Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK
Correspondence
David W. Holden
d.holden{at}imperial.ac.uk
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ABSTRACT |
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Present address: Departamento de Biología Celular, Fisiología y Genética, Facultad de Ciencias, Universidad de Málaga, Campus Teatinos, Málaga 29071, Spain.
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INTRODUCTION |
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Serovars of Salmonella enterica encode two distinct virulence-associated TTSSs located within Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are involved in different aspects of S. enterica pathogenicity. The SPI-1 TTSS of S. enterica serovar Typhimurium (S. typhimurium) translocates at least eight effector proteins that control several processes, including host cell invasion, an apoptotic-like effect in macrophages, and trans-epithelial migration of neutrophils (Hersh et al., 1999; Zhou & Galán, 2001
). The SPI-1 TTSS is expressed optimally in growth conditions that reflect those in the lumen of the small intestine, including low oxygen, high osmolarity and slight alkalinity (pH 8) (Bajaj et al., 1996
). The S. typhimurium SPI-2 TTSS is required for systemic infection of mice, and intracellular replication in both macrophages and epithelial cells (Cirillo et al., 1998
; Hensel et al., 1995
, 1998
; Ochman et al., 1996
; Shea et al., 1996
). SPI-2 TTSS gene expression is induced inside the host cell and requires the two-component regulatory system SsrAB, also encoded within SPI-2 (Cirillo et al., 1998
). SsrAB controls the expression of genes encoding the components of the SPI-2 TTSS, as well as genes encoding SPI-2 effectors located both in SPI-2 and elsewhere in the chromosome (Beuzón et al., 2000
; Brumell et al., 2003
; Cirillo et al., 1998
; Knodler et al., 2002
; Miao & Miller, 2000
; Worley et al., 2000
). Two effector proteins, SlrP and SspH1, which have been shown to be translocated via both SPI-1 and SPI-2 TTSS, are expressed constitutively, in a SsrAB-independent manner (Miao & Miller, 2000
).
The expression of SsrAB is regulated by the OmpREnvZ two-component system. OmpR binds directly to the ssrAB promoter (Lee et al., 2000). In Escherichia coli, OmpREnvZ has been shown to be responsible for both activation and repression of gene expression, in response to changes in osmolarity and pH (Heyde & Portalier, 1987
). The OmpREnvZ system is required for Salmonella replication and survival within macrophages (Lee et al., 2000
) and is necessary for full virulence in mice (Chatfield et al., 1991
; Dorman et al., 1989
).
Several studies have analysed SPI-2 TTSS gene expression in different conditions, thought to reflect the environment within the Salmonella-containing vacuole (SCV). Bacteria grown in different minimal media express SPI-2 TTSS genes when reaching stationary phase (Beuzón et al., 1999; Deiwick & Hensel, 1999
; Deiwick et al., 1999
; Lee et al., 2000
; Miao et al., 2002
). In addition, low osmolarity in the growth medium has been shown to play a role in the induction of SPI-2 TTSS gene expression (Lee et al., 2000
). Low concentrations of Mg2+, Ca2+ or
in growth media have also been reported to stimulate SPI-2 TTSS gene expression (Deiwick et al., 1999
), although more recent studies have failed to reproduce the effect of low concentrations of Mg2+ (Lee et al., 2000
; Miao et al., 2002
). It has been shown that the SCV undergoes acidification to a pH between 4·0 and 5·0 (Rathman et al., 1996
). Using transcriptional fusions of several SPI-2 genes to a gene encoding the green fluorescent protein (GFP), Cirillo et al. (1998)
reported that inhibition of SCV acidification abolished SPI-2 TTSS gene expression inside the host cell. Using fusions to different reporters Lee et al. (2000)
and Miao et al. (2002)
found that acidic pH induced SPI-2 TTSS gene expression in vitro but Deiwick et al. (1999)
found no significant differences in SPI-2 gene expression in response to pH changes.
In this study, we have determined the effect of [Ca2+], osmolarity and pH on SPI-2 gene expression and the relative influence exerted by the SsrAB and OmpREnvZ two-component regulatory systems in this process. Our results show that the effects of these signals on SPI-2 TTSS gene expression are completely dependent on SsrAB but only partially dependent on OmpREnvZ. Futhermore, the effect of OmpREnvZ on SPI-2 TTSS gene expression requires a functional SsrAB system, which indicates that the effect of OmpREnvZ on SPI-2 gene expression is mediated through ssrAB.
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METHODS |
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Preparation of protein samples.
Bacterial cell densities were determined by measurement of the OD600. To ensure that protein from equal numbers of cells was analysed, in all experiments protein samples were adjusted to OD600 values such that each fraction from a 10 ml culture of OD600 0·6 was taken up in 100 µl protein-denaturing buffer for gel electrophoresis.
PAGE and Western analysis of proteins.
Protein samples were dissolved in the appropriate volume of protein-denaturing buffer containing 62·5 mM Tris/HCl pH 6·8, 2 % SDS, 5 % -mercaptoethanol, 10 % glycerol and 0·02 % bromophenol blue and held at 100 °C for 5 min. Proteins were immediately separated on a 12 % SDS-polyacrylamide gel (Laemmli, 1970
). Proteins were transferred from gels to Immobilon-P membranes (Millipore) using a semi-dry blotting apparatus (Bio-Rad) with the buffer described by Kyhse-Andersen (1984)
. Westerns were developed using the ECL detection system under the conditions recommended by the manufacturer (Amersham Life Science). Rabbit anti-SseB (Beuzón et al., 1999
) or anti-RecA (a gift from Kenji Adzuma, The Rockefeller University, New York, USA) polyclonal antibodies, or mouse monoclonal anti-GFP (Clontech) were used as primary antibodies. Donkey anti-rabbit or anti-mouse horseradish-peroxidase-conjugated antibodies (Amersham Life Science) were used as secondary antibodies. Affinity purification of anti-SseB antibody was performed using the method described by Ruiz-Albert et al. (2003)
.
Preparation of bacteria for flow cytometric analysis.
S. typhimurium 12023 wild-type and mutant strains carrying pID835 or pID836 plasmids were centrifuged at 4000 g, and pellets were resuspended in PBS. In each experiment, strain 12023 and strain 12023 carrying pFVP25.1 were used as negative and positive controls for fluorescence, respectively. For each sample, 105 cells were analysed on a FACS Calibur cytometer (Becton Dickinson). GFP was detected at 525 nm in the FL1 channel. Data were analysed with CellQuest software. Flow cytometric data were analysed as follows. The geometric mean of the fluorescence of each strain in three independent experiments was calculated. The fold increase in fluorescence of the ssrA, ompR, or ssrA ompR mutant strains versus that of the wild-type was calculated by dividing the geometric mean fluorescence of the wild-type strain by the geometric mean fluorescence of the mutants.
Antibodies and reagents.
Anti-Salmonella goat polyclonal antibody CSA-1 was purchased from Kirkegaard and Perry Laboratories and was used at a dilution of 1 : 400. Texas red sulfonyl chloride (TRSC)-conjugated donkey anti-goat antibody was purchased from Jackson Immunoresearch Laboratories and used at a dilution of 1 : 400.
Cell culture.
RAW 264.7 cells were obtained from ECACC (ECACC 91062702). HeLa cells (clone HtTA1) were kindly provided by Dr H. Bujard (Heidelberg, Germany). Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal calf serum (FCS) and 2 mM glutamine at 37 °C in 5 % CO2.
Bacterial infection of HeLa cells.
HeLa cells were seeded onto glass coverslips (12 mm diameter) in 24-well plates at a density of 5x104 cells per well, 24 h before infection. Bacteria were incubated for 16 h at 37 °C with aeration, diluted 1 : 33 in fresh LB broth and incubated in the same conditions for 3·5 h. Cultures were diluted in Earle's buffered salt solution (EBSS) pH 7·4 and added to the HeLa cells at a m.o.i. of 100 : 1. The infection was allowed to proceed for 15 min at 37 °C in 5 % CO2. The monolayers were washed once with DMEM containing FCS and 100 µg gentamicin ml-1 and incubated in this medium for 1 h, after which the gentamicin concentration was decreased to 16 µg ml-1.
Immunofluorescence.
For immunofluorescence, cell monolayers were fixed in 3·7 % paraformaldehyde in phosphate-buffered saline (PBS) pH 7·4, for 15 min at room temperature and washed three times in PBS. Antibodies were diluted in 10 % horse serum, 1 % bovine serum albumin, 0·1 % saponin in PBS. Coverslips were washed twice in PBS containing 0·1 % saponin, incubated for 30 min with primary antibodies, washed twice with 0·1 % saponin in PBS and incubated for 30 min with secondary antibodies. Coverslips were washed twice in 0·1 % saponin in PBS, once in PBS and once in H2O, and mounted on Mowiol. Samples were analysed using an Olympus BX50 fluorescence microscope or a Zeiss LSM510 confocal laser scanning microscope. For staining with anti-SseB antibody, cells were permeabilized for 10 min by incubation with 0·1 % Triton X-100 in PBS, prior to incubation with the antibody.
Bacterial infection of macrophages for flow cytometric analysis.
Macrophages were seeded at a density of 4x105 cells per well in 24-well tissue culture plates, 24 h before use. Bacteria were cultured at 37 °C with shaking until they reached an OD600 of 2·0. The cultures were diluted to an OD600 of 1·0 and opsonized in DMEM containing FCS and 10 % normal mouse serum for 20 min. Bacteria were added to the monolayers at a m.o.i. of 100 : 1, centrifuged at 170 g for 5 min at room temperature and incubated for 25 min at 37 °C in 5 % CO2. Macrophages were washed once with DMEM containing FCS and 100 µg ml-1 gentamicin and incubated in this medium for 1 h. The medium was replaced with DMEM containing FCS and 16 µg ml-1 gentamicin for the rest of the experiment. Bafilomycin A1 was added to cell monolayers 15 min prior to the addition of the bacteria, to a final concentration of 100 nM, where indicated. At 2 h after bacterial uptake, cells were lysed with 0·1 % Triton X-100 for 10 min, and used for flow cytometry.
Flow cytometric analysis of infected macrophages.
Infected macrophage lysates were resuspended in a mixture of 250 µl 0·1 % Triton X-100 and 250 µl PBS and kept on ice for immediate analysis. For each sample, 104 bacterial-sized particles were analysed on a FACS Calibur cytometer (Becton Dickinson). GFP was detected at 525 nm in the FL1 channel. Data were analysed with CellQuest software.
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RESULTS |
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Regulation of sifA and sifB expression in vitro
Mg minimal (MgM) salts medium (see Methods) has been used to stimulate SPI-2 TTSS gene expression and to attempt to identify the signals that trigger expression of the SPI-2 TTSS inside the SCV (Deiwick & Hensel, 1999; Deiwick et al., 1999
; Miao et al., 2002
; Beuzón et al., 1999
). Growth of bacteria in this medium results in a strong induction of SPI-2 TTSS expression. Therefore, this medium was used in this study as a basis to further investigate the signals stimulating SPI-2 gene expression. The pH of bacterial cultures after overnight growth in MgM medium buffered to pH 7·5 before inoculation remained unchanged.
Wild-type S. typhimurium carrying either PsifA : : gfp or PsifB : : gfp transcriptional fusions were grown overnight in MgM medium. Expression from each plasmid was detected by flow cytometry (Fig. 2a, b) and immunoblotting using an anti-GFP antibody (Fig. 3
a, lower panel). As observed in infected cells (Fig. 1a
), we found that expression driven by the sifB promoter was higher than that driven by PsifA (Fig. 2a, b
, upper panels).
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Contribution of different environmental signals to SPI-2 TTSS gene expression in vitro
We next investigated the contribution of [Mg2+] to SPI-2 TTSS gene expression by growing bacterial strains in MgM medium containing either 8 µM or 200 µM MgCl2. Flow cytometry was carried out using wild-type bacteria harbouring either PsifA : : gfp or PsifB : : gfp fusions, after overnight growth in MgM medium at pH 7·5. No significant differences in fluorescence were detected for either fusion after growth in 8 µM or 200 µM MgCl2 (Fig. 3a, upper panel). Similarly, no significant differences were observed by immunoblotting either with an anti-GFP antibody using wild-type bacteria carrying either PsifA : : gfp or PsifB : : gfp fusions (Fig. 3a
, lower panel), or with an anti-SseB antibody using wild-type bacteria (Fig. 3b
).
Deiwick et al. (1999) reported that, after growth in MOPS-salts medium (O'Neal et al., 1994
), the presence of 200 µM Mg2+ inhibited the expression of ssaB, and this effect could be reverted by lowering the [PO3-4]. However, in the presence of 8 µM Mg2+, ssaB expression was independent of [PO3-4]. In agreement with these results, no significant differences were observed in PsifA : : gfp or PsifB : : gfp expression in response to changes in [PO3-4] in medium containing 8 µM Mg2+ (data not shown). However, under our assay conditions, no inhibitory effect of high [Mg2+] was observed (Fig. 3a, b
). This prevented us from confirming a possible effect of PO3-4 starvation in reverting an inhibitory effect of high [Mg2+].
Removal of 38 mM glycerol from the medium had no significant effect on sifA : : gfp or sifB : : gfp expression, when analysed by flow cytometry (data not shown). Similarly, reducing [(NH4)2SO4] in the growth medium by 10- or 100-fold did not have a significant effect on sifA : : gfp or sifB : : gfp expression (data not shown).
Together, these results indicate that changes in the concentrations of Mg2+, PO3-4, glycerol and (NH4)2SO4 have no significant effect on SPI-2 gene expression, after overnight growth in MgM medium.
The absence of Ca2+ leads to SPI-2 TTSS gene expression via SsrAB
It has been shown previously that the absence of Ca2+ in Tris-buffered MgM medium induces expression from the SPI-2 promoters PssaB and PsseA (Deiwick et al., 1999). Consistent with these results, the fluorescence of wild-type bacteria carrying either PsifA : : gfp or PsifB : : gfp increased by six- to eightfold and eightfold, respectively, after growth in MgM medium, compared to the expression after growth in MgM medium containing 2 mM Ca2+ (data not shown and Fig. 4
b). Similarly, SseB was undetectable in wild-type bacteria grown overnight in MgM medium in the presence of 2 mM Ca2+, but was detected after growth in MgM medium without Ca2+ (Fig. 4c
).
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Low osmolarity induces SPI-2 TTSS gene expression via SsrAB
It has been shown previously that high osmolarity (generated by the presence of 20 % sucrose or 0·5 M NaCl) in MgM medium represses expression from the SPI-2 promoter PssaH (Lee et al., 2000). S. typhimurium is not able to use sucrose as a carbon source, but the presence of Casamino acids allows normal growth of bacterial cells in MgM medium containing 20 % sucrose. Expression of PsifA : : gfp and PsifB : : gfp was eight- and tenfold higher, respectively, in MgM medium than in the same medium containing 20 % sucrose (Fig. 5
a, upper panel, and 5b). The addition of the osmoprotectant glycine betaine partially prevented the inhibitory effect of 20 % sucrose on both PsifA : : gfp and PsifB : : gfp expression, showing that the effect of sucrose is mainly osmotic (Fig. 5a
, upper panel, and 5b). SseB levels were also higher in the absence than in the presence of 20 % sucrose (Fig. 5c
).
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It has been shown that S. typhimurium reacts to a sudden increase in osmolarity by taking up large amounts of K+ from the environment (Kempf & Bremer, 1998). Jung et al. (2001)
showed that the EnvZ-autokinase activity of purified and reconstituted EnvZ is stimulated in the presence of increasing [KCl] and the amount of phosphorylated OmpR in the reconstituted signal cascade increases over time in the presence of KCl, suggesting that [K+] in the medium could act as the signal detected by OmpREnvZ when changes in osmolarity occur. Although the [K+] in the SCV is not known, [K+] in Staphylococcus aureus-containing vacuoles in neutrophils has been reported to be in the 200300 mM range (Reeves et al., 2002
). Therefore, we investigated if changes in [K+] affect the expression of sifA, sifB and sseB. However, no effects comparable to those caused by changes in osmolarity were detected. The increase of [KCl] from 5 mM to 200 mM caused only a minor increase in the level of SseB detected by immunoblotting (data not shown). No significant differences were observed in PsifA : : gfp or PsifB : : gfp expression either by flow cytometry or by immunoblotting using an anti-GFP antibody, at 5 mM or 200 mM KCl (data not shown). Despite these results, we cannot rule out a role for [K+] in mediating osmolarity-dependent activation of OmpREnvZ, since the extracellular increase of [K+] might not result in an increase in its uptake into the periplasm.
SsrAB and OmpREnvZ are both required to mediate the effect of acidic pH on SPI-2 TTSS gene expression
Flow cytometry and immunoblot experiments were performed to analyse the effect of pH on PsifA : : gfp and PsifB : : gfp expression, by comparing the expression of each gene in bacterial cultures grown in MgM medium at pH 7·5 or at pH 4·5. However, the results obtained were too variable to allow any conclusions to be drawn. On the other hand, levels of SseB were consistently higher in MgM medium at pH 4·5 than at pH 7·5 (Fig. 6c). The effect of pH on the expression of sifA : : gfp and sifB : : gfp was then analysed in infected macrophages, where acidification of the SCV can be prevented by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, which acidifies endosomal and lysosomal compartments (Cirillo et al., 1998
). PsifA : : gfp and PsifB : : gfp expression by intracellular wild-type bacteria was strongly inhibited in macrophages pre-treated with bafilomycin A1, as shown by immunofluorescence and flow cytometry, respectively (Fig. 6a, b
). Flow cytometric analysis showed that the expression of PsifB : : gfp was twofold higher in intracellular bacteria inside non-treated macrophages than inside those treated with bafilomycin A1 (Fig. 6b
). These results are in agreement with those obtained by Cirillo et al. (1998)
for vacuole acidification-dependent induction of ssaJ, sscB and spiA (ssaC) expression. The absence of detectable PsifA : : gfp expression by both microscopy and flow cytometry when the fusion construct was carried by intracellular ssrA, ompR, and ssrA ompR mutant bacteria prevented further analysis of this promoter.
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DISCUSSION |
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The main finding of this work is that the effects on SPI-2 gene expression caused by the absence of Ca2+ and low osmolarity are transmitted predominantly through SsrAB, whereas acidic pH is sensed by both SsrAB and OmpREnvZ (Fig. 7). Lee et al. (2000)
reported that the effect of osmolarity and acidic pH on ssaH transcription was decreased in an ompR mutant. In agreement with these results, we found that OmpREnvZ is required for the full expression of SPI-2 genes in response to these signals. Furthermore, this effect fully depends on a functional SsrAB system, since the phenotype of an ssrA ompR double mutant in all three conditions is identical to that of the ssrA single mutant. These results indicate that the effect of OmpREnvZ on the expression of SPI-2 genes, other than ssrAB, is not a result of direct binding of OmpR to their promoters but is indirect, probably as a consequence of its binding to the ssrAB promoter (Lee et al., 2000
).
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Whether SsrA can sense different environmental signals directly is an open question. It is formally possible that SsrB could be activated independently from SsrA. It is also possible that at least one of the signals is sensed by another sensorregulator and that this is then linked through SsrAB. This is the case for the PmrAB-regulated genes, whose expression is modulated by the [Mg2+] and [Fe3+] in the environment by a regulatory cascade of two-component regulatory systems, where the first one (PhoPQ) senses [Mg2+] and the second (PmrAB) senses [Fe3+] (Groisman, 2001). Supporting this notion, the expression of sifB : : gfp in MgM medium in an ssrA single or ssrA ompR double mutant strain remained eightfold higher than in LB, where sifB : : gfp expression is severely repressed (data not shown), suggesting that additional regulatory system(s) contribute to sifB expression. It was initially proposed that PhoPQ (Groisman, 2001
), required for intra-macrophage replication and systemic growth within the mouse (Fields et al., 1986
), could be involved in the control of SPI-2 gene expression (Deiwick et al., 1999
; Worley et al., 2000
). However, it has been recently shown that PhoPQ and the SPI-2 TTSS are functionally independent (Beuzón et al., 2001
; Miao et al., 2002
). One possible candidate is the transcriptional regulator SlyA, which is required for virulence and survival in macrophages (Libby et al., 1994
). slyA mutants are sensitive to oxidative products of the respiratory burst (Buchmeier & Libby, 1997
). Another candidate for the additional regulation of SPI-2 TTSS genes is the alternative sigma factor RpoE (
E). rpoE mutant strains are highly attenuated in mice (Humphreys et al., 1999
). Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant is defective in its ability to survive and proliferate in both cell lines (Humphreys et al., 1999
), and has also an increased sensitivity to the respiratory burst (Testerman et al., 2002
).
What is the physiological significance of the roles of Ca2+, low osmolarity and acidic pH in regulating SPI-2 TTSS gene expression? Using fluorescence lifetime imaging microscopy, it has been shown that inside macrophage lysosomes [Ca2+] is at least five times lower (400 µM) than the concentration outside the cell (2 mM) (Christensen et al., 2002). Although the [Ca2+] in the lumen of the SCV is unknown, the strong repression of SPI-2 TTSS gene expression observed in vitro at a [Ca2+] equivalent to that found outside the cell (2 mM) could explain the absence of SPI-2 gene expression in extracellular bacteria (Beuzón et al., 2000
). Low osmolarity represses SPI-1 TTSS gene expression (Bajaj et al., 1996
), whereas it induces SPI-2 TTSS gene expression in vitro (this work; Lee et al., 2000
). An opposite regulation by osmolarity on SPI-1 TTSS gene expression (expressed extracellularly and required for invasion) and SPI-2 TTSS (expressed intracellularly and required for intracellular replication) could help to ensure that these functionally distinct systems are expressed independently and only when and where they are required during the infection process. It has also been shown that the majority of vacuoles containing S. typhimurium acidify from pH 6·0 to between pH 4·0 and 5·0 within 60 min after formation (Rathman et al., 1996
). Although it is clear that acidic pH induces SPI-2 TTSS-mediated secretion in vitro, the effect of acidic pH on inducing SPI-2 TTSS gene expression has been controversial (Beuzón et al., 1999
; Deiwick et al., 1999
; Lee et al., 2000
; Miao et al., 2002
). In this study, we found it impossible to ascertain whether acidic pH has a significant effect on SPI-2 TTSS gene expression in vitro. However, in infected macrophages, acidic pH has a clear effect on PsifB : : gfp expression (Fig. 6
), confirming the requirement of vacuolar acidification for SPI-2 gene expression reported by Cirillo et al. (1998)
. If the effect of pH on SPI-2 TTSS expression is not as strong as its effect on secretion, or the effective range of action is very narrow, it is possible that, even in pH-controlled in vitro conditions, undetectable variations of pH from experiment to experiment could result in a lack of reproducibility which might explain the different results obtained (Deiwick et al., 1999
; Lee et al., 2000
; Miao et al., 2002
). In conclusion, [Ca2+], osmolarity and pH could account for SPI-2 TTSS intracellular expression in vivo. These signals are predominantly transmitted through SsrAB and may involve additional sensing proteins and regulators of the SPI-2 system.
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ACKNOWLEDGEMENTS |
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Received 4 April 2003;
revised 20 June 2003;
accepted 20 June 2003.