Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK1
Author for correspondence: Robert K. Poole. Tel: +44 114 222 4447. Fax: +44 114 272 8697. e-mail: r.poole{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: cytochrome bd, oxidase, Escherichia coli, haems, membrane transport
Abbreviations: p, protonmotive force; ABC, ATP-binding cassette; CCCP, carbonyl cyanide m-chlorophenylhydrazone
a Present address: Department of Microbiology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
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INTRODUCTION |
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Strains having mutations in cydD also lack periplasmic cytochromes c (Poole et al., 1994 ; Goldman et al., 1996
), and do not assemble cytochrome b562 (Goldman et al., 1996
). CydD is not required for the synthesis of haem D or the assembly of the CydA and CydB subunits into the membrane (Bebbington & Williams, 1993
). We therefore hypothesized that the CydDC transporter may be required for the export of haem(s) to the periplasm for maturation of soluble cytochromes c and b and perhaps assembly into periplasmically exposed components of cytochrome bd (Poole et al., 1993
, 1994
). Indeed, the haem-binding site for cytochrome b558 (histidine-186) is predicted to be at the periplasmic face, with the implication that haem attachment may occur there (Spinner et al., 1995
). Interestingly, Osborne & Gennis (1999)
have reanalysed the probable topology of subunit I of cytochrome bd in several bacteria and conclude that the putative ligand for haem b595 is also close to the periplasmic edge of the membrane, and further that the active site of the oxidase comprising this haem and haem d are located near the periplasmic surface. It is not clear whether such a disposition requires haem insertion from outside. The assembly of cytochrome bo', the major alternative oxidase, is unaffected by a mutation in cydD1 (G. M. Cook & R. K. Poole, unpublished data). Some doubt has been cast on the hypothesis that CydDC is a haem transporter by experiments with haem reporters (Goldman et al., 1996
): expression of heterologous haemproteins in the periplasm of E. coli results in assembly of the holoprotein (i.e. with haem) even in cydC mutants, suggesting that a pathway for haem export must exist in the absence of the CydDC ABC transporter. An alternative hypothesis for CydDC function is that it is required for maintenance of a redox environment in the periplasm appropriate for haem ligation (Goldman et al., 1996
), but again no firm role has been proposed and no substrate for the transporter has been identified.
Efforts to understand periplasmic cytochrome c assembly in E. coli have focused on the eight Ccm (cytochrome c maturation) proteins (Thöny-Meyer et al., 1994 ; Grove et al., 1996
; Thöny-Meyer, 1997
; Throne-Holst et al., 1997
; Schulz et al., 1999
). One model is that the products of the ccmABC genes, and perhaps ccmD as well, constitute an ABC transporter that exports haem to the periplasm, where it is incorporated into cytochrome c apoproteins (Thöny-Meyer, 1997
). Similar transporters have been proposed in other bacteria: in Paracoccus denitrificans, the ccmABC genes encode the subunits of an ABC transporter with the composition (CcmA)2CcmBCcmC (Page et al., 1997
). The helABC and helD genes (haem export for ligation) of Rhodobacter capsulatus (Goldman et al., 1997
) are also thought to encode an ABC transporter necessary for cytochrome c assembly. No firm evidence for the transport processes catalysed by such pathways has however been presented. Furthermore, since ccmABC mutants of P. denitrificans are able to assemble periplasmic cytochromes b, and exogenously added haem fails to restore assembly of periplasmic cytochromes c (Page et al., 1997
), this hypothesis appears untenable. Recently, Schultz et al. (1999)
have shown in E. coli that CcmA and CcmB (but not CcmC) do constitute an ABC transporter but that it is not essential for cytochrome c maturation. (In this paper, we embrace this finding and refer to the ABC transporter encoded by the ccm operon in E. coli as CcmAB, not CcmABC.) It is proposed that CcmC (a transmembrane protein) and CcmD serve to transfer haem to CcmE, an essential haem chaperone that passes haem to periplasmic apocytochromes. Several lines of evidence suggest that CcmAB is not a haem transporter (Schulz et al., 1999
) but no direct measurements of haem transport have been reported.
Movement of haem into and across lipid bilayers plays a critical role not only in the biogenesis of periplasmic haemproteins in bacteria, but also in that of eukaryotic extramitochondrial haemproteins, such as globins. However, transmembrane movement of haem is likely to be slow (Light & Olson, 1990 ), predicting the need for haem transport systems. Nevertheless, despite concerted efforts from several groups and claims for the existence of bacterial haem transporters, no clear candidate has emerged, although putative haem exporters have been found in several bacterial genera (Beckman et al., 1992
; Thony-Meyer et al., 1994; Aguilar & Soberon, 1996
; Page et al., 1997
). This may be in part a result of technical difficulties in measuring transport kinetics of haem, which self-aggregates in aqueous solutions and interacts non-specifically with cellular components. Some workers have used haemCO, which is monomeric in dilute aqueous solutions (Light & Olson, 1990
), whilst others have used haem bound to a carrier protein (e.g. Stojiljkovic & Hantke, 1994
). We are unaware of direct attempts to determine rates of haem transport across E. coli isolated cytoplasmic membranes and report such experiments here.
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METHODS |
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Cytochrome d assays.
For identification and quantitation of cytochrome d, cells were grown in Luria broth (Miller, 1972 ) to an OD600 of 0·91·0 (measured after correction for dilution where necessary). Harvested cells were washed in 0·1 M potassium phosphate buffer (pH 7·0) and used to record reduced minus oxidized and CO difference (CO-reduced minus reduced) spectra at room temperature as previously described (Poole et al., 1989
). Cytochrome d concentrations were calculated from the former using an absorption coefficient of 18·5 mM-1 cm-1 from A630650 (Kita et al., 1984
). The protein content of cell suspensions was assayed by the method of Markwell et al. (1978)
.
Preparation of everted membrane vesicles.
Cells were grown aerobically to the mid-exponential phase of growth, either in minimal MOPS medium supplemented with glucose or lactose or in LB. Defined medium was used only for studies of lactose transport, where variation of carbon source was required (see Results). Cells were harvested by centrifugation and the cell pellet washed with pre-cooled 100 mM potassium phosphate buffer (pH 7·0) containing 10 mM disodium EDTA, and resuspended in the same buffer (5 ml per litre of original culture). To prepare everted vesicles, the procedure of Thanassi et al. (1997) was used. In brief, cells were disrupted by one passage through a French pressure cell at 5000 p.s.i. (34·5 MPa). Pancreatic DNase was added to 0·1 mg ml-1, and the mixture was kept on ice for 1 h or until the viscosity decreased significantly. After centrifugation at 27000 g for 10 min, the vesicles were sedimented from the supernatant suspension by centrifugation at 150000 g for 1 h. Vesicles were gently resuspended in the same buffer and collected again by centrifuging under the same conditions. They were then resuspended in 100 mM potassium phosphate buffer (pH 7·2) containing 5 mM MgCl2 to a concentration of 15 mg protein ml-1. Aliquots (200 µl) of the vesicles were stored at -70 °C after rapid freezing in liquid nitrogen.
[14C]Haemin and [14C]lactose transport assays.
We have adopted the following nomenclature: the term haem is used to describe the chelate complex of Fe(II) with protoporphyrin IX, the prosthetic group of, for example, cytochrome b or haemoglobin; haemin is the chloride of the Fe(III) form, and haematin is the hydroxide of the Fe(III) form. [14C]Haemin [approx. 130 mCi mmol-1 (4810 MBq mmol-1)] was purchased from Professor Stan Brown, University of Leeds Innovations Ltd (Woodhouse Lane, Leeds LS2 3AR, UK). Haemin transport assays were based on previously established methods (Stojiljkovic & Hantke, 1994 ; Tompkins et al., 1997
). [14C]Haemin (1 µCi; 37 kBq) was solubilized in a drop of 0·1 M NaOH and diluted with 50 mM potassium phosphate buffer (pH 7·2) containing bovine serum albumin (BSA; 10 mg ml-1) as a carrier for haemin to avoid polymerization, self-aggregation, and non-specific interaction with cellular components. [14C]Lactose (57 mCi mmol-1; 2109 MBq mmol-1; Amersham) was added to achieve a final concentration of 400 µM in the transport assay. Everted vesicles were thawed slowly on ice and diluted to a protein concentration of 0·51·0 mg ml-1 in 50 mM potassium phosphate buffer (pH 7·2) containing 5 mM MgCl2. Vesicles were added to glass tubes containing 200 µl of the same pre-warmed phosphate buffer and pre-incubated for 15 min without shaking in a water bath at 30 °C. To initiate transport, radiolabelled [14C]haemin or [14C]lactose was added to achieve final concentrations of 5·7 µM or 400 µM, respectively. Vesicles were energized for 15 min prior to [14C]haeminBSA or [14C]lactose addition with either 10 mM D-lactate or ATP (25 mM). Vesicles were de-energized with either carbonyl cyanide m-chlorophenylhydrazone (CCCP; 50 µM) to collapse the proton gradient (
p), monensin (100 µM) to collapse a sodium gradient, or sodium vanadate (100 µM), a specific inhibitor of ATP-driven transport systems (Pressman, 1976
). After 020 min, transport was terminated by the addition of ice-cold LiCl (2 ml, 100 mM) and rapid filtration (0·45 µm pore size cellulose-nitrate filter). The filter was washed twice with 2·0 ml of the LiCl solution, dried for 20 min at 105 °C, and radioactivity measured by liquid scintillation counting. To minimize non-specific binding of [14C]haeminBSA to filters, the filters were pre-soaked in 100 mM LiCl containing BSA (10 mg ml-1).
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RESULTS |
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Because CydDC is an ABC transporter presumed to be energized by ATP, ATP (5 mM) was added to the transport assay 10 min before [14C]haeminBSA. When either ATP or sodium lactate was added to the membrane vesicles prior to [14C]haeminBSA addition, both the initial [14C]haeminBSA binding and the subsequent transport occurred at rates (again determined between 2 and 12 min) that did not differ significantly from that seen where neither ATP nor lactate had been added (Fig. 1b, Table 2
). It is probable that the differences between the three uptake experiments shown in Fig. 1(b)
and below (Fig. 3
) are due to the experimental difficulties of working with solutions of such a hydrophobic substrate, and are not due to major differences in the proportion of vesicles that are everted (see Discussion). We conclude that the uptake of [14C]haeminBSA is not dependent on an externally provided energy source. Furthermore, none of sodium vanadate (100 µM), monensin (100 µM) or CCCP (50 µM) had any significant effect on the rate of [14C]haeminBSA uptake by wild-type cells (results not shown), suggesting that an energized membrane is not required for haemin uptake and that ATP-driven transporter activity does not play a major role in haemin transport in these vesicles.
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[14C]HaeminBSA transport in everted membrane vesicles from ABC transporter mutants
The ATP-independence of the haem movements described above strongly suggests that the activities of ABC-type transporters, such as CydDC or CcmAB, do not contribute significantly. This was confirmed by experiments with vesicles prepared from the cydD1 mutant AN2343, which revealed similar patterns of [14C]haeminBSA uptake as in the isogenic wild-type strain (Fig. 1c). The rate of [14C]haeminBSA uptake as determined between 2 and 12 min was 0·06 nmol min-1 (mg protein)-1 (Table 2
). As observed for the wild-type strain, unlabelled haeminBSA did not displace a significant fraction of the label from vesicles prepared from the cydD1 mutant (Fig. 3b
).
To test the role of the CcmAB proteins in haemin transport, everted vesicles were prepared from cells grown anaerobically on glycerol and nitrate, conditions required for the induction of the ccmAB genes. Everted vesicles from either MC1061 (wild-type) or EC21 (ccmA) accumulated [14C]haeminBSA with time and there was little difference between the strains in terms of the rate of [14C]haeminBSA uptake as calculated between 2 and 12 min (Fig. 3c
, Table 2
). The uptake was biphasic, and bound [14C]haeminBSA could not be chased in either strain and was not dependent on an exogenous energy source (Table 2
).
Effect of the reducing environment on cytochrome bd assembly
Goldman et al. (1996) reported that cydC mutants have a more oxidized periplasm than that of wild-type cells and cydAB mutants. These results are consistent with the CydDC transporter exporting a reductant to the periplasm that is required either for haem ligation and/or to ensure that the redox environment of the periplasm is appropriate for haem ligation. To test this hypothesis, a number of reducing pathways in E. coli were tested for their effect on cytochrome d levels. Wild-type cells contained approximately 0·04 nmol haem d (mg protein)-1 as measured in reduced minus oxidized and CO difference spectra (not shown). In contrast, cytochrome d levels in an isogenic cydD1 mutant were undetectable. Mutations in the upstream gene trxB did not reduce the level of cytochrome d significantly compared to the wild-type, confirming previous results (Poole et al., 1994
). Furthermore, mutations in grxA (glutaredoxin), trxA (thioredoxin), or both alleles, (grxA trxA) did not cause a loss of cytochrome d (not shown).
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DISCUSSION |
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However, there is conflicting evidence regarding the ability of haemin to penetrate the cytoplasmic membrane and the requirement for specific haem transporters, whether for inward or outward movement (with reference to the cell) in this process. First, respiration-deficient membrane vesicles from a hemA mutant of E. coli, which cannot synthesize haem, can be reconstituted in vitro by adding haematin and ATP (Haddock & Schairer, 1973 ). If cytoplasmic membranes are intrinsically impermeable to haem, this result implies either that all haemproteins required for respiration are exposed to the outer faces of the membrane vesicles, or that haem transport is driven by ATP. Respiration is not reconstituted, however, by adding haematin to cells during growth (Haddock & Schairer, 1973
), presumably because of the requirement for outer-membrane haem receptor and transport systems in E. coli (Stojiljkovic & Hantke, 1992
). Stojiljkovic & Hantke (1992)
expressed the Yersinia enterocolitica haemin receptor HemR in E. coli K-12, thus conferring the ability to use haemin as a porphyrin source. Additional gene products appear necessary for using haemin as an iron source, since Y. enterocolitica mutants defective in hemU and hemV (encoding a putative haem-specific permease and ATPase, respectively) are severely impaired in haemin utilization, yet still show weak growth stimulation by haemin (Stojiljkovic & Hantke, 1994
). This suggests leakage of haemin through the cytoplasmic membrane. Furthermore, Torres & Payne (1997)
have demonstrated that introduction of the gene for an outer-membrane protein is sufficient to confer haem utilization in E. coli K-12, and growth in vivo of pathogenic E. coli strains is stimulated by haemin or haemoglobin, perhaps again indicating the presence of a cytoplasmic membrane transporter for haem (Stojiljkovic & Hantke, 1994
) or perhaps that only the outer membrane presents a significant barrier to uptake to the cytoplasm. Nevertheless, it is tacitly assumed that E. coli has a mechanism(s) for haem translocation from the cytoplasm to the outer face of the cytoplasmic membrane, where it is required for periplasmic cytochrome assembly (Beckman et al., 1992
; Goldman et al., 1998
; Thony-Meyer et al., 1994; Thony-Meyer, 1997
). Such a mechanism(s) has so far remained unidentified.
The present study has used everted vesicles prepared after cell disruption in the French pressure cell. It is widely recognized that this procedure results in a population of vesicles that is predominantly inverted with respect to the in vivo orientation. We consider it unlikely that the small variations seen between experiments (as in Figs 1 and 3
) are due to variation in the proportion of everted vesicles, since specific transport activity has been shown by others to be insensitive to experimental variables, specifically French cell pressure, for example, between 4000 and 14000 p.s.i. The procedure of Thanassi et al. (1997)
used here gives vesicles that exhibit negligible uptake of proline (which is transported inwards in vivo), which is stimulated 60-fold when vesicles are prepared by lysosyme-EDTA treatment (McMurry et al., 1980
). Other criteria for the everted nature of vesicles isolated after French press disruption include high rates of uptake of Ca2+ (which is exported by intact cells), accessibility of substrates to enzymes whose activity is normally cryptic, the direction of respiration- and ATPase-driven H+ translocation, exposure of inaccessible antigenic determinants, and morphology (Rosen & Tsuchiya, 1979
). On these bases it seems highly probable that the haemmembrane interactions described here are with everted vesicles.
We found that interaction of haem with the membrane does not require ATP, an oxidizable substrate, or p. [14C]HaeminBSA uptake appears to be a biphasic process. The first rapid phase is probably haemlipid association; the second slower phase is driven by the concentration gradient of haemin across the cell membrane and probably represents haem movement across the bilayer, possibly resulting in ultimate dissociation from the inner face of the membrane (Fig. 4
). The lack of saturation kinetics suggests either that the process is not catalysed by specific transport proteins or that higher concentrations of haemin are required for saturation. These results agree with those obtained for haemCO and unilamellar lipid vesicles (Light & Olson, 1990
): transmembrane movement is slow and distinct from the initial binding process. Possible involvement of the cydDC and ccmAB gene products in haemin transport could not be demonstrated, the rates of haemin uptake being comparable in wild-type and mutant strains. Thus, the CydDC and CcmAB transporters do not seem to be necessary for haem transport, but our experiments do not rule out the possibility that such proteins accelerate haem transport in vivo or the existence of other haem-binding membrane components. The lack of involvement of CydDC in haem transport might seem to support the work of Goldman et al. (1996)
, who expressed various haem apoproteins in the E. coli periplasm and showed that mutations in cydC did not prevent periplasmic holoprotein assembly, even in the absence of exogenous haemin. However, the experiments of Light & Olson (1990)
clearly demonstrate the possibility of haem movement into and out of the bilayer in the absence of transport proteins; dissociation from the bilayer is readily demonstrated when liposomes with embedded haem are mixed with apomyoglobin. Thus the experiments of Goldman et al. (1996)
, which utilized artefactually high periplasmic concentrations of apoproteins, may have provided a trap for haem dissociating outwards from the bilayer into the periplasm. The mechanism for such haem movement in E. coli is becoming clearer. CcmE is a protein N-terminally anchored to the cytoplasmic membrane, which accumulates in a haem-bound form in ccmF mutants defective in cytochrome c assembly (Reid et al., 1998
). The haem is bound covalently by a histidine residue and is released in the presence of apocytochrome c and other Ccm proteins needed for assembly (Schulz et al., 1998
). Schulz et al. (1999)
have shown that only CcmC is absolutely required for the charging of CcmE with haem, at least when CcmC is highly expressed. It could not be excluded from those experiments, however, that haem translocation normally occurs via the CcmAB transporter, only that high abundance of CcmC allows haem to cross the membrane in the absence of CcmAB. Perhaps the strong haem binding of CcmC (evidenced by the persistence of haem in SDS gels) could pull or extract haem from the lipid bilayer, as demonstrated for the reaction of apomyoglobin with liposomes (Light & Olson, 1990
).
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Phospholipid membranes have a high affinity for haem, and the interaction of phospholipid with haemin, which can be considered an aromatic, soap-like molecule (Rose et al., 1985 ), has a major hydrophobic component. It is therefore pertinent to compare possible mechanisms of haem transport with what is known about hydrophobic drugs. These have also been shown to be taken up by a biphasic process (see Bolhuis et al., 1997
for a review). Normally this is observed as a fast entry into the outer leaflet of the membrane followed by a slower movement across the bilayer to the inner leaflet of the membrane (Bolhuis et al., 1997
). The ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug-resistant transporters which might function as a hydrophobic vacuum cleaner to remove drugs from the inner leaflet and extrude them from the membrane. There is a direct relationship between the amount of hydrophobic drug associated with the cytoplasmic leaflet of the lipid bilayer and the rate of transport, as observed for drug extrusion by the lactococcal multidrug transporter LmrP (Bolhuis et al., 1997
). According to the flippase model (Higgins & Gottesman, 1992
), the function of the ABC transporter might be to pump substrate (perhaps haem?) from the inner to the outer leaflet (Fig. 4
), from where the haem may diffuse to the periplasm, haem chaperone or membrane-associated apoprotein. In our experiments, inability to chase labelled haemin may reflect tight binding to the CydDC subunits even though they are incapable of active translocation. The idea of flipping is not new, and this term was used by Rose et al. (1985)
in the context of haem transport. Interestingly, though, the experiments of Light & Olson (1990)
with unilamellar lipid vesicles reveal a second slow phase in both the association and dissociation directions that might equate with the transmembrane movement role envisaged for ABC transporters. It is important to note that the present results do not rule out energization-dependent haem translocation from an aqueous phase on one side of the membrane to the aqueous phase on the other. Our results show only that haemin (proffered as a BSA complex) is bound to the membrane, and rendered incapable of being chased, independently of membrane energization or the involvement of CydDC or CcmAB. It is feasible, for example that, in vivo, haem binds to the inner leaflet of the cytoplasmic membrane bilayer and may cross to the outer leaflet, and only then might its passage into the periplasm (or the interior of a vesicle) require the ATPase activity of the transporter.
Could haem export to the periplasm proceed without a specific transporter? Although the transmembrane movement of haemCO in liposomes having long unsaturated acyl chains is very slow (the release phase having a rate constant of about 0·001 s-1; Light & Olson, 1990 ), such rate constants may underestimate the in vivo rates for natural haem substrates. The rate may be raised further by protein-induced discontinuities in apolar regions of the membrane similar to that seen in vitro at temperatures near the phase-transition temperature. Finally, perhaps even a half-time of 10 min for haem flipping as estimated for egg lecithin liposomes might be sufficient for haem assembly and function in the periplasm.
Despite results from many laboratories, the substrates for the transporters encoded by both the cydDC and ccmAB gene products are unknown. Recent work from Ferguson and co-workers has suggested that the putative CcmABC haem transporter in P. denitrificans does not transport either haem or c-type apocytochromes and suggests a low-molecular-mass thiol or oxidized thiol as substrate (Page et al., 1997 ). However, cysteine, glutathione or 2-mercaptoethanesulfonic acid could not complement a
ccmA mutation in E. coli (Schulz et al., 1998
). Similar proposals for the nature of the transported substrate have been put forward by Poole et al. (1994)
and Schulz et al. (1999)
. Essentially, the substrate is considered to be a reductant that keeps haem and/or apoprotein cysteine residues in the periplasm reduced for haem ligation, despite the oxidizing environment of the periplasm. Disulfide bond formation plays a key role in protein folding in the periplasm of Gram-negative bacteria (Raina & Missiakis, 1997
). It is notable then that: (a) the gene upstream of the cydDC operon is trxB, encoding thioredoxin reductase (Russel & Model, 1988
), and (b) in many cases, genes encoding an ABC exporter are adjacent to the gene encoding the transported product (Salmond & Reeves, 1993
). In the cytoplasm, a reducing balance is maintained using three glutaredoxins (products of grxA, grxB and grxC) as well as the thioredoxin (trxA/trxC)/thioredoxin reductase (trxB) system (for a review, see
slund & Beckwith, 1999
). Furthermore, Goldman et al. (1996)
have reported that cydC mutants have a more oxidized periplasm than do wild-type and cydAB mutants. These results are consistent with the CydDC proteins exporting a reductant to the periplasm that is required either for haem ligation and/or to ensure a redox environment of the periplasm appropriate for haem ligation. However, the present results show that none of trxB, trxA or grxA is required for cytochrome bd assembly, although trxA function is needed for the transfer of electrons from the cytoplasm to periplasmic DsbC for cytochrome c assembly (Reid et al., 1998
).
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ACKNOWLEDGEMENTS |
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Received 11 August 1999;
revised 5 November 1999;
accepted 8 November 1999.