Department of Microbiology, #300, 6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z31
Author for correspondence: Robert E. W. Hancock. Tel: +1 604 822 2682. Fax: +1 604 822 6041. e-mail: bob{at}cmdr.ubc.ca
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ABSTRACT |
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Keywords: PhoP-PhoQ, antimicrobial cationic peptides, aminoglycoside resistance, Pseudomonas aeruginosa
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INTRODUCTION |
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The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen. Chronic infections due to this organism are prevalent in cystic fibrosis patients and are frequently recalcitrant to treatment. In addition to displaying high levels of intrinsic antibiotic resistance, P. aeruginosa frequently converts to a mucoid state resulting in a rapid adaptive resistance that accounts for the high failure rate of antibiotic therapy in eradicating these infections.
Sequencing of the P. aeruginosa PAO1 genome has recently been completed (http://www.pseudomonas.com) and should provide some insight into the pathogenesis of this bacterium. One striking feature of the predicted protein complement of P. aeruginosa is the presence of a disproportionately large number of regulatory proteins. Recently we identified the PhoP-PhoQ homologues in P. aeruginosa (Macfarlane et al., 1999 ). The genes for this regulatory system lie immediately downstream of the oprH gene, which encodes a small outer-membrane protein OprH that is highly expressed under Mg2+-starvation conditions. The three genes oprH-phoP-phoQ form an operon that is under the joint control of PhoP and Mg2+ ion concentrations. Similar to its counterpart in S. typhimurium, P. aeruginosa PhoP-PhoQ is highly expressed under Mg2+-starvation conditions and is involved in resistance to polymyxin B. P. aeruginosa PAK has been shown to exhibit LPS structural modifications when grown under Mg2+-deficient conditions (Ernst et al., 1999
), and polymyxin resistance in P. aeruginosa, like that in S. typhimurium, may thus be related to altered outer-membrane permeability. In S. typhimurium, PhoP-PhoQ mediates resistance through activation of PmrA-PmrB, and a PhoP-null strain is therefore rendered supersusceptible to polymyxin B. However, our PhoP-null strain of P. aeruginosa PAO1 (strain H851) retained polymyxin B resistance under Mg2+-deficient growth conditions, and a PhoQ-null strain (H854) exhibited constitutive polymyxin B resistance. In accord with the large number of regulators in P. aeruginosa, the involvement of another regulatory system(s) in the resistance of this organism appears likely. However, in contrast to the situation in S. typhimurium, PhoP is not required for activation of this system(s) under low-Mg2+ conditions.
In this paper we have further characterized the oprH-phoP-phoQ operon and demonstrated that the two-component regulatory system PhoP-PhoQ influences the resistance of P. aeruginosa to both cationic antimicrobial peptides and aminoglycoside antibiotics.
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METHODS |
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Construction of strains H855 (oprH::xylE-GmR), H895 (oprH73) and H896 (
oprHC).
To construct strain H855, a 1·2 kb SmaIHincII fragment containing the oprH gene was excised from plasmid pAK9 (Macfarlane et al., 1999 ) and cloned into the SmaI site of the gene-replacement vector pEX100T (Schweizer & Hoang, 1995
) to give pEXH. The xylE-GmR cassette from plasmid pX1918GT (Schweizer & Hoang, 1995
) was then cloned between the two PstI sites that are situated 18 bp apart within the oprH gene. Gene replacement in strain H103 using this construct was carried out as described previously for strains H851 and H854 (Macfarlane et al., 1999
). Southern blot analysis following standard protocols (Ausubel et al., 1987
) and using probes complementary to either oprH or xylE confirmed the presence of the xylE-GmR cassette within oprH in strain H855. Plasmid pEMH4a was constructed by subcloning a 0·7 kb SstIXbaI fragment carrying the oprH gene from pBHR20 (Rehm & Hancock, 1996
) into pUCP21 (West et al., 1994
).
To construct the oprH deletion strains H895 and H896, the gene-replacement vectors pEXH73 and pEXH
C were constructed as follows. Primer
oprH2 was designed with the sequence 5'-CTGCTGGTGTCGGAGGCATTCTCGTAGGTC-3' that was complementary to nt 263234 of oprH. The three underlined bases replaced CAT in the coding sequence and introduced a BsmI site. A 410 bp fragment containing the 5' end of oprH plus 144 bp of vector sequence was PCR amplified from pEMH4a using primer
oprH2 and a second primer, pUCPlac, that was complementary to the vector lac promoter. A fragment of 557 bp from the 5' end of oprH was removed from pEXH by SmaI/BsmI digestion and replaced with the HincII/BsmI-digested PCR amplicon. The resulting plasmid, pEXH
73, contained the oprH gene with a 219 bp in-frame deletion (nt 709927 inclusive). For plasmid pEXH
C, primer
oprH1 (5'-CTGAGCAAGAATGCCTC|ACCAACGCCAGCACCGAG-3') was designed that contained a 28 bp deletion (nt 930957 of oprH) indicated by the vertical line. A 337 bp fragment containing the 3' end of oprH was PCR amplified from pGB22 (Bell et al., 1991
) using this primer and RT-PCR3' (Macfarlane et al., 1999
). pGB22 was digested with SmaI and religated to remove the vector KpnI site and give plasmid pEMH2. Replacement of a KpnIBsmI fragment containing the 3' end of oprH in pEMH2 with the KpnIBsmI-digested PCR amplicon yielded plasmid pEMH2
C. The 28 bp deletion in the oprH gene resulted in a frameshift that would prematurely terminate translation 83 bp upstream of the regular oprH TAA stop codon. Finally, the modified oprH gene was excised from pEMH2
C by SmaIHincII digestion and cloned into the SmaI site of pEX100T to give pEXH
C.
Gene replacement in strain H103 using pEXH73 and pEXH
C was carried out as described previously (Macfarlane et al., 1999
) with the exception that single crossover events were identified by selection on BM2-glucose minimal medium containing carbenicillin alone. Double crossover events were identified as sucrose-resistant, carbenicillin-sensitive colonies. Mid-exponential phase cultures of these strains were screened for OprH expression by Western immunoblot using anti-OprH specific antiserum (Macfarlane et al., 1999
). Genomic DNA was subsequently isolated from OprH strains and the appropriate deletion in oprH was confirmed by PCR.
MIC determinations and killing assays.
Aminoglycoside MIC values were determined using the standard two-fold microtitre broth dilution protocol (Amsterdam, 1991 ) starting with mid-exponential phase cultures. Peptide MIC values were determined using a modified version of this protocol (Wu & Hancock, 1999
) to prevent adhesion of the peptides to the walls of the microtitre plates that would artificially elevate the MIC values. All MIC values were read after a 48 h incubation due to the slow growth rate of strain H854. Killing assays were carried out as described previously (Macfarlane et al., 1999
) with the following exceptions. For CP28, cultures were diluted 1:100 into 30 mM sodium phosphate buffer, pH 7, containing 150 mM NaCl and 8 µg CP28 ml-1. For streptomycin, cultures were diluted 1:200 into pre-warmed LB (Miller) broth containing 16 µg streptomycin ml-1 and killing was carried out at 37 °C.
Streptomycin-assisted lysozyme lysis assays.
Aliquots (1 ml each) of mid-exponential phase cultures (OD600 0·40·8) grown in LB (Miller) broth were centrifuged and washed once with 30 mM sodium phosphate buffer, pH 7. The cells were then resuspended in 1 ml of the same buffer and placed in a cuvette. Streptomycin (450 µg ml-1) and lysozyme (50 µg ml-1) were added simultaneously to the cuvette and cell lysis was monitored by following the decrease in optical density at 600 nm.
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RESULTS |
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Strain H855 was transformed with plasmids carrying the oprH, phoP, phoQ and phoP-phoQ genes (pEMH4a, pEMR3, pEMQ1a and pEMPQ2a, respectively; Table 1). Cultures of H855 alone and harbouring each of these plasmids were grown to mid-exponential phase in both high- and low-Mg2+ media and the catechol 2,3-dioxygenase activity expressed from the xylE transcriptional fusion was measured. The results, shown in Table 2
, indicated that transcription of oprH was still Mg2+ regulated in strain H855 with the level of catechol 2,3-dioxygenase activity measured under low-Mg2+ growth conditions being between 16- and 23-fold higher than that measured under high-Mg2+ conditions. The xylE-GmR cassette in strain H855 is flanked by omega fragments (Schweizer & Hoang, 1995
); therefore the oprH::xylE-GmR construct was assumed to exert a polar effect on the downstream phoP-phoQ genes. Since we have shown that transcription of oprH is dependent on the presence of PhoP (Macfarlane et al., 1999
), the observed Mg2+ regulation of the transcriptional fusion in strain H855 strongly suggested that phoP-phoQ was being weakly transcribed from a second promoter. Presumably the level of this transcript was too low to be detected in the Northern blots and primer-extension experiments described above, even when the autoradiograms were overexposed (data not shown).
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The OprH-null strain H855 retains Mg2+-regulated polymyxin B resistance
The resistance of the OprH-null strain H855 to the polycationic antibiotic polymyxin B was determined by a killing assay. Cultures of this strain with and without the OprH, PhoP and PhoQ expression plasmids were grown to mid-exponential phase in BM2-glucose minimal medium and treated with 8 µg polymyxin B ml-1. The number of survivors after 5 min was determined by a plate count (Table 3). Polymyxin B susceptibility in strain H855 remained similar to that of the wild-type strain H103 under both high- and low-Mg2+ growth conditions. Plasmids carrying the phoQ (pEMQ1a), phoP-phoQ (pEMPQ2a) or oprH (pEMH4a) genes had no effect on the polymyxin B resistance of strain H855. In contrast, constitutive overexpression of PhoP resulting from the presence of pEMR3 (phoP) led to constitutive polymyxin B resistance in this strain. This was a similar effect to that of pEMR3 on the polymyxin B resistance of the PhoP-null strain H851 (Macfarlane et al., 1999
).
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MIC determinations revealed increased resistance (two- to fourfold) to a number of aminoglycoside antibiotics for the three mutant strains grown under high-Mg2+ conditions (Table 5). As was the case for cationic peptides, high divalent cation concentrations also interfered with the action of aminoglycosides (Zimelis & Jackson, 1973
), which accounts for the increased MIC values for streptomycin measured under high-Mg2+ conditions compared to those in LB medium, which contains only moderate concentrations of Mg2+. Although all three mutant strains carry the aacC1 gene encoding the acetyltransferase-3-1 (AAC(3)I) on the xylE-GmR cassette, this enzyme has been shown to have very narrow substrate specificity (Phillips & Shannon, 1984
) and, therefore, is unlikely to affect resistance to other aminoglycosides. Of the aminoglycosides used in these experiments, streptomycin lacks the requisite substituent for modification by AAC(3)I, and no modification of kanamycin A or amikacin by this enzyme has been observed (Phillips & Shannon, 1984
).
We chose to study the resistance of the strains H851, H854 and H855 to streptomycin in more detail. Since the increased resistance in all of these strains (fourfold over wild-type) was observed in LB medium as well as high-Mg2+ BM2-glucose, we conducted killing assays in the former medium. After treatment with 16 µg streptomycin ml-1, wild-type P. aeruginosa H103 was completely killed within 30 min (Table 4). All three mutant strains showed higher streptomycin resistance than the wild-type, but the extent of this resistance varied. The PhoP-null (H851) and OprH-null (H855) strains showed the highest levels of streptomycin resistance (100% and 70% survival, respectively, after 30 min), while the PhoQ-null strain H854 showed a level of resistance intermediate to these two strains and the wild-type (15% survival after 30 min).
To determine whether altered outer-membrane permeability could account for the observed increase in aminoglycoside resistance, we tested the ability of streptomycin to promote lysozyme lysis of the wild-type, and PhoP-, PhoQ- and OprH-null mutants. Lysozyme targets the peptidoglycan layer, but normally is unable to cross the outer-membrane barrier. Addition of aminoglycoside antibiotics disrupts the outer membrane sufficiently to allow the lysozyme molecules access to the cell. As can be seen from Fig. 3, all three mutant strains (H851, H854 and H855) were less susceptible to lysozyme lysis in the presence of 450 µg streptomycin ml-1 than wild-type H103. Of these mutants, the PhoQ-null strain H854 showed the highest level of resistance to streptomycin-assisted lysozyme lysis, and the OprH-null strain H855 the lowest. Treatment of cultures with 450 µg streptomycin ml-1 in the absence of lysozyme caused minimal cell lysis and no lysis was seen in cells treated with lysozyme alone (data not shown).
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Western blot analysis of mid-exponential phase cultures of both strains H895 and H896 grown under Mg2+-limiting (inducing) conditions indicated that no proteins were expressed that reacted with anti-OprH specific antiserum (data not shown). This suggests that if a mutant OprH protein was produced by these strains, it was rapidly degraded within the cell.
The unmarked deletions in strains H895 and H896 should permit normal transcription of phoP-phoQ from the inducible promoter upstream of oprH and thus allow us to determine the effects of an OprH-null mutation without concomitant effects on PhoP-PhoQ expression. The MIC values for several antibiotic compounds were determined for the two oprH deletion strains grown in BM2-glucose minimal medium supplemented with 2 mM (high) MgSO4. The values for polymyxin B, the aminoglycosides streptomycin, kanamycin and amikacin, and the -helical cationic peptide CP28 are given in Table 5
. No difference in the MIC value for any of these five antibiotics was observed between strains H895, H896 and the wild-type P. aeruginosa strain H103. These results strongly suggest that the increased resistance of strain H855 (oprH::xylE-GmR) to aminoglycosides was due to polar effects of the xylE-GmR cassette on the transcription of phoP-phoQ and not to the loss of OprH expression.
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DISCUSSION |
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OprH is not involved in polymyxin B resistance
In addition to the P. aeruginosa phoP and phoQ mutants, we also constructed an analogous chromosomal knockout of oprH, the first gene of the oprH-phoP-phoQ operon. Killing assays with polymyxin B revealed levels of resistance in this oprH::xylE-GmR strain (H855) similar to those of the wild-type strain H103 (Table 3), supporting our previous suggestion that OprH is not essential for polymyxin B resistance but may play an accessory role in stabilizing the outer membrane under Mg2+-starvation conditions (Macfarlane et al., 1999
). The only plasmid-borne gene to affect the polymyxin B resistance of strain H855 was phoP (pEMR3) (Table 3
), the presence of which resulted in significant resistance in high-Mg2+ medium. This pattern of polymyxin B resistance closely resembles that of the PhoP-null strain H851 (Macfarlane et al., 1999
), and thus supported our initial assumption that the polar nature of the xylE-GmR insertion in oprH rendered strain H855 PhoP- and PhoQ-null. This assumption was based on our inability to detect a second promoter for phoP-phoQ by Northern blotting (Macfarlane et al., 1999
) or primer-extension analysis (data not shown) of RNA transcripts in the wild-type strain H103, or by dot-blot analysis of RNA from strain H855 using phoP and phoQ primers (data not shown). Quantitation of the RNA dot blots indicated that if any phoP-phoQ transcript was present in strain H855, it occurred at a level at least twofold lower than that of any phoP-containing transcripts in strain H103 under high-Mg2+ (uninduced) conditions (data not shown).
The region between the oprH and phoP genes in the P. aeruginosa genome is 79 bp in length. A possible ribosome-binding site occurs 5 bp upstream of the phoP ATG start codon, but no promoter consensus sequences were identified. There are also no repeat sequences in this region resembling those found upstream of oprH. However, the Mg2+ regulation of catechol 2,3-dioxygenase expressed from the oprH::xylE transcriptional fusion in strain H855 (Table 2), together with our observation that transcription of oprH is wholly dependent on the presence of PhoP (Macfarlane et al., 1999
), provided indirect evidence for a very low level of phoP-phoQ transcription in this strain from an as yet unidentified second promoter. The presence of two promoters one constitutive and one inducible allowing a basal level of transcription is a common feature of two-component regulatory systems and has been reported for phoP-phoQ in E. coli (Kato et al., 1999
), and for both phoP-phoQ and pmrA-pmrB in S. typhimurium (Gunn & Miller, 1996
; Soncini et al., 1995
). For the latter system, the second promoter lies within the 3' region of pmrC, the first gene of the pmrCAB operon (Gunn & Miller, 1996
). The oprH-phoP intergenic region was also inspected for possible secondary structures. Although no classic rho-independent terminator sequences were identified, an inverted repeat sequence predicted to be capable of forming a hairpin structure (predicted
G=-14·3 kcal mol-1) was identified 36 bp downstream of the oprH stop codon. Northern blot analysis of strains H103 (Macfarlane et al., 1999
) and H854 (Fig. 1
) revealed high levels of oprH transcripts and diminishing amounts of oprH-phoP and oprH-phoP-phoQ transcripts. Formation of a hairpin structure that functions as a transcriptional attenuator downstream of oprH and provides additional regulation of oprH-phoP-phoQ transcription would be consistent with these results. It is worth noting also that our results do not eliminate the possibility that P. aeruginosa oprH-phoP-phoQ may be subject to some form of global regulation, for example by another two-component regulatory system, that acts in conjunction with PhoP-mediated regulation.
PhoP-PhoQ selectively regulates cationic peptide resistance
The polymyxin B and CP28 resistance of the OprH-null strain H855 supported our conclusions that PhoP is not essential for resistance to these two antibiotics under Mg2+-starvation conditions. Although transcription of phoP-phoQ presumably occurs at a very low level in strain H855, phoP transcription cannot be induced through the promoter upstream of oprH and levels of PhoP protein, therefore, would be minimal in this strain.
Interestingly, a phoP::Gm mutant of P. aeruginosa PAK has recently been reported that is supersusceptible to polymyxin B, but resistant to the cationic peptide C18G, under Mg2+-starvation conditions (Ernst et al., 1999 ). The possibility that the Gm cassette inserted into the PAK phoP gene was non-polar and allowed transcription of phoQ could account for the discrepancy in polymyxin B resistance between the phoP mutants in the two strains (Macfarlane et al., 1999
).
The results of MIC determinations for cationic peptides other than CP28 (Table 5) indicated that P. aeruginosa resistance to a second
-helical peptide CP29 and the protegrin-like peptide IB-367 (currently undergoing clinical trials against P. aeruginosa lung infections in cystic fibrosis patients; see http://www.intrabiotics.com) probably involves a pathway similar to that for polymyxin B and CP28 resistance. However, resistance to the indolicidin analogue CP11CN, although still affected by mutations in PhoP-PhoQ, appears to be subject to different regulatory mechanisms. Increased resistance to this peptide was seen only in the two mutants, H851 and H855, that could not induce expression of PhoP. Resistance to polyphemusin, on the other hand, appeared to be independent of PhoP-PhoQ. While this was the only peptide we tested that was unaffected by the antagonistic effects of Mg2+ ions, resistance still appeared to be Mg2+ regulated (data not shown), indicating that other regulatory systems in P. aeruginosa must also respond to extracellular Mg2+ ion concentrations.
Although the mechanism of action of antimicrobial cationic peptides remains uncertain, the -helical peptides CP28 and CP29, like polymyxin B, are believed to initially interact with and disrupt the outer membrane of Gram-negative bacteria (Hancock & Chapple, 1999
). In S. typhimurium, increased resistance to polymyxin B and certain antimicrobial peptides is observed under Mg2+-starvation conditions and is partly due to structural alterations of the outer membrane resulting from activation of genes in the PhoP-PhoQ regulon. Similarly, a phoP mutant of P. aeruginosa PAK was shown to lack certain modifications to core lipid A that were seen in the wild-type strain under Mg2+-starvation conditions (Ernst et al., 1999
). Resistance of P. aeruginosa PAO1 to polymyxin B and CP28 likewise may be due to structural alterations to the outer membrane that are regulated in part by PhoP-PhoQ.
The small differences in MIC values measured for the oprH, phoP and phoQ mutants compared to wild-type indicate that PhoP-PhoQ is not a major factor in P. aeruginosa cationic peptide resistance. Our results are consistent with an indirect role for this regulatory system in the Mg2+ regulation of cationic peptide resistance. This conclusion contrasts peptide resistance in P. aeruginosa to that in S. typhimurium, where the PhoP-PhoQ two-component regulatory system has been proposed to play a key role. However, although killing assays comparing S. typhimurium phoP mutants with wild-type have showed substantial differences in peptide susceptibility (Fields et al., 1989 ), examination of the susceptibility of S. typhimurium strain MS7953 (phoP::Tn10; Fields et al., 1989
) to the peptides CP28, CEME and CP11CN revealed decreases in MIC values of only two- to eightfold relative to the values for wild-type (Falla & Hancock, 1997
; Piers et al., 1994
).
Aminoglycoside-resistance regulation
The differing effects of the P. aeruginosa phoP and phoQ mutations on cationic peptide resistance are indicative of the complex regulation of antibiotic resistance in this bacterium. The observed increase in resistance to aminoglycosides for all three mutants (PhoP-, PhoQ- and OprH-null) is a further indication of the intricacies of the PhoP-PhoQ regulatory system. The only feature common to strains H851, H854 and H855 is the inability to induce expression of phoQ. Strains H895 and H896, which carry deletions in oprH but which should allow fully inducible expression of phoP-phoQ, showed identical aminoglycoside resistance to the wild-type strain H103 (Table 5). The increased resistance observed in strain H855 (oprH::xylE-GmR), therefore, can be directly attributed to the polar effect of the xylE-GmR cassette on the downstream phoP-phoQ genes rather than to loss of OprH expression. Similarly, previous results that implicated OprH in P. aeruginosa resistance to aminoglycosides (Hancock et al., 1981
; Young et al., 1992
) can now be explained in terms of polar effects of the tet cassette inserted into oprH in strain H703 (Young et al., 1992
) on phoP-phoQ expression. Our results imply that PhoQ is responsible, either directly or through interaction with other regulatory systems, for the downregulation of resistance to the aminoglycosides streptomycin, kanamycin and amikacin. In this case, such a downregulation effect must be overridden by another system under Mg2+-starvation conditions, as these conditions are known to induce both PhoP-PhoQ expression (Macfarlane et al., 1999
) and resistance to streptomycin and gentamicin (Hancock et al., 1981
). Interestingly, in killing assays with streptomycin, the overexpression of PhoP in the PhoQ-null mutant H854 was seen to partially counteract the positive effect of a PhoQ-null phenotype (Table 4
). Hence it is feasible that high levels of PhoP expression in the absence of PhoQ affect the other system(s) involved in streptomycin resistance, most probably through cross-talk.
Decreased permeability of the outer membrane to the aminoglycoside streptomycin was observed for all three mutants (Fig. 3). However, the high concentrations of streptomycin (>100-fold higher than the MIC) that were required for this experiment probably do not reflect the normal killing mechanism, and are consequently more indicative of a general increase in outer-membrane stability in the mutants. A greater resistance to perturbation of the outer membrane was observed in strain H854, which overexpresses OprH, than in strains H851 and H855 (both unable to express OprH), lending support to our suggestion of an outer-membrane-stabilization role for OprH.
We noted earlier that an exceptionally large number of two-component regulatory systems have been revealed in P. aeruginosa by the Pseudomonas genome sequencing project. Our results support the concept of P. aeruginosa antibiotic resistance being the result of a complex interplay of several of these systems. Further studies aimed at unravelling such interactions, as well as defining genes that fall into the PhoP-PhoQ regulon, should greatly aid our understanding of antibiotic resistance in this important pathogen.
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ACKNOWLEDGEMENTS |
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Received 6 April 2000;
revised 19 June 2000;
accepted 29 June 2000.