Discipline Project (#B203 and B505) is appreciated. The donation of pME6000, pME6863 and E. coli S17-1 from Professor Dieter Haas (University of Lausanne, Switzerland) and C. violaceum CV026 from Professor Paul Williams (University of Nottingham, UK) is also gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2010.05.006.
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