CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia1
Author for correspondence: Irene Horne. Tel: +61 2 6246 4110. Fax: +61 2 6246 4173. e-mail: irene.horne{at}ento.csiro.au
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ABSTRACT |
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Keywords: organophosphate hydrolase, coroxon
Abbreviations: OP, organophosphate; MBP, maltose-binding protein
b The GenBank accession number for the hocA gene is AF469117.
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INTRODUCTION |
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Synthetic organophosphates (OPs) are widely used as insecticides. OPs contain three phosphoester linkages, and hydrolysis of any one of the phosphoester bonds dramatically reduces the toxicity of the pesticides by virtue of their inability to inactivate acetylcholinesterase. The OPs can be divided into two groups: those containing a P=O bond (oxon OPs) and those containing a P=S bond (thion OPs). Several enzymes capable of detoxifying OPs have been isolated from micro-organisms capable of using OPs as carbon sources (Dumas et al., 1989 ; Cheng et al., 1996
) and are being evaluated for application in various pesticide bioremediation processes.
The most widely characterized phosphotriesterase enzyme identified is the OPH protein from Flavobacterium sp. ATCC 27551 (Mulbry & Karns, 1989 ). This bacterial strain is capable of using the OP diazinon (O,O-diethyl O-[6-methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) as a carbon source (Sethunathan & Yoshida, 1973
). A similar gene was also identified in a Pseudomonas strain capable of using methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) as a carbon source (Chaudhry et al., 1988
). OPH is a 31 kDa protein that is metal-requiring, with Zn(II) identified in the native enzyme and a reaction mechanism that does not involve a phosphorylated intermediate (Lewis et al., 1988
). The OPAA (organophosphate acid anhydrolase) proteins from Alteromonas spp. are much larger, at approximately 60 kDa. These proteins are also metalloenzymes [with Mn(II)] and have sequence similarity with prolidases (Cheng et al., 1996
). However, OPs have not been reported to serve as a nutrient source for these organisms. Two further OP-hydrolytic enzymes have been identified in Nocardia-related species isolated for their ability to use coumaphos (3-chloro-4-methyl-7-coumarinyl diethyl phosphorothioate) as a carbon source (Shelton & Somich, 1988
). Mulbry (1992)
identified a hydrolytic enzyme (AdpB) from a Nocardia strain which possessed very different physical properties to OPH, including the lack of a requirement for metal ions. Mulbry (2000)
subsequently identified a phosphotriesterase in the related Nocardiodes simplex NRRL B-24074. This enzyme was suggested to be a metalloenzyme, and had a molecular mass of 45 kDa.
Many studies have identified bacterial strains capable of using OPs as phosphorus sources (Cook et al., 1978 ; Rosenberg & Alexander, 1979
), but as yet no genes involved in OP metabolism have been characterized from these organisms. A recent study by Horne et al. (2002b)
described the isolation of a Pseudomonas monteilii strain (C11) capable of using the oxon OP coroxon (3-chloro-4-methyl-7-coumarinyl diethyl phosphate) as a phosphorus source (with glucose as a carbon source). P. monteilii C11 appeared to possess a single enzyme capable of hydrolysing coumaphos and coroxon, and this enzyme was associated with the soluble fraction of the cell. OP-hydrolytic activity appeared to be due to a novel phosphotriesterase enzyme that was not a metalloenzyme, and this activity was regulated by the presence of phosphate in the medium. Here, we describe the isolation of this novel phosphotriesterase gene/enzyme system and present a preliminary characterization of its properties.
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METHODS |
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Assays and biochemical techniques.
Protein concentrations in crude extracts and pure proteins were determined by previously described methods (Bradford, 1976 ; Gill & von Hippel, 1989
). SDS-PAGE (10%; acrylamide/bisacrylamide 30:1) was performed according to the method of Laemmli (1970)
. Hydrolysis of coroxon, coumaphos, O,O-dimethylumbelliferyl phosphate, 4-methylumbelliferyl phosphate, 4-methylumbelliferyl acetate and 4-methylumbelliferyl ß-D-galactopyranoside was measured by examining the formation of fluorescent hydrolysis products (Roth, 1969
; Harcourt et al., 2002
). Parathion (O,O-diethyl p-nitrophenyl phosphorothioate), paraoxon (O,O-diethyl p-nitrophenyl phosphate), methyl parathion and bis-p-nitrophenyl phosphate hydrolyses were determined colorimetrically by measuring the formation of p-nitrophenol (Dumas et al., 1989
). All assays were performed in 50 mM Tris/HCl (pH 7·5) at 25 °C. The effect of EDTA as a metal-chelator was tested by dialysis of pure protein against three changes of EDTA (5 mM) in 50 mM Tris/HCl (pH 7·5). When included in assays, MnSO4, MgSO4, ZnCl2 and CoCl2 were added to a final concentration of 1 mM. All assays were performed at least twice on two separate samples. Standard errors are displayed throughout the text. Cell density measurements were performed as previously described (Sutherland et al., 2000
).
Construction of a plasmid for HocA overexpression and purification.
To construct a maltose-binding protein (MBP)HocA overexpression plasmid, the hocA gene was amplified using PCR. The upstream and downstream oligonucleotide primers, hoc5 (5'-GTCTAAGGATCCATGAAAGAAGAACTAAAAACC-3') and hoc3 (5'-GTCTAAAAGCTTTTACCAGTTTAGCTTTAG-3') contained, respectively, a BamHI restriction site at the hocA start codon and a HindIII restriction site at the stop codon (underlined bases). The PCR fragment was subsequently cloned into the BamHIHindIII restriction sites of pMAL-c2X (New England Biolabs) to generate the recombinant plasmid pmalhoc2. The correct sequence of the insert was confirmed. Optimal production of MBPHocA was obtained when mid-exponential-phase cells (OD595 0·6) were induced with 0·1 mM IPTG for 5 h at 37 °C. Harvested cells were disrupted by sonication and the soluble fraction loaded onto an amylose resin (New England Biolabs) equilibrated with 50 mM Tris/HCl (pH 7·5). MBPHocA was eluted with 10% maltose in 50 mM Tris/HCl (pH 7·5). The purity of fractions was assessed by SDS-PAGE, and fractions that appeared to be homogeneous were pooled. Approximately 720 µg pure protein was obtained from 500 ml culture.
Construction of hocAlacZ transcriptional fusions.
A 3·0 kb ClaIXbaI lacZ-containing fragment from pEM32m (Machowski et al., 2000 ) was ligated with similarly digested pBluescript DNA to create plasmid pBSlac. The 5' region of hocA (containing approximately 162 bp upstream of the hocA start codon) and the upstream region were amplified by PCR from pBSRK7(1), using the vector primer T3 (5'-AATTAACCCTCACTAAAGGG-3') and the RK7T3r primer [5'-GATCCTCGAGTAAGGCTGATTGTTCAAGTTC-3', containing a XhoI restriction site at the 5' end (underlined)]. This generated a 500 bp PCR fragment that contained a XhoI site at either end, the second generated from vector sequence. This PCR fragment was digested with XhoI and ligated into similarly digested pBSlac, and the correct orientation confirmed by digestion with PstI to produce a 3·2 kb fragment and a 2·9 kb fragment. This plasmid was called pC11lac. A 6·9 kb HindIII fragment containing the mobilization genes (mob) and tetracycline-resistance cassette from pR459II was ligated into pC11lac to create the integrative lacZ fusion plasmid, pC11lacmob. A replicative transcriptional hocAlacZ fusion was also constructed. A 3·5 kb PstI fragment from pC11lac, containing the 5' region of hocA with lacZYA, was cloned into the broad-host-range plasmid pDSK519, to create pDSKC11lac. Both integrative and replicative lacZ fusion vectors were transferred into P. monteilii C11 using E. coli S17-1.
Construction of a defective HocA mutant.
The 5' PCR fragment generated by amplification (see above) using the T3 primer and RK7T3r (containing an XhoI restriction site at the 5' end), and using pBSRK7(1) as a template was ligated into pGEM-T Easy (Promega) to generate the plasmid pGhocA5. A 3' region of hocA was also amplified by PCR with pBSRK7(1) as a template, using the primers RK7T3f (5'-GAACTTGAACAATCAGCCTTA-3') and the T7 vector primer (5'-TAATACGACTCACTATAGGGAGA-3'). This generated an 800 bp fragment that was cloned into pGEM-T Easy to create pGhoc3(3). Digestion of pGhoc3(3) with SpeI released the 800 bp PCR fragment, which was then ligated with SpeI-digested pGhocA5. Transformants were screened for correct orientation by a positive PCR amplification using the hoc3 and hoc5 primers. One was chosen and designated pGhocA5+3. A 1·2 kb BamHI fragment from pGhocA5+3 that contained both the 5' and 3' fragments and only one XhoI site (between the 5' and 3' fragments) was ligated with similarly digested pJP5603, to create pJPhoc. The streptomycin-resistance cassette from pUI1188Sp was cloned into the unique XhoI site in pJPhoc to create pJPhocSp, which was transferred into P. monteilii C11 using E. coli S17-1 pir; kanamycin-sensitive, streptomycin-resistant exconjugants were selected. One exconjugant was chosen and shown by PCR to lack an intact hocA gene. Rather than the 0·5 kb PCR product produced for the wild-type strain (C11), a PCR fragment of 2·5 kb was obtained, demonstrating the insertion of the 2 kb streptomycin-resistance cassette in hocA. The PCR was performed using the hoc5 and hoc3 primers.
Chemicals.
Methyl parathion and parathion were obtained from Riedel-de Haan. Coumaphos (3-chloro-4-methyl-7-coumarinyl diethyl phosphorothioate) was a gift from Bayer. Coroxon was purchased from Alltech. Paraoxon, bis-p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, 4-methylumbelliferyl acetate and 4-methylumbelliferyl ß-D-galactopyranoside were obtained from Sigma. O,O-Dimethyl 4-methylumbelliferyl phosphate was synthesized by Professor Alan Devonshire (A. L. Devonshire and others, unpublished).
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RESULTS |
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The HocA protein demonstrated no significant sequence similarity to any of the previously identified OP-hydrolytic enzymes, OPH/OpdA (GenBank accession numbers M20392 and AY043245, respectively), OPAA-2 (GenBank accession number U29240) or AdpB (GenBank accession number M91040). Some sequence similarity was observed with a putative cytoplasmic protein from Salmonella typhimurium LT2 that was identified in the genome sequencing project (GenBank accession number AE008708). This putative protein showed 41% sequence identity at the amino acid level to HocA over the entire protein, and 59% sequence similarity. The function of this putative Salmonella protein is not known. No other strong sequence similarity to any other proteins in the GenBank, PDB, EMBL, SWISS-PROT and Prosite databases were observed using the BLASTP program. However, some similarity (43%) was seen with isocitrate dehydrogenase from Methanococcus jannaschii (Bult et al., 1996 ) over the entire protein (23% identity), while the central third showed 60% similarity (36% identity) with phospholipase C from Arabidopsis thaliana (Sato et al., 2000
). Both of these proteins have some involvement with phosphate. Phosphatidyl-inositol-specific phospholipase C catalyses the cleavage of the membrane lipid, phosphatidylinositol or its phosphorylated derivatives, to produce diacylglycerol (Griffith & Ryan, 1999
). Isocitrate dehydrogenase is an enzyme that is regulated by phosphorylation of a serine residue in its active site (Hurley et al., 1990
). Key residues in isocitrate dehydrogenase and phospholipase C that bind phosphate are not conserved in HocA, nor are key catalytic residues. It is likely that these sequence similarities have no functional significance. No conserved domains or motifs were identified in this protein using either EMBL or Prosite, and so this appears to be a novel protein with no obvious homologues.
P. monteilii HOC, which contains an insertional mutation in hocA, showed limited phosphotriesterase activity with coroxon as a substrate (1·6±0·3% activity of the wild-type). Furthermore, P. monteilii HOC was unable to grow with coroxon as a phosphorus source. This confirms that HocA is a protein required for P. monteilii C11 to grow with coroxon as a phosphorus source.
Purification and substrate specificity of HocA
To confirm that hocA did indeed encode a phosphotriesterase, the enzyme activity of an MBP fusion was examined. The purified protein possessed phosphotriesterase activity against the substrates coumaphos, coroxon, O,O-dimethylumbelliferyl phosphate, paraoxon, parathion and methyl parathion. The kinetics of hydrolysis against the oxon OPs coroxon, paraoxon and O,O-dimethylumbelliferyl phosphate are shown in Fig. 2a. Reactions with the thion OPs did not appear to follow MichaelisMenten kinetics (Fig. 3
). This may be due to the Km of the enzyme being quite low, and the substrate concentrations tested being greater than the Km. Specific activities for these substrates are approximately 3050% of the activity with oxon OPs at the same substrate concentration (Fig. 2b
). While it is possible that fusion with MBP interfered with these activities, the activity of untagged HocA expressed from the lacZ promoter in E. coli DH10ß (pBSRK7(1)) demonstrated the same Km for coroxon as for the fusion protein, so we find this unlikely. In a previous study, the specific phosphotriesterase activity in P. monteilii C11 was 1·33 nmol min-1 (mg protein)-1 in crude cell extracts (Horne et al., 2002b
). Given that pure HocA has a specific activity of 0·12 nmol min-1 (nmol enzyme)-1, this would suggest that there is approximately 11·1 nmol HocA (mg protein)-1 in cells grown under those conditions. The HocA protein did not show phosphatase, phosphodiesterase or carboxylesterase activity against 4-methylumbelliferyl phosphate, bis-p-nitrophenyl phosphate or 4-methylumbelliferyl acetate, respectively.
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Phosphate levels regulate hocA expression
Previously, coumaphos-hydrolytic activity in P. monteilii C11 appeared to be regulated by phosphate levels (Horne et al., 2002b ). In many organisms, including the pseudomonads, genes of the Pho regulon are either induced or repressed by the PhoP-R sensorregulator pair upon phosphate starvation. The PhoR protein binds to a distinctive segment of DNA (5'-TTGCAGTCTCGCTGTCACAA-3'). The region contained in the 1·2 kb EcoRIBamHI fragment did not possess any obvious promoter region or regulatory DNA sequence such as the Pho DNA-binding sequence. To examine the regulation of hocA expression, both an integrative and a replicative lacZ fusion were constructed. With the integrated hocAlacZ fusion, transcription from upstream regions not contained in the 1·2 kb EcoRIBamHI fragment can contribute to hocA expression (and therefore ß-galactosidase expression). The ß-galactosidase activity from this fusion was examined in media containing either excess phosphate or 4-methylumbelliferyl phosphate (limited phosphate). The ß-galactosidase activity in cells grown without phosphate was twice that of cells grown with phosphate (109·4±1·3 nmol min-1 mg-1 compared with 51·7±0·2 nmol min-1 mg-1). This suggests that expression of the hocA gene is regulated by phosphate levels in the medium.
A replicative lacZ fusion (pDSKC11lac) was used to determine whether the 1·2 kb EcoRIBamHI fragment contained immediate upstream sequences involved in phosphate regulation. Greater ß-galactosidase activity was observed from this construct in P. monteilii C11 compared with the integrative hocAlacZ fusion, presumably because of a copy-number effect. Therefore, no overall comparison can be made between the integrative and replicative lacZ fusions with respect to the extent to which upstream promoters contribute to overall hocA expression. However, it is clear that the replicative plasmid conveyed identical ß-galactosidase activity in cells grown with either phosphate (141·9±7·7 nmol min-1 mg-1) or 4-methylumbelliferyl phosphate (142·2±3·8 nmol min-1 mg-1). This suggests that regions involved in phosphate regulation are contained on DNA sequences much further upstream of hocA than those cloned in this study. The hocA gene may be contained in a much larger operon that is regulated by phosphate. Alternatively, the regulatory region is much further upstream. For example, the Pho box is almost 600 bp upstream of the phosphate-regulated phoA gene of P. aeruginosa (Stover et al., 2000 ). It was not possible to identify such upstream regulatory regions in this study, as the cloned fragment containing the hocA gene contained only 162 bp of sequence upstream of the start codon.
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DISCUSSION |
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The expression of hocA in P. monteilii C11 was regulated by phosphate levels. While this suggests a role for HocA in phosphate metabolism, it is highly unlikely that the native role is to hydrolyse phosphotriesters, because OPs have only been in existence for the last 50 years. HocA may have evolved from a pre-existing phosphatase or phosphodiesterase as it has been shown that a phosphotriesterase (OPH) could be altered by only one amino acid to possess phosphodiesterase activity (Shim et al., 1998 ), demonstrating how a minor change can allow a protein to acquire a new activity. Likewise, HocA may have been a pre-existing phosphodiesterase that has acquired phosphotriesterase activity.
It is common for phosphatases and phosphodiesterases to be inhibited by their phosphorus-containing hydrolysis product (von Tigerstrom & Stelmaschuk, 1986 ). End-product inhibition of HocA activity was observed by the phosphorus-containing moeity. The severe end-product inhibition of HocA by diethyl phosphate probably contributed to the lack of parathion/paraoxon hydrolysis by P. monteilii C11 observed in our previous study (Horne et al., 2002b
). Furthermore, the activity is quite low for these substrates and may not be seen until the protein is in sufficient concentrations, as seen here with purified enzyme. The unusual kinetics observed for HocA with the thion OPs may be a result of end-product inhibition by the phosphorothioates, diethyl thiophosphate and dimethyl thiophosphate, the hydrolysis products of coumaphos and methyl parathion, respectively. Alternatively, the enzyme has a low Km for the thion OPs, and substrates were tested at concentrations far exceeding the Km of the enzyme. We find this unlikely, as this would be the first report of a phosphotriesterase having an extremely low Km for thion OPs relative to the oxon versions. In general, the converse is true and the enzymes have a higher affinity and catalytic activity for the naturally occurring P=O-containing OPs rather than the bigger, unnatural P=S-containing OPs.
P. monteilii C11 was isolated by virtue of its ability to utilize OPs as a source of phosphorus. Phosphotriesterase activity is the first step in obtaining phosphate from coroxon. The doubling time of P. monteilii C11 was not increased when inorganic phosphate, rather than coroxon, was provided as the sole source of phosphorus (data not shown), suggesting that phosphotriesterase activity is not a rate-limiting step in providing inorganic phosphate for P. monteilii C11. This implies that there is no requirement for a kinetically better phosphotriesterase in this organism. HocA (the only enzyme in this organism responsible for phosphotriesterase activity) is less efficient at hydrolysing OPs than are other phosphotriesterases isolated from organisms capable of using OPs as a carbon source. In general, phosphotriesterase activity would be the first step in the use of OPs as a carbon source. The OPH enzyme of Flavobacterium sp. ATCC 27551 can hydrolyse paraoxon at rates close to the limits of diffusion. We find it unlikely that the kinetics of HocA would be able to support growth of an organism with OPs as a carbon source. Bacteria require much higher levels of carbon than phosphorus for growth. Stanier et al. (1986) suggested that a Pseudomonas putida strain required 7 mM carbon, whereas 100-fold less phosphorus is needed for growth; therefore an enzyme that is providing the only source of phosphorus can be 100-fold less active than an enzyme providing the only source of carbon. In accordance with this, the kcat values of OPH for both coroxon and paraoxon appear to be 100-fold higher than those of HocA (based on the results of Horne et al., 2002a
). We propose that the isolation of enzymes from organisms using OPs as carbon sources rather than phosphorus sources potentially yields more efficient enzymes.
The kinetics of the OPH/OpdA proteins suggest that they are far better suited to bioremediation than HocA. However, HocA does not require a metal ion. It might, therefore, be better suited than OpdA for bioremediation in certain environments such as heavily polluted waters containing inhibitory metals or metal-chelating agents. It might be possible to improve HocA by in vitro evolution (MacBeath et al., 1998 ) for better kinetics and reduced end-product inhibition.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bowen, A. R. St G. & Pemberton, J. M. (1985). Mercury resistance transposon Tn813 mediates chromosome transfer in Rhodopseudomonas sphaeroides and intergeneric transfer in pBR322. In Plasmids in Bacteria , pp. 105-115. Edited by D. R. Helsinki, S. N. Cohen, D. B. Clewell, D. A. Jackson & A. Hollaender. New York:Plenum.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Bult, C. J., White, O., Olsen, G. J. & 20 other authors (1996). Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273, 10581073.
Burge, C. & Karlin, S. (1997). Prediction of complete gene structures in human genomic DNA. J Mol Biol 268, 78-94.[Medline]
Chaudhry, G. R., Ali, A. N. & Wheeler, W. B. (1988). Isolation of a methyl parathion degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp. Appl Environ Microbiol 54, 288-293.[Medline]
Cheng, T.-C., Harvey, S. P. & Chen, G. L. (1996). Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme. Appl Environ Microbiol 62, 1636-1641.[Abstract]
Cook, A. M., Daughton, C. G. & Alexander, M. (1978). Phosphorus-containing pesticide breakdown products: quantitative utilization as phosphorus sources by bacteria. Appl Environ Microbiol 36, 668-672.[Medline]
Dumas, D. P., Caldwell, S. R., Wild, J. R. & Raushel, F. M. (1989). Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J Biol Chem 264, 19659-19665.
Eraso, J. M. & Kaplan, S. (1994). prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides. J Bacteriol 176, 32-43.[Abstract]
Folkesson, A., Advani, A., Sukupolvi, S., Pfeifer, J. D., Normark, S. & Lofdahl, S. (1999). Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease. Mol Microbiol 33, 612-622.[Medline]
Gardiner, A. T., MacKenzie, R. C., Barrett, S. J., Kaiser, K. & Cogdell, R. G. (1996). The purple photosynthetic bacterium Rhodopseudomonas acidophila contains multiple puc peripheral antenna complex (LH2) genes: cloning and initial characterization of four /ß pairs. Photosynth Res 49, 223-235.
Gill, S. C. & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182, 319-326.[Medline]
Griffith, O. H. & Ryan, M. (1999). Bacterial phosphatidyl inositol-specific phospholipase C: structure, function and interaction with lipids. Biochim Biophys Acta 1441, 237-254.[Medline]
Harcourt, R. L., Horne, I., Sutherland, T. D., Hammock, B. D., Russell, R. J. & Oakeshott, J. G. (2002). Development of a simple and sensitive fluorimetric method for isolation of coumaphos-hydrolysing bacteria. Lett Appl Microbiol 34, 263-268.[Medline]
Horne, I., Sutherland, T. D., Harcourt, R. L., Russell, R. J. & Oakeshott, J. G. (2002a). Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Appl Environ Microbiol (in press).
Horne, I., Harcourt, R. L., Sutherland, T. D., Russell, R. J. & Oakeshott, J. G. (2002b). Isolation of a Pseudomonas monteilli strain with a novel phosphotriesterase. FEMS Microbiol Lett 206, 51-55.[Medline]
Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, D. E.Jr & Stroud, R. M. (1990). Regulation of an enzyme by phosphorylation at the active site. Science 249, 1012-1016.[Medline]
Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988). Improved broad-host range plasmids for DNA cloning in Gram negative bacteria. Gene 70, 191-197.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105-132.[Medline]
Laemmli, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lewis, V. E., Donarski, W. J., Wild, J. R. & Raushel, F. M. (1988). Mechanism and stereochemical course at phosphorus of the reaction catalysed by a bacterial phosphotriesterase. Biochemistry 27, 1591-1597.[Medline]
MacBeath, G., Kast, P. & Hilvert, D. (1998). Redesigning enzyme topology by directed evolution. Science 279, 1958-1961.
Machowski, E. E., McAdam, R. A., Derbyshire, K. M. & Mizrahi, V. (2000). Construction and application of mycobacterial reporter transposons. Gene 253, 67-75.[Medline]
Mulbry, W. W. (1992). The aryldialkyl phosphatase-encoding gene adpB from Nocardia sp. strain B-1: cloning, sequencing and expression in Escherichia coli. Gene 121, 149-153.[Medline]
Mulbry, W. (2000). Characterization of a novel organophosphorus hydrolase from Nocardiodes simplex NRRL B-24074. Microbiol Res 154, 285-288.[Medline]
Mulbry, W. W. & Karns, J. S. (1989). Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and the protein. J Bacteriol 171, 6740-6746.[Medline]
Penfold, R. J. & Pemberton, J. M. (1992). An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118, 145-146.[Medline]
Rainey, F. A., Dorsch, M., Morgan, H. W. & Stackebrandt, E. (1992). 16S rDNA analysis of Spirochaeta thermophila in phylogenetic position and implications for the systematics of the order Spirochaetales. Syst Appl Microbiol 15, 197-202.
Rosenberg, A. & Alexander, M. (1979). Microbial cleavage of various organophosphorus insecticides. Appl Environ Microbiol 37, 886-891.[Medline]
Roth, M. (1969). Fluorimetric assay of enzymes. Methods Biochem Anal 17, 189-285.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sato, S., Nakamura, Y., Kaneko, T., Katoh, T., Asamizu, E. & Kotani, H. (2000). Structural analysis of Arabidopsis thaliana chromosome 5. DNA Res 7, 31-63.[Medline]
Sethunathan, N. & Yoshida, T. (1973). A Flavobacterium that degrades diazinon and parathion. Can J Microbiol 19, 873-875.[Medline]
Shelton, D. R. & Somich, C. J. (1988). Isolation and characterization of coumaphos-metabolising bacteria from cattle-dip. Appl Environ Microbiol 54, 2566-2571.
Shim, H., Hong, S.-B. & Raushel, F. M. (1998). Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase. J Biol Chem 273, 17445-17450.
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1, 784-791.
Sinclair, M. I., Maxwell, P. C., Lyon, B. R. & Holloway, B. W. (1986). Chromosomal location of TOL plasmid in Pseudomonas putida. J Bacteriol 168, 1302-1308.[Medline]
Stanier, R. Y., Ingraham, J. L., Wheelis, M. L. & Painter, P. R. (1986). General Microbiology, 5th edn. Englewood Cliffs, NJ: Prentice Hall.
Stover, C. K., Pham, X.-Q. T., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959964.[Medline]
Sutherland, T. D., Horne, I., Lacey, M. J., Harcourt, R. L., Russell, R. J. & Oakeshott, J. G. (2000). Enrichment of an endosulfan-degrading mixed bacterial culture. Appl Environ Microbiol 66, 2822-2828.
von Tigerstrom, R. G. & Stelmaschuk, S. (1986). Purification and characterization of the outer membrane associated alkaline phosphatase of Lysobacter enzymogenes. J Gen Microbiol 132, 1379-1387.
Wanner, B. L. (1983). Overlapping and separate controls of the phosphate regulon in Escherichia coli K-12. J Mol Biol 166, 283-308.[Medline]
Williams, P. A. & Murray, K. A. (1974). Metabolism of benzoate and the methylbenzoates by Pseudomonas putida (arvilla) mt-2: evidence for the existence of a TOL plasmid. J Bacteriol 120, 416-423.[Medline]
Received 28 January 2002;
revised 3 May 2002;
accepted 21 May 2002.
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