Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B. cereus food-poisoning strain

Julien Brillard1,{dagger} and Didier Lereclus1,2,{ddagger}

1 Institut Pasteur, Génétique et Physiologie des Bacillus Pathogènes, Département de Microbiologie Fondamentale et Médicale, 25 rue du Dr Roux, 75724 Paris cedex 15, France
2 INRA, Unité Génétique Microbienne et Environnement, La Minière, 78285 Guyancourt cedex, France

Correspondence
Didier Lereclus
lereclus{at}jouy.inra.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.


{dagger}Present address: UMR A408 ‘Sécurité et qualité des produits d'origine végétale’, INRA, Site Agroparc, 84914 Avignon cedex 9, France.

{ddagger}Present address: INRA – Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Bacillus cereus group consists of Gram-positive, rod-shaped, motile and spore-forming bacteria. The group includes B. cereus sensu stricto and the closely related species Bacillus anthracis, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus mycoides and Bacillus pseudomycoides (Daffonchio et al., 2000; Helgason et al., 2000; Nakamura, 1998). B. cereus is an opportunistic human pathogen, causing local and systemic infections, and is most often observed as a causative agent of food poisoning (Granum, 2001). Because of its endospore production, B. cereus is present in various natural environments, and may be isolated at a high frequency from various kinds of contaminated food products.

Food-borne diseases caused by B. cereus are classified as emetic and diarrhoeal syndromes. The emetic syndrome is due to one toxin, the emetic toxin (cereulide), which causes vomiting (Agata et al., 1995a). The diarrhoeal syndrome might be caused by several enterotoxins. The HBL enterotoxin is a three-component haemolysin that consists of one binding component (B) and two lytic proteins (L1 and L2) (Beecher & Wong, 1997). This toxin has haemolytic and dermonecrotic activities, and it increases vascular permeability and causes fluid accumulation in rabbit ileal loops (Beecher et al., 1995). Another important enterotoxin, NHE (non-haemolytic enterotoxin), is a three-component complex that was originally identified in a B. cereus strain responsible for a food-poisoning outbreak (Lund & Granum, 1996). Other enterotoxin genes, bceT, entFM, entS and entI, have also been described (Agata et al., 1995b; Asano et al., 1997). However, it was recently suggested that the bceT gene product does not contribute to food-borne diseases (Choma & Granum, 2002) and may in fact be a cloning artefact (Hansen et al., 2003). B. cereus produces several other secreted proteins, including phospholipases and proteases, that may contribute to B. cereus pathogenicity associated with food-borne diseases. The expression of most of these putative virulence factors is controlled by the pleiotropic transcriptional activator PlcR (Agaisse et al., 1999; Gohar et al., 2002). Its recognition site is believed to be a highly conserved palindromic sequence: TATGNAN4TNCATA. This global regulator has been shown to contribute to B. cereus virulence in mice, in insects (Salamitou et al., 2000) and in rabbit endophthalmitis (Callegan et al., 2003).

Cytotoxin K (CytK) may also be involved in B. cereus food poisoning. It was first characterized in B. cereus strain 391-98, a strain isolated from cases of food-borne disease and responsible for the death of three people (Lund et al., 2000). Interestingly, none of the other, commonly described, enterotoxin genes (for example hbl and nhe) was detected in this strain, further implicating CytK as a major virulence factor. However, several B. cereus isolates possess the cytK gene, as is the case for the hbl or nhe genes (Guinebretière et al., 2002). Thus, the presence of a particular gene presumably involved in virulence is not in itself sufficient to confer pathogenicity. Presumably, the transcription level of the gene is important in virulence.

The cytotoxin CytK is a pore-forming toxin that belongs to a family of {beta}-barrel channel-forming toxins (including Staphylococcus aureus leucocidins and Clostridium perfringens {beta}-toxin). It is necrotic and haemolytic (Lund et al., 2000), and also cytotoxic for intestinal epithelia and therefore probably causes diarrhoeal syndrome (Hardy et al., 2001). The cytK promoter region of B. cereus 391-98 contains a putative PlcR box which, however, does not conform to the consensus as currently defined (Lund et al., 2000).

To understand the strong pathogenicity of B. cereus strain 391-98, in which cytK is the only known enterotoxin gene (Lund et al., 2000), we studied the transcriptional activity of the cytK promoter region. We also found that PlcR regulated the cytK promoters of both this strain and the reference strain ATCC 14579 whether the PlcR box was the native sequence or carried mutations.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Strains and growth conditions.
The wild-type strain B. cereus ATCC 14579 (laboratory collection) was used as a reference strain. The wild-type strain B. cereus 391-98 (provided by M.-L. De Buyser, AFSSA, Maison-Alfort, France) is a clinical isolate that caused the death of three people in 1998 (Lund et al., 2000). It is the only known B. cereus food-poisoning strain lacking both hbl and nhe enterotoxin genes (Guinebretière et al., 2002). Two additional wild-type strains were used: B. thuringiensis 407Cry (Lereclus et al., 1989), an environmental strain pathogenic to insects; and B. cereus F4430, another clinical isolate (provided by M.-H. Guinebretière, INRA, Avignon, France). These two strains are known to carry the cytK gene, but also hbl and nhe enterotoxin genes. B. cereus ATCC 14579 {Delta}plcR (Salamitou et al., 2000), a PlcR-deficient kanamycin-resistant strain, was used to check for the PlcR dependence of expression of the cytK genes. Escherichia coli K-12 strain TG1 [{Delta}(lac–proAB) supE thi hsd-5 (F' traD36 proA+ proB+ lacIq lacZ{Delta}M15)] was used as a host for the construction of plasmids and for cloning experiments. E. coli strains SCS110 [rpsL (Strr) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44 {Delta}(lac-proAB) (F' traD36 proA+ proB+ lacIq lacZ{Delta}M15) (Stratagene) and ET12567 (F dam-13 : : Tn9 dcm-6 hsdM hsdR recF143 zjj-202 : : Tn10 galK2 galT22 ara14 pacY1 xyl-5 leuB6 thi-1) were used to generate unmethylated plasmid DNA for B. cereus transformation.

E. coli and B. cereus cells were routinely grown in Luria broth (LB) medium with vigorous agitation at 37 °C. The antibiotic concentrations used for bacterial selection were: 100 µg ampicillin ml–1 (for E. coli), 10 µg erythromycin ml–1 (for B. cereus) and 200 µg kanamycin ml–1 (for B. cereus). Bacteria with the Lac+ phenotype were identified on LB agar containing 40 µg X-Gal ml–1.

DNA manipulation.
Plasmid DNA was extracted from E. coli and B. cereus by a standard alkaline lysis procedure on QIAprep spin columns (Qiagen), with the following modification in the first step of the lysis procedure for B. cereus: incubation at 37 °C for 1 h with 5 mg chicken egg white lyzosyme (14 300 U mg–1). Chromosomal DNA was extracted from B. cereus cells harvested in mid-exponential phase as described previously (Msadek et al., 1990). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs). Oligonucleotide primers were synthesized by Proligo-Genset. PCR was performed in a GeneAmp PCR system 2400 thermal cycler (Perkin-Elmer), using Pwo DNA polymerase (Roche) or Pfx DNA polymerase (Invitrogen), both high-fidelity polymerases. Amplified DNA fragments were purified by using the QIAquick PCR purification Kit (Qiagen) and separated on 0·7 % agarose gels after digestion. Digested DNA fragments were extracted from agarose gels with a centrifugal filter device (montage DNA gel extraction kit; Millipore). All constructions were confirmed by DNA sequencing.

Electroporation was used to transform E. coli (Dower et al., 1988) and B. cereus (Lereclus et al., 1989) as previously described.

Construction of the cytK'–lacZ transcriptional fusion.
The cytK'–lacZ transcriptional fusions were constructed by cloning a BamHI/HindIII DNA fragment harbouring the putative cytK promoter between the BamHI and HindIII sites of pHT304-18'Z (Agaisse & Lereclus, 1994). The 402 bp (B. cereus ATCC 14579) and 381 bp (B. cereus 391-98) DNA fragments were generated by PCR amplification of chromosomal DNA with the primers Pc79-F and Pc79-R, and Pc98-F and Pc98-R, respectively (Table 1). The recombinant plasmids, designated Pc79'-Z (cytK promoter originating from B. cereus ATCC 14579) and Pc98'-Z (cytK promoter originating from B. cereus 391-98), were introduced into B. cereus ATCC 14579 wild-type and {Delta}plcR mutant strains by electroporation. The transformants were resistant to erythromycin (10 µg ml–1).


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Table 1. Primers

 
To create substitutions in the PlcR box, the regions upstream and downstream from the PlcR box of cytK were amplified using Pc79-M1-R and Pc79-F, and Pc79-M1-F and Pc79-R, respectively. These primers carry the desired mutations (see Table 1). After amplification, a mixture of the two DNA fragments was subjected to a further 20 cycles of amplification with additional polymerase and dNTPs. This final fragment was then purified, digested with BamHI and HindIII and inserted between the corresponding sites of pHT304-18'Z, leading to Pc79M1'-Z (Fig. 1). Similar experiments were performed to create a double substitution in cytK PlcR box, leading to Pc79M2'-Z. Pc98M1'-Z and Pc98M2'-Z were also constructed to create a single or a double substitution, respectively, in the cytK PlcR-box region from B. cereus strain 391-98 (Fig. 1). All the DNA fragments were verified by DNA sequencing (GenomeExpress).



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Fig. 1. Schematic representation of PlcR box regions from B. cereus ATCC 14579 and B. cereus 391-98 used for lacZ transcriptional fusions, with or without substitutions in the PlcR recognition sites. The previously defined consensus is underlined. Hatched boxes and doted boxes represent the cytK promoter region of strain ATCC 14579 and 391-98, respectively. The names of the plasmids harbouring these DNA fragments are indicated. The positions of the primers used in this study are indicated by arrows.

 
{beta}-Galactosidase assay.
Cells of B. cereus strains harbouring plasmids with lacZ transcriptional fusions were cultured in LB medium in the absence of antibiotics at 37 °C with vigorous shaking. {beta}-Galactosidase activity was followed in three to five independent cultures for each construction. {beta}-Galactosidase specific activities were measured in triplicate samples from each culture as described previously (Msadek et al., 1990) and are expressed in units of {beta}-galactosidase per mg protein (Miller units). Total proteins in the sample were determined using the Bradford method (Bio-Rad protein assay).

Mean values of {beta}-galactosidase activity measured 2 h after the entry in stationary phase of growth for three to five distinct cultures were analysed by the Student t-test in order to determine P values for differences in expected values versus actual values.

Mapping of the 5' end of cytK mRNA by primer extension.
Total RNA was extracted from wild-type B. cereus ATCC 14579 and 391-98 cells grown in LB at 37 °C with shaking, as described previously (Agaisse & Lereclus, 1996). The cytK transcription start site of both strains was determined by primer extension using ExtsnCytK79 and ExtsnCytK98 oligonucleotides (Table 1), as described previously (Agaisse & Lereclus, 1996). DNA sequencing was performed by the dideoxy chain-termination method with the primers ExtsnCytK79 and ExtsnCytK98, and using the corresponding PCR product as the template, with the T7 sequenase PCR product sequencing kit (USB).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Kinetics of cytK transcription
We constructed fusions between cytK-promoter regions from strains 391-98 and ATCC 14579 and the lacZ reporter gene to determine their levels of transcription (see Fig. 1 and Methods). During B. cereus growth, cytK transcription began at the onset of the stationary phase of growth (T0), reaching a maximum about 2 h after T0 (T2) (Fig. 2); this general pattern applied for the promoters from both strains. In experiments with four independent cultures, the mean (±SE) {beta}-galactosidase activity at T2 was 1393±234 Miller units for Pc79'-Z and 2705±383 Miller units for Pc98'-Z (Fig. 3a, b). The expression level was significantly higher (P<0·001, Student's t-test) for Pc98'-Z (cytK-promoter region from B. cereus strain 391-98). Thus, in the B. cereus reference strain, Pc98'-Z is twice as strong a promoter as Pc79'-Z.



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Fig. 2. Expression of B. cereus ATCC 14579 and B. cereus 391-98 cytK genes in strain ATCC 14579. Filled triangles, expression of Pc98'-Z. Filled circles, expression of Pc79'-Z. Open symbols, OD600 of cultures of bacteria carrying the corresponding plasmids. Cells were grown at 37 °C in LB medium. Time zero indicates entry into stationary phase. Standard deviations of triplicate measurements are shown for {beta}-galactosidase activity. Graphical representation of one experiment, representative of four independent experiments.

 


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Fig. 3. Transcriptional activity of cytK promoter regions from B. cereus ATCC 14579 (a) or from B. cereus 391-98 (b). {beta}-Galactosidase activity of cytK'–lacZ fusions was measured in B. cereus ATCC 14579 2 h after entry into the stationary phase. Mean values±standard deviations were calculated on three to five independent experiments. For Fig. 3(a), asterisks indicate that the mean values were significantly different from Pc79'-Z (P<0·001) according to Student's t-test.

 
Unfortunately, experiments could only be performed in the strain ATCC 14579. All attempts to transform B. cereus 391-98 failed despite various conditions used. However, similar differences between the levels of expression of the two promoters were also observed in two other strains. Indeed, in the clinical strain B. cereus F4430, the mean (±SE) {beta}-galactosidase activity at T2 was 229±37 Miller units for Pc79'-Z and 1250±285 Miller units for Pc98'-Z (the difference between the data is significant, P<0·001, Student's t-test). In the environmental strain B. thuringiensis 407Cry, also belonging to the B. cereus group, the {beta}-galactosidase production was 1197±84 Miller units for Pc79'-Z and 1539±197 Miller units for Pc98'-Z. In this last strain, the difference between the data is significant (P<0·05, Student's t-test), although to a lesser extent than in the two B. cereus strains. Altogether these results suggest that the difference of expression between Pc79'-Z and Pc98'-Z is not strain specific.

Expression of cytK is regulated by PlcR
The B. cereus 391-98 cytK promoter region contains a putative PlcR box (Fig. 1): TATGCAATTTCGCATA (the underlined nucleotides are the most highly conserved in all the PlcR boxes) (Lund et al., 2000). However, this sequence is not palindromic and does not conform to the consensus as currently defined, TATGNAN4TNCATA (Agaisse et al., 1999). In contrast, the cytK promoter in B. cereus strain ATCC 14579 contains a PlcR recognition site conforming to the consensus (Fig. 1).

To determine whether this divergence of the PlcR box has an effect on regulation by PlcR, we introduced the cytK transcriptional fusions into the B. cereus ATCC 14579 {Delta}plcR mutant. No transcriptional activity above 10 Miller units was detected for either Pc79'-Z or Pc98'-Z (data not shown). This indicates that cytK transcription is PlcR-dependent in both cases.

To elucidate the role played by the PlcR box in the difference of cytK expression between strains, we introduced substitutions as shown in Fig. 1. {beta}-Galactosidase expression was followed in three cultures for each of these constructs (mean values at T2 are presented in Fig. 3). Both the single point mutation (Pc79M1'-Z) and the exchange of two adjacent nucleotides (Pc79M2'-Z, such that the sequence is the same as that of the PlcR box in Pc98'-Z) in the PlcR box of cytK promoter region from B. cereus strain ATCC 14579 substantially reduced, but did not abolish, cytK expression (Fig. 3a).

In contrast, restoration of the consensus as currently defined for the PlcR box of Pc98'-Z (Pc98M1'-Z) and substitution with a PlcR box similar to that found in the reference strain (Pc98M2'-Z) did not significantly affect expression (Fig. 3b).

cytK promoter characterization
Primer extension experiments were performed in both strains to determine the cytK transcription start point (Fig. 4). We then determined the putative –10 and –35 {sigma}A boxes of both cytK promoter regions (Fig. 5). The putative –10 {sigma}A boxes in the two strains are identical, and are in accordance with the –10 {sigma}A consensus of Bacillus subtilis. However, the –35 {sigma}A boxes of the two cytK promoter regions are different. The –35 {sigma}A region of the cytK promoter from B. cereus strain 391-98 is very similar to the consensus for B. subtilis but that from B. cereus ATCC 14579 is not (Fig. 5).



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Fig. 4. Determination of the transcriptional start site of cytK originating from B. cereus ATCC 14579 and B. cereus 391-98. Cells were grown at 37 °C in LB medium and harvested 1 h after entry into the stationary phase. Total RNA (80 µg for B. cereus ATCC 14579 or 3 µg for B. cereus 391-98) was subjected to primer extension analysis using oligonucleotides ExtsnCytK79 or ExtsnCytK98, respectively. The same oligonucleotides were used to prime dideoxy sequencing reactions from the corresponding regions obtained by PCR amplification (lanes C, T, A, G). Positions of the transcriptional start sites are indicated by an asterisk.

 


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Fig. 5. Alignment of the cytK promoter regions from B. cereus ATCC 14579 and B. cereus 391-98. PlcR recognition sites are boxed. Transcription start sites are indicated in bold underlined letters (beside the arrows) and putative RBSs are indicated in bold letters. Putative –10 and –35 {sigma}A recognition sites are underlined, and the consensus sites for B. subtilis are given below.

 
The primer extension experiment also revealed that the abundance of cytK mRNA differs between the two strains. The quantities of total RNA used for the primer extension experiment (Fig. 4) were 3 µg for B. cereus 391-98 and 80 µg for B. cereus ATCC 14579, and the signal for the 5'-end of cytK mRNA from B. cereus 391-98 was stronger. The findings were similar in two repeat experiments. Thus, this semi-quantitative approach indicates that the cytK mRNA signal is much stronger in the clinical strain 391-98.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cytotoxin CytK was first characterized from the B. cereus strain 391-98 (Lund et al., 2000). None of the other enterotoxin genes commonly found (hbl, nhe) have been detected in this strain, and it is therefore likely that CytK production or overproduction caused the severe diarrhoeal syndrome observed during the outbreak. Our results indicate that cytK transcription detected in the reference strain is twofold higher with the promoter from B. cereus strain 391-98 than with that from B. cereus ATCC 14579 (Fig. 2). The amount of cytK mRNA in the clinical isolate is also much higher than that in the reference strain ATCC 14579, as observed during primer extension experiments (Fig. 4). Thus, the transcriptional activities of cytK as assessed using lacZ fusions in the reference strain seem to be underevaluated compared to the cytK transcription in the clinical strain. This might be due to a difference in PlcR expression or activity between the two strains.

This difference in cytK transcription is in accordance with the greater amount of CytK detected in the extracellular proteome of B. cereus 391-98 (6·9 % of total exported protein; M. Gohar, personal communication) than that of ATCC 14579 (0·4 %) as previously reported (Gohar et al., 2002). However, this high CytK production is not a general characteristic of clinical B. cereus isolates. Indeed, the amount of CytK protein was determined by proteomic approaches in a set of 25 B. cereus strains of various origins (clinical and non-clinical strains). In the 391-98 strain, CytK amount is at least three times higher than in the other strains (M. Gohar & R. Gravelline, unpublished results).

Lund et al. (2000) have shown that in B. cereus 391-98, the cytK promoter region contains a putative PlcR box. However, this sequence did not match the currently defined consensus: TATGNAN4TNCATA (Agaisse et al., 1999; Økstad et al., 1999). In contrast, the cytK promoter region in B. cereus ATCC 14579 contains a PlcR recognition site that matches the consensus. The kinetics of expression from the two promoter regions are characteristic of PlcR-regulated genes (Fig. 2), and in both cases, expression was shut off in a PlcR-deficient mutant. This indicates that cytK transcription is PlcR-dependent in the two B. cereus strains. Thus, the PlcR box in the cytK promoter region in B. cereus 391-98 is functional, despite diverging from the currently defined consensus.

Our identification of a non-palindromic PlcR box is consistent with what has been found for the inhA2 gene, which has also been shown to be PlcR regulated, despite not conforming to the currently defined consensus (Fedhila et al., 2003); the divergence in the inhA2 gene is the first nucleotide of the PlcR box. Thus, the PlcR recognition-site consensus is not necessarily palindromic and PlcR may recognize divergent sequences. Consequently, the PlcR regulon of B. cereus is probably much larger than that proposed by Ivanova et al. (2003).

Introduction of substitutions into the PlcR box of cytK promoter region from B. cereus strain ATCC 14579 reduced significantly, but did not abolish, expression from the cytK promoter (Fig. 3a). The standard PlcR box found in the promoter region of cytK from ATCC 14579 is therefore more efficient than the one containing substitutions. However, restoration of the consensus in the PlcR box of Pc98'-Z did not significantly change the expression level (Fig. 3b). This indicates that the higher transcription from the promoter region of cytK from 391-98 is not due to the sequence of the PlcR box. The difference in transcriptional activity between the two cytK promoter regions may be due to the differences between –35 {sigma}A boxes.

In conclusion, our work indicates that B. cereus 391-98, responsible for an infectious outbreak, produces large amounts of cytotoxin CytK due to high levels of transcription. This high CytK expression may account for the high virulence of strain 391-98. However, there is only 88 % of identity between the CytK amino acid sequence of the two strains. We therefore can not rule out that such differences may also play a role in cytotoxic activity. Finally, we also found that the PlcR recognition site is more heterogeneous than previously believed, allowing the possibility to identify other PlcR-regulated virulence factors.


   ACKNOWLEDGEMENTS
 
We thank Michel Gohar for communicating results concerning the B. cereus 391-98 proteome, and for critical reading of the manuscript. We also thank V. Broussolle and C. Lavire for helpful discussion. J. B. was funded by a grant ‘Aliment-Qualité-Sécurité: Caractérisation de la virulence de Bacillus cereus from the Ministère de la Recherche and the Ministère de l'Agriculture et de la Pêche.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 28 January 2004; revised 21 April 2004; accepted 21 May 2004.



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