SEARCH

SEARCH BY CITATION

Keywords:

  • Polychlorinated biphenyl;
  • bphK;
  • Burkholderia LB400;
  • Bioscreen C®;
  • Pseudomonas F113;
  • Glutathione S-transferase

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

A bphK gene encoding glutathione S-transferase (GST) activity is located in the bph operon in Burkholderia sp. strain LB400 but its role in polychlorinated biphenyl (PCB) metabolism is unknown. This gene was over-expressed in Escherichia coli and an in vivo assay based on growth of E. coli containing GST activity was used to identify potential novel substrates for this enzyme. Using this assay, 4-chlorobenzoate (4-CBA) was identified as a substrate for the BphK enzyme. High pressure liquid chromatography analysis and chloride ion detection showed removal of 4-CBA and an equivalent increase of chloride in cell extracts when incubated with this enzyme. These results would indicate that this BphK enzyme has dechlorination activity in relation to 4-CBA and may have a role in protection of other Bph enzymes against certain chlorinated metabolites of PCB degradation.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The glutathione S-transferases (GSTs: EC 2.5.1.18) are a family of multifunctional dimeric proteins that catalyse the conjugation of the sulfur atom of glutathione (GSH) with a large variety of electrophilic compounds of both endobiotic and xenobiotic origin [1]. Although GSTs have been isolated from micro-organisms little is known about their biological functions, structure or regulation [2,3]. While many bacterial GSTs seem to be implicated in the biodegradation of xenobiotics [4,5], in most cases their precise role is unclear. However, a dichloromethane dehalogenase isolated from Methylobacterium sp. strain DM4 is a member of the GST superfamily. This enzyme catalyses a typical GST-type reaction in which glutathione attacks dichloromethane to release chloride and form a thioether intermediate [6]. Similarly a tetrachlorohydroquinone dehalogenase from Sphingomonas chlorophenolica, which has a low but significant sequence identity to microbial GSTs, is involved in the reductive dehalogenation of tetrachlorohydroquinone [7].

A gene (bphK) encoding a protein with a significant sequence similarity to prokaryotic and eukaryotic GSTs was found at a central location within the bph gene cluster of Burkholderia sp. LB400 [8]. However, this gene does not appear to be essential for growth on biphenyl [9]. GST activity of the protein product of the bphK gene in Burkholderia sp. LB400 was demonstrated by Hofer et al. [8] and it was found to conjugate a limited number of electrophiles [9].

The bph operon from Burkholderia sp. LB400, which is responsible for the polychlorinated biphenyl (PCB)-degrading ability of the strain, was previously cloned into Pseudomonas fluorescens F113 to generate the genetically modified (GM) root coloniser, P. fluorescens F113pcb [10]. In this strain and LB400, 4-chlorobiphenyl breakdown results in 4-chlorobenzoate (4-CBA). It has been shown that chlorinated metabolites of PCB breakdown can inhibit 2,3-dihydroxybiphenyl 1,2-dioxygenase, a key enzyme of the pathway [11]. It is also reported that 4-CBA can be converted by other microbes in soil to protoanemonin which negatively affects the survival of 4-chlorobiphenyl-cometabolising micro-organisms [12,13].

The most common substrate for the study of GSTs is 1-chloro-2,4-dinitrobenzene (CDNB). An in vivo assay for the detection of GST activity was devised by Hin-Cheung et al. [14] using a eukaryotic GST. In this study the Hin-Cheung assay was adapted to identify other potential substrates for the GST in P. fluorescens F113PCB when the gene responsible for this activity was cloned into Escherichia coli. Polymerase chain reaction (PCR) amplification and subsequent cloning was used to confirm the presence of an allele of the LB400 bphK gene in E. coli. 4-CBA was identified as a potential substrate and further analysis revealed that BphK had a potential dechlorination function with relation to this substrate.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

2.1Bacterial cultures

Cultures of E. coli JM109 (pGEM) and E. coli JM109 (pNG4) were grown for 24 h at 37°C in TSB (tryptone soya broth, Oxoid, Hampshire, UK) containing 100 μg ml−1 ampicillin (amp) and a final concentration of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cultures of P. fluorescens F113PCB and P. fluorescens F113rif were grown for 24 h at 30°C in TSB containing 50 μg ml−1 rifampicin (rif). Biphenyl was added to the broths containing P. fluorescens F113PCB.

2.2Preparation of cellular extracts

The cultures (300 ml) were grown under the appropriate conditions for 48 h to an OD600 of approximately 1.9. The cells were harvested by centrifugation at 9680×g for 15 min and the resulting pellet was washed twice in 0.1 M potassium phosphate buffer (pH 6.8) with 1.0 mM EDTA. The final pellet was resuspended in 25 ml of buffer. Cells were disrupted by sonication with a Soniprep 150 (Sanyo) using four 30-s bursts at an amplitude of 17 μm at 0°C. Disrupted cells were centrifuged for 1 h at 47 810×g at 4°C. The cell extracts were maintained at 5°C until assayed (typically within 2–8 h).

2.3In vitro GST activity assay

The GST activity was assayed based on the method of Habig and Jakobi [15]. The reaction mixture consisted of a final concentration of 1 mM CDNB, 1 mM GSH and 200 μl of cell extract (1–5 mg of protein) made up to a final volume of 3 ml using 0.1 M phosphate buffer pH 6.8 containing 1.0 mM EDTA. The reaction was initiated by the addition of GSH. CDNB–GSH conjugation was monitored spectrophotometrically at 340 nm for 2 min. Protein concentration was determined by the method of Bradford [16] using bovine serum albumin as a standard. Activities were expressed in units per milligram of protein. One unit was defined as the activity that catalyses the conversion of 1 μmol of substrate per minute.

2.4PCR amplification and cloning of the bphK gene

Genomic DNA isolation was carried out using a Wizard Genomic Kit (Promega). The following PCR primer set was designed based on the adjacent sequences of the bphC and bphH genes of the bph operon to amplify the bphK gene from P. fluorescens F113PCB: forward primer K1 5′-CAACAAAGCATGAACAACAACC-3′ and reverse primer K2 5′-ATGGCGTTTCTCATTCCAGG-3′. The resulting PCR product was cloned into the cloning site of pGEM T-Easy according to the manufacturer's instructions (Promega). The ligation mixture was then transformed into E. coli JM109 cells and recombinant plasmids selected and screened on agar plates containing 100 μg ml−1 amp and X-gal (Sigma). White colonies were selected as containing the recombinant plasmid and characterised with respect to inserts. One clone with a plasmid of the expected size was chosen for further study, E. coli JM109 (pNG4). A control strain was constructed consisting of pGEM T-Easy vector transformed into E. coli JM109 cells giving E. coli JM109 (pGEM).

2.5Rapid assay for in vivo GST activity using the Bioscreen® automated turbidimetric system

This in vivo GST activity was an adaptation of the method of Hin-Cheung et al. [14]. 350 μl TSB containing CDNB from a 20 mM stock and 50 μl of an overnight culture was added to each well in the microplate giving a final volume per well of 400 μl. The OD600 was read every hour up to 24 h using an automated turbidimetric system (Bioscreen®, Finland). This assay was repeated using a range of chlorinated compounds including 4-CBA, 3-CBA, 4-chlorobiphenyl.

2.64-CBA and chloride ion detection

50 μl of a 10 mM 4-CBA solution and 50 μl of a 10 mM GSH solution was added to 900 μl of cellular extract which was prepared as described previously. The reaction was terminated by the addition of 20 μl of 5 M H2SO4 followed by centrifugation at 4300×g for 5 min. Concentrations of 4-CBA were quantitatively analysed by high pressure liquid chromatography (HPLC) with a C18 polar end capped column (250×4.6 mm: Phenomenex, UK) as described by van den Tweel et al. [17]. The mobile phase was methanol–water–acetic acid (60:40:1), the flow rate was 1 ml min−1 and detection was by UV absorbance at 254 nm. A retention time of 10.9 min was observed under these conditions. The inorganic chloride ion concentration was measured using the spectrophotometer assay as described by Bergmann and Sanik [18].

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

3.1The bphK gene in F113PCB confers GST activity

P. fluorescens F113PCB and its isogenic parent strain, P. fluorescens F113rif, were tested for the production of GST using the GST activity assay [15] as described previously. P. fluorescens F113PCB showed significantly higher GST activity than the unmodified P. fluorescens F113rif (Table 1) in the presence of the inducer biphenyl. The GST activity of P. fluorescens F113PCB when grown without the addition of biphenyl was 0.0046 U protein mg−1, which is similar to the value obtained for P. fluorescens F113rif that does not contain the bph operon (data not shown). This indicates that the addition of biphenyl is necessary for the expression of bphK from the bph operon in this GM strain.

Table 1.  GST activitya in crude extracts from P. fluorescens F113PCB and E. coli recombinants grown under inducing conditions (biphenyl for Pseudomonas, IPTG for E. coli)
  1. aValues shown are mean values±S.D. for two replicates.

StrainTotal activity (U)Total protein (mg)Specific activity (U mg protein−1)
P. fluorescens F113PCB0.053±0.0054.53±0.010.0116±0.002
P. fluorescens F113rif0.031±0.0038.25±0.050.0037±0.005
E. coli JM109 (pNG4)1.726±0.022.46±0.0010.702±0.01
E. coli JM1090.016±0.00310.31±0.0050.0016±0.007

3.2Cloning and over-expression of bphK in E. coli

Primers K1 and K2 were designed based on DNA sequences flanking the bphK gene from the LB400 bph operon. A PCR using these two oligonucleotides amplified a 850-bp fragment from P. fluorescens F113PCB genomic DNA indicating the presence of the entire gene in the GM strain. This amplicon was cloned into pGEM-T and transformed into E. coli JM109. DNA sequencing analysis (data not shown) confirmed that a 609-bp gene similar to bphKLB400 was present in these clones. However, this bphK had a single base change from A to G at position 136 (compared to bphKLB400) leading to an amino acid change from isoleucine to valine at position 46 in the translated protein. This change did not appear to affect the activity of the enzyme as measured in a GST assay. The sequence for this bphK allele was submitted to the EMBL database (accession number AJ505824). One recombinant plasmid, pNG4, where bphK was under the control of the pGEM-T lac promoter was chosen for further study.

Examination of cloned bphK in E. coli JM109 (pNG4) showed a high GST activity indicating that a functional gene was present. This activity was inducible by IPTG as the specific activity in the absence of IPTG was only 0.011 U protein mg−1. Under inducing conditions the level of GST activity in this strain is about 70 times greater than that of P. fluorescens F113PCB and this clone provided a useful source of the BphK protein for subsequent analysis.

3.3Identification of potential novel alternative substrates using a GST in vivo activity assay

It has previously been reported by Bochner et al. [19] that CDNB inhibited the growth of E. coli by possibly depleting the GSH in the cell. In the presence of a functional GST this effect becomes more acute, as CDNB is a substrate for this enzyme with glutathione acting as a co-substrate. An in vivo assay for the detection of GST activity based on this method was devised by Hin-Cheung et al. [14] using a eukaryotic GST.

A range of CDNB concentrations was examined for E. coli JM109 (pNG4) and tested against growing cells of E. coli JM109 (pGEM) (negative control) (all results not shown). Growth of E. coli JM109 (pNG4) was inhibited at a CDNB concentration of 700 μM (Fig. 1). An automated turbidimetric system, Bioscreen C®, was used to develop a rapid semi-automated assay for the determination of the presence or absence of a functional GST by monitoring growth of bacterial cells in the presence of a low level of CDNB. This assay was used to screen other potential substrates including 4-CBA, 3-CBA, benzoic acid, trichloroacetic acid, biphenyl, 4-chlorobiphenyl and 2,4-dichlorophenoxyacetic acid for the enzyme (data not shown). 4-CBA showed a similar effect on E. coli growth as CDNB and was therefore identified as a potential substrate (Fig. 2).

image

Figure 1. Optimisation of rapid CDNB in vivo assay. Growth of E. coli JM109 (pGEM) cells (?) and E. coli JM109 (pNG4) cells (◯) at a concentration of 500 μM CDNB. Growth of E. coli JM109 (pGEM) cells (▴) and E. coli JM109 (pNG4) cells (▵) at a concentration of 700 μM CDNB.

Download figure to PowerPoint

image

Figure 2. Analysis of alternative substrates for BphK using in vivo assay. Growth of E. coli JM109 (pGEM) cells (?) and E. coli JM109 (pNG4) cells (□) at a concentration of 1 mM 4-CBA.

Download figure to PowerPoint

3.4BphK shows dechlorination activity against 4-CBA

E. coli JM109 (pNG4) cellular extracts over-expressing bphK when incubated with 4-CBA were found to remove the compound as measured by HPLC over a 12-h period. In addition, as 4-CBA levels decreased the levels of chloride ion released increased almost stoichiometrically (Fig. 3).

image

Figure 3. Removal of 4-CBA (?) and formation of chloride (□) by E. coli JM109 (pNG4) cell extracts.

Download figure to PowerPoint

In contrast, E. coli extracts lacking bphK (JM109 (pGEMT-Easy)) showed no removal of 4-CBA or production of chloride (results not shown). This activity was dependent on the addition of GSH to the reaction mixture. The in vivo GST assay was carried out without the addition of GSH and GST activity was observed. This would indicate a dechlorination activity for BphK in relation to the substrate 4-CBA in the presence of glutathione.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

Based on PCR amplification of bphK from F113PCB and in vitro GST assays detailed in Table 1 the presence of a functional bphK gene in P. fluorescens F113PCB was shown and over-expression of the cloned gene in E. coli JM109 (pNG4) was achieved under the lac promoter. The low background GST activity found in P. fluorescens F113 and E. coli is likely due to endogenous GST genes. A recent study demonstrated that recently sequenced Pseudomonas genomes including P. fluorescens PFO1 have between 10 and 15 GST homologues [3]. The single base pair change detected in the F113PCB bphK allele may reflect a genuine mutation in that GM strain or have resulted during PCR-mediated cloning. However, based on a Clustal W amino acid alignment of similar GSTs, this is not a conserved residue (data not shown) in the protein.

The Hin-Cheung et al. [14] method was successfully adapted to detect the presence or absence of GST activity in vivo both in Pseudomonas and in E. coli. This in vivo assay is therefore a simple alternative method to detect the presence or absence of a functional GST. The Bioscreen C® automated turbidimetric instrument can run 200 samples at a time making this method efficient for screening large numbers of samples. This assay, for example, would be extremely advantageous in mutagenesis studies that are routinely used to study GST structure and function [20] or screening for novel substrates as described in this report.

This E. coli expression system and in vivo assay was successfully used to elucidate other potential substrates for BphK. Using the in vivo assay, 4-CBA showed a similar reduction in E. coli growth as CDNB. 4-CBA is an end product of the breakdown of 4-chlorobiphenyl by the bph operon in which bphK is centrally located. This in vivo assay has successfully identified a new substrate for this GST and could be used to identify other potential substrates. The activity seems to be specific as 3-CBA does not appear to be a substrate on the basis of this assay.

Further studies using 4-CBA as a substrate for BphK in cell extracts in the presence of GSH indicate that it has a dechlorination function in relation to this substrate. HPLC, used to detect the removal of 4-CBA, showed no peak that could indicate a product for the 4-CBA conversion. Further studies using 4-CBA as a substrate for BphK in cell extracts in the presence of GSH indicate that it has a dechlorination function in relation to this substrate. The in vitro activity of GST in relation to 4-CBA is approximately 30 000 times slower than the GST activity in relation to CDNB. Also the level of BphK enzyme in P. fluorescens F113PCB is approximately 70 times lower than the bphK gene cloned into E. coli. Therefore the biological significance of the dechlorination activity may be limited in the case of 4-CBA. The BphK enzyme however did show dechlorination activity and further studies using this enzyme and other chlorinated compounds may give results with more biological significance.

Dehalogenation by GSTs has previously been shown by La Roche and Leisinger [6] and Anandarajah et al. [7]. This indicates a possible function for bphK not ascertained previously. It may be of relevance that F113RifPCB strains are able to grow on biphenyl in the presence of 4-CBA but not when 3-CBA is present in the medium (data not shown). 4-CBA is a dead end metabolite so the dechlorination of it is an advantage to the strain particularly as Blasco et al. [11] have shown that it can be converted to an antibiotic that can kill the strain. It is suggested that bphK may have a role in protecting the strain against the toxic inhibitory end metabolites of PCB degradation by virtue of this dechlorination activity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

This work was in part funded by the Higher Education Authority of Ireland PRTLI programme and EU Contracts BIO4-CT-97-2227, QLK3-CT2000-00164 and QLK3-CT-2001-00101.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References
  • [1]
    Wilce, M.C.J., Parker, M.W. (1994) Structure and function of glutathione S-transferase. Biochim. Biophys. Acta 1205, 118.
  • [2]
    Vuilleumier, S. (1997) Bacterial glutathione S-transferase: What are they good for. J. Bacteriol. 179, 14311441.
  • [3]
    Vuilleumier, S., Pagni, M. (2002) The elusive roles of bacterial glutathione S-transferases: new lessons from genomes. Appl. Microbiol. Biotechnol. 58, 138146.
  • [4]
    Lloyd-Jones, G., Lau, P.C.K. (1997) Glutathione S-transferase encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons. Appl. Environ. Microbiol. 63, 32863290.
  • [5]
    Favoloro, B., Tamburro, A., Trofino, M.A., Bologna, L., Rotilio, D., Heipieper, H.J. (2000) Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress. Biochem J. 346, 553559.
  • [6]
    La Roche, S.D., Leisinger, T. (1990) Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family. J. Bacteriol. 172, 164171.
  • [7]
    Anandarajah, K., Kiefer, P.M., Donohoe, B.S., Copley, S.D. (2000) Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol. Biochemistry 39, 53035311.
  • [8]
    Hofer, B., Backhaus, S., Timmis, K.N. (1994) The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes. Gene 144, 916.
  • [9]
    Bartels, F., Backhaus, S., Moore, E.R.B., Timmis, K., Hofer, B. (1999) Occurrence and expression of glutathione S-transferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria. Microbiology 145, 28212834.
  • [10]
    Brazil, G., Kenefick, L., Callanan, M., Haro, A., de Lorenzo, V., Dowling, D.N., O'Gara, F. (1995) Construction of a rhizosphere pseudomonad with the potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere. J. Bacteriol. 179, 19241930.
  • [11]
    Blasco, R., Mallavarapu, M., Wittich, R.M., Timmis, K.N., Pieper, D.H. (1997) Evidence that formation of protoanemonin from metabolites of 4-chlorobiphenyl degradation negatively affects the survival of 4-chlorobiphenyl-cometabolizing microorganisms. Appl. Environ. Microbiol. 63, 427434.
  • [12]
    Sondossi, M., Sylvestre, M., Ahmad, D. (1992) Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway. Appl. Environ. Microbiol. 58, 485495.
  • [13]
    Dai, S., Vaillancourt, F.H., Maaroufi, H., Drouin, N.M., Neau, D.B., Snieckus, V., Bolin, J.T., Eltis, L.D. (2002) Identification and analysis of a bottleneck in PCB biodegradation. Nat. Struct. Biol. 9, 934939.
  • [14]
    Hin-Cheung, L., Yann-Pyng, T.S., Yen-Sheng, L.T., Chen-Pei, T.D. (1995) A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli. J. Biol. Chem. 270, 99109.
  • [15]
    Habig, W.H., Jakobi, W.B. (1981) Assay for differentiation of glutathione S-transferases. Methods Enzymol. 77, 398405.
  • [16]
    Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248254.
  • [17]
    van den Tweel, W.J.J., Kok, J.B., de Bont, J.A.M. (1987) Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo- and 4-iodobenzoate by Alcaligenes denitrificans NTB-1. Appl. Environ. Microbiol. 53, 810815.
  • [18]
    Bergmann, J.G., Sanik, J. (1957) Determination of trace amounts of chlorine in naphtha. Anal. Chem. 29, 241243.
  • [19]
    Bochner, B.R., Lee, P.C., Wilson, S.W., Cutler, C.W., Ames, B.N. (1984) AppppA and related adenylylated nucleotides are synthesized as a consequence of oxidation stress. Cell 37, 225232.
  • [20]
    Favoloro, B., Tamburro, A., Angelucci, S., De Luca, D., Meliono, S., Di Ilio, C., Rotilio, D. (1998) Molecular cloning and expression and site directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi. Biochem J. 335, 573579.