1 Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6
Correspondence
Derek W. Hood
dhood{at}molbiol.ox.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF503507.
Present address: National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
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INTRODUCTION |
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Disruption of the lex2B gene in strain DL42 led to loss of reactivity of this strain to an LPS-specific monoclonal antibody (mAb) 5G8. A correlation between repeat numbers within lex2A and mAb 5G8 reactivity was found (Jarosik & Hansen, 1994) but the specific function of the lex2 locus has not been elucidated since both the structure of the LPS of this strain and the composition of the epitope reactive with mAb 5G8 are unknown. In the present study we investigate the role of the lex2 locus in H. influenzae RM7004 and RM153, strains for which the details of the LPS structure are known (Masoud et al., 1997
, 2003
), and elucidate the function of Lex2B.
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METHODS |
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Southern blot and colony blot analyses were performed as described by Sambrook et al. (1989) using Hybond-N nylon membranes (Amersham). Following blotting, the membranes were incubated at 65 °C for 23 h in hybridization buffer as described by Sambrook et al. (1989)
but lacking formamide. Hybridizations, with added labelled DNA probe, were performed overnight at 65 °C and filters were subsequently washed twice in 2x SSC/0·1 % SDS for 15 min at 65 °C then once in 0·2x SSC/0·1 % SDS for 15 min at 65 °C.
PCR amplification was carried out in buffer containing 50 mM KCl, 10 mM Tris/HCl pH 8, 0·01 % (w/v) gelatin and 2·5 mM MgCl2. Thirty cycles of PCR were performed, each cycle consisting of 1 min periods of denaturation at 94 °C, annealing at 47 °C and extension at 72 °C.
Cloning and DNA sequence analysis of the lex2 locus.
The 801 bp PCR product amplified from chromosomal DNA of H. influenzae strain RM7004 using primers lex5' and lex3', designed from the published lex2 sequence of strain DL42 (GenBank U05670) (Table 2), was cloned into pCR2.1 (Invitrogen), generating plasmid pCR2.1lex2. The 432 bp SspIRsaI fragment corresponding to an internal fragment of lex2B was purified and labelled with [
-32P]dCTP (Amersham Pharmacia), by random-primed labelling (Boehringer Mannheim). This hybridization probe was used for Southern analysis of chromosomal DNA of strains RM7004 and RM153, yielding signals on the blot corresponding to band sizes of 1·9 kb and 5·5 kb for EcoRI- and PstI-digested DNA respectively. The corresponding fragments were cloned by recovering DNAs of the appropriate sizes from an agarose gel and ligating into EcoRI- or PstI-digested, dephosphorylated pBluescript SK- (Stratagene) then transforming into E. coli. The transformants obtained were screened by colony hybridization, using the 432 bp lex2B probe. DNA was prepared from positively hybridizing colonies and verified by restriction digestion. Clones containing the 1·9 kb EcoRI insert were designated pB7004Elex2 and pB153Elex2 and clones containing the 5·5 kb PstI fragment from strain RM7004 were designated pB7004Plex2.
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For direct sequencing of 5'-GCAA repeat tracts in lex2A, DNA fragments generated by PCR amplification with a biotinylated form of primer lex2for and primer lex3' (Table 2) were denatured in 150 mM NaOH and single-stranded DNA was purified by the use of streptavidin-coated Dynabeads (Dynal) as described by High et al. (1993)
. The DNA obtained was sequenced by the dideoxy chain termination method using a modified T7 polymerase (Sequenase II kit: United States Biochemical Corp.) and primer lex2seq (Table 2
).
DNA sequences were collated and analysed using Chromas (Conar McCarthy, Australia) and the GCG GAP program (Devereux et al., 1984). Sequence homologies were determined using the GenBank DNA and protein sequence databases through the National Centre for Biotechnology Information BLAST network server (Altschul et al., 1997
).
Generation of a lex2B chromosomal mutant in H. influenzae.
A kanamycin-resistance (kanR) cassette, released by digestion of pUC4K (Pharmacia) with HincII, was inserted at a unique site cut by StyI then end-filled with Klenow enzyme and dNTPs (Sambrook et al., 1989), located 209 bp downstream of the initiation codon of lex2B in plasmid pCR2.1lex2. This knockout construct was transformed into H. influenzae strains RM7004 and RM153; correct transformants were confirmed by PCR amplification and Southern analysis.
Construction of lex2lacZ fusion reporter strains.
To engineer lacZ fusion constructs the following oligonucleotides were used with restriction sites (underlined) incorporated (HindIII site in PR1, KpnI site in PR2 and PR3, XhoI site in PR4 and PR6, EagI site in PR5 and PR7): PR1 (5'-TATCCCAAGCTTGATTCAGTTTGGTTTGCAGGA-3'), PR2 (5'-TCGGGGTACCTGATGTACTATTTAAGTCACTCT-3'), PR3 (5'-TAGTCGGGGTACCAACATAATCTCTCCATAGTT-3'), PR4 (5'-AATGCACTCGAGGAGATTATGTTTATTACACCT-3'), PR5 (5'-GCGTGCCGGCCGTTTCTCATAAATAGTTGATTC-3'), PR6 (5'-AATGCACTCGAGATAGTAGACAAAGCAGTTG-3') and PR7 (5'-GCGTGCCGGCCGAGCAAAATCTCAAGGTAGAG-3'). A previously described plasmid vector, pGZ-MCS (De Bolle et al., 2000), was used as a source of the lacZ gene for reporter gene constructs. PR1 and PR2 were used to amplify a region of chromosomal DNA from strain RM7004 from 547 bp upstream of the initiation codon of lex2A (within purL) to the nucleotide immediately adjacent to and downstream of the final repeat of lex2A. The 741 bp PCR product was digested with HindIII/KpnI then cloned into HindIII/KpnI-digested pGZ-MCS to make clone pGZPr1.2. The 3' end of the KpnI site in pGZ-MCS forms the second codon of the lacZ gene. PR4 and PR5 were used to amplify by PCR a region of chromosomal DNA from strain RM7004 from 6 bp downstream of the stop codon of lex2A to 93 bp upstream of the stop codon of lex2B. The 678 bp PCR product digested with EagI/XhoI was ligated into EagI/XhoI-digested pGZPr1.2 generating clone pGZPr1.2.4.5 The kanR gene derived from pUC4K by digestion with BamHI was cloned into the BamHI site of pGZPr1.2.4.5, located immediately downstream of lacZ and upstream of the XhoI site. The clones obtained, designated pGZPr1.2.4.5k, were verified by DNA sequencing.
A second construct was prepared in a similar manner to pGZPr1.2.4.5k with lacZ fused in-frame with the ATG initiation codon of lex2B. PR1 and PR3 were used to amplify by PCR a region of chromosomal DNA from strain RM7004 from 547 bp upstream of the initiation codon of lex2A to 2 nucleotides downstream of the initiation codon of lex2B. The 872 bp HindIII/KpnI-digested PCR product was cloned into HindIII/KpnI-digested pGZ-MCS, generating clone pGZPr1.3. PR6 and PR7 were used to amplify by PCR a region of chromosomal DNA from strain RM7004 from 16 bp downstream of the stop codon of lex2B to 62 bp downstream of the initiation codon of HI0755. The 671 bp XhoI/EagI-digested PCR product was cloned into XhoI/EagI-digested pGZPr1.3. The kanR gene was inserted into the BamHI site in an identical manner to that described above to give clone pGZPr1.3.6.7k.
Constructs pGZPr1.2.4.5k and pGZPr1.3.6.7k were transformed into strain RM7004 to give strains RM7004lex2AlacZ and RM7004lex2BlacZ respectively.
Construction of non-polar lex2A mutant.
A lex2A construct with the repeats removed, leaving a single BglII restriction enzyme site and retaining the majority of the reading frame in-frame with the start codon, has been engineered as described elsewhere (De Bolle et al., 2000). To interrupt the lex2A reading frame, the BglII site was digested, the ends were filled by Klenow enzyme and dNTPs (Sambrook et al., 1989
), then the plasmid was religated. This procedure adds 4 bp to the reading frame, making a frameshift mutation. The construct, pRM7004
GCAAlex2AEF, was transformed into strain RM7004 to produce strain RM7004
GCAAlex2A- containing a non-polar mutation of lex2A.
RNA isolation and transcript analysis.
Total RNA was isolated from strain RM7004 using the Promega SV Total RNA Isolation kit as detailed by the supplier. An extra DNase treatment was included as the last step of the isolation procedure. cDNA was prepared from 0·5 µg RNA using MLV reverse transcriptase (Gibco-BRL) and random primers (Promega) under conditions recommended by the supplier. PCR was performed under the conditions described above and using 1 µl reverse-transcribed (RT) RNA as template. Controls were carried out using primers frdBup (5'-CTTATCGTTGGTCTTGCCGT-3') and frdBdwn (5'-TTGGCACTTTCCACTTTTCC-3') to amplify frdB, a constitutively expressed metabolic gene.
LPS analysis.
For colony immunoblotting, a single colony of the appropriate H. influenzae strain was diluted in PBS and dilutions spread on agar plates to obtain single colonies. Following overnight growth, colonies were transferred to nitrocellulose membranes then incubated with hybridoma culture supernatants containing LPS-specific mAbs 4C4 or 5G8 (provided by E. J. Hansen, University of Texas), as described by Roche et al. (1994).
For gel fractionation and Western immunoblotting, whole-cell lysates were prepared from overnight cultures as described by Serino & Virji (2000). The lysates were fractionated by Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis (T-SDS-PAGE) (Lesse et al., 1990
) and LPS profiles were detected by staining with silver (Quicksilver; Amersham) (Roche et al., 1994
). LPS was transferred to nitrocellulose membranes for Western analysis (Towbin et al., 1979
) then incubated with mAb 4C4 as described by Weiser et al. (1997)
.
Determination of LPS structure.
Water-insoluble LPS was extracted using the hot-aqueous phenol method of Westphal & Jann (1965) and O-deacylated LPS was then prepared for analysis by ESI-MS on a VG Quattro mass spectrometer (Micromass) as described by Masoud et al. (1997)
. NMR analysis was carried out as described by Lysenko et al. (2000)
.
Investigation of phase variation of strains RM7004lex2AlacZ and RM7004lex2BlacZ.
Three single blue colonies of the appropriate strain from a fresh plate were independently serially diluted in PBS to obtain between 500 and 1000 c.f.u. per plate. After growth on plates including X-Gal, one single blue and one single white colony derived from each of the three founder colonies were isolated then serially diluted in PBS to yield approximately 1500 c.f.u. per plate. The proportion of blue and white variants on each plate was established.
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RESULTS |
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The chromosomal regions encompassing the lex2 locus in H. influenzae strains RM7004 and RM153 were cloned. The 1·9 kb EcoRI fragments in plasmids pB7004Elex2 and pB153Elex2 were sequenced for each strain (Fig. 2). The lex2A and lex2B ORFs of strain RM7004 (GenBank AF503507) were 282 bp and 745 bp in length respectively, separated by 13 bp of intergenic sequence. A single putative promoter sequence was found upstream of lex2A and sequences exhibiting partial hyphenated dyad symmetry, which may play a role in transcriptional termination, were identified downstream of the putative translational termination codon of lex2B, as reported previously (Jarosik & Hansen, 1994
). These features suggest that lex2A and lex2B may form an operon. The nucleotide sequences of lex2 from our serotype b strains and strain DL42 were more than 99 % identical. However, 12 and 20 5'-GCAA repeats were identified in lex2A, 65 bp downstream of the initiation codon, of strains RM7004 and RM153 respectively, instead of the 18 repeats found in strain DL42. Thus, strain RM7004 contained a permissive, and strain RM153 a non-permissive, number of repeats for correct translation of lex2A. Comparisons of the deduced amino acid sequences of lex2A and lex2B with those of other proteins in the public databases revealed sequence homology for Lex2B only. Lex2B showed similarity to a number of glycosyltransferase enzymes from other organisms, the highest homology being 60 % similarity to Lob1 from Haemophilus somnus (Inzana et al., 1997
).
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The lex2 locus contained 71 mol% A+T (including the 5'-GCAA repeat region), in contrast to genes HI0753 and HI0754 which, like the mean composition of the Rd genome sequence, contain 61 mol% A+T. This would be consistent with a hypothesis that the lex2 locus has been laterally transferred from another species and has integrated into the genome by homologous recombination with the resultant loss of HI0753 and HI0754.
Transcription and translation of lex2A and lex2B
The position of lex2A and lex2B after DNA sequence analysis predicted that the two genes would be co-transcribed. Using primer pairs lex5'/lex3' and oligo1/lex3', weak but reproducible bands (0·8 kb and 0·4 kb respectively) were obtained by RT-PCR of RNA isolated from strain RM7004 (data not shown), confirming that the two genes are transcribed together. No bands were obtained in PCR controls using total RNA as a template.
To investigate the possible interdependence of translation between the closely sited reading frames within the lex2 locus, translational fusions of both lex2A and lex2B to a -galactosidase (lacZ) reporter gene were constructed. In plasmid pGZPr1.2.4.5k, the lacZ gene was fused in-frame with the twelfth and final repeat of the 5'-GCAA tract of the strain RM7004 lex2A sequence. The lacZ ORF could only be translated from the lex2A initiation codon (Fig. 2B
). Plasmid pGZPr1.3.6.7k was prepared with the lacZ gene fused in-frame with, and adjacent to, the initiation codon of lex2B (Fig. 2B
). The plasmid constructs, pGZPr1.2.4.5k and pGZPr1.3.6.7k, were then used to transform strain RM7004 to generate strains RM7004lex2AlacZ and RM7004lex2BlacZ respectively. Several independent blue or white colonies of transformed strains RM7004lex2AlacZ and RM7004lex2BlacZ after growth on media containing X-Gal were each dispersed then replated. The former strain showed clear evidence of phase variation. The mean proportion of white variants generated from a blue founder colony was calculated to be 1 in 571 whilst the mean proportion of blue variants generated from a white founder colony was 1 in 1263. Upon plating, strain RM7004lex2BlacZ gave rise to only blue colonies. No white phase variants were detected in 10 000 colonies screened, consistent with lex2B being translated independently of lex2A.
Analysis of lex2B function
lex2B, encoding a predicted glycosyltransferase, was inactivated in strains RM7004 and RM153 to give mutants RM7004lex2B and RM153lex2B respectively. Upon immunoblotting, colonies of both mutants and the corresponding wild-type strains demonstrated no reactivity with mAb 5G8. This is contrary to the variable reactivity in colony phenotype demonstrated by other strains when reacted with mAb 5G8, in previous studies (Jarosik & Hansen, 1994). Differences in the pattern of reactivity between strains were observed with mAb 4C4; this antibody binds a digalactoside moiety on H. influenzae LPS (Virji et al., 1990
). The majority of colonies of wild-type strain RM7004 (>98 %) reacted (R) with mAb 4C4 and a negative (off; O) phenotype was represented at a level of less than 1 % (Fig. 3
A). Occasional stronger-reacting (S) colonies were also observed at a frequency of less than 0·5 %. Colonies of strain RM7004lex2B, derived from several independent transformants, showed only the R and O phenotypes upon reaction with mAb 4C4, with the majority of colonies being of the R phenotype (Fig. 3B
). A majority of colonies of both strains RM153 and RM153lex2B were of the O phenotype when reacted with mAb 4C4 and 0·2 % were of an R phenotype (Fig. 3C, D
). Thus, insertional inactivation of lex2B in strain RM7004 abolished the ability of this strain to generate the LPS phenotype, allowing the highest levels of mAb 4C4 binding, presumably as a result of reduced digalactoside incorporation into the LPS.
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Analysis of the role of the lex2A gene
Strain RM7004lex2AlacZ contains a mutated lex2A gene through deletion of a majority of the reading frame and insertion of the lacZ gene but retains an intact lex2B gene. SDS-PAGE and colony immunoblot analyses showed that the LPS from this strain was equivalent to that of strain RM7004lex2B (Fig. 4A). Upon reacting with mAb 4C4, no S variants of RM7004lex2AlacZ were detected amongst 10 000 colonies screened. Due to the close proximity of the two genes, there remained a possibility that there was a polar effect from the disrupted lex2A gene on transcription of lex2B. Localized disruption of the coding sequence of lex2A was introduced into strain RM7004 using plasmid pRM7004
GCAAlex2AEF. This construct was generated by replacing the repeat tract of lex2A with a BglII site then digesting with BglII and filling the recessed ends to introduce a frameshift once religated. This renders lex2A out of frame whilst minimizing any direct polar effect on lex2B transcription. Removal of the 5'-GCAA repeats from lex2A does not alter lex2 function (data not shown). Upon SDS-PAGE (Fig. 4
) and colony immunoblotting (data not shown), LPS from this strain, RM7004
GCAAlex2A-, behaved in an equivalent manner to that of strain RM7004lex2AlacZ described above. These data were consistent with our proposal that lex2A is somehow necessary for the phenotypic expression of Lex2B activity.
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DISCUSSION |
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Mutation of lex2B in strain RM7004 resulted in the synthesis of truncated LPS that was probably altered in its digalactoside content, as shown by the reduction in the level of reactivity of glycoforms to mAb 4C4. ESI-MS analysis of the LPS of strain RM7004lex2B confirmed the loss of three hexose sugars. The preferred order of LPS oligosaccharide biosynthesis in strain RM7004 is substitution of each heptose by a single hexose sugar followed by addition of further hexoses from the second heptose to complete a Gal--1-4-Gal-
-1-4-Glc-
-1-4-Glc-
extension (Masoud et al., 2003
; J. C. Richards, unpublished). This occurs before adding Gal-
-1-4-Gal-
-1-4-Glc-
-1 to the
-glucose linked to the first heptose to complete the fully extended LPS (Masoud et al., 2003
). NMR analysis of LPS from the lex2B mutant confirmed that the terminal globoside trisaccharide was absent from the HepI oligosaccharide extension, and that the glucose linked to the first heptose was now a terminal residue. Thus, we have convincing evidence for the role of Lex2B as the glycosyltransferase that adds the second
-glucose to the oligosaccharide extending from the first heptose of the LPS of strain RM7004.
lex2 is the fifth of the published H. influenzae phase-variable LPS-associated loci to have its function elucidated; a majority of these genes encode glycosyltransferases. In the original study by Jarosik & Hansen (1994), the function of the lex2 locus was correlated with phase-variable reactivity of bacteria with mAb 5G8. In the present study, we did not find a useful correlation between our strains and binding of this antibody. However, mAb 4C4, previously reported to bind a digalactoside of H. influenzae LPS (Virji et al., 1990
), did show strain-dependent phase-variable expression of LPS epitopes dependent upon lex2 activity. The generation of more than one mAb 4C4-reactive phenotype for strain RM7004 noted in this study and reported by Roche et al. (1994)
until now has not been understood. Our findings lead us to propose that the common reactive phenotype (R) results from the binding of mAb 4C4 to predominantly one digalactoside, presumed to be that extending from the second heptose, and that the S phenotype is generated by the binding of mAb 4C4 to cells predominantly expressing both digalactosides extending from the first and second heptose sugars. In a large population of cells only a minority of glycoforms of strain RM7004 include two digalactosides (Masoud et al., 2003
), presumably due to phase variation and other microheterogeneity associated with H. influenzae LPS synthesis. In contrast to strain RM7004, only a minority of variant colonies of strain RM153 react with mAb 4C4. Since lex2A is maintained predominantly out of frame in strain RM153, extension beyond the glucose attached to HepI is prohibited in this strain. Indeed, we have shown that strain RM153 variant colonies that gain reactivity to mAb 4C4 and contain a lex2A gene with an in-frame number of repeats (21 copies of 5'-GCAA) exhibit an LPS profile upon SDS-PAGE that resembles that of strain RM7004 (data not shown).
Previous studies have indicated that digalactoside expression on LPS enhances the ability of H. influenzae to escape the bactericidal activity of serum in in vitro assays, and facilitates intravascular survival in the infant rat model of infection (Kimura & Hansen, 1986; Kimura et al., 1987
; Cope et al., 1991
; Maskell et al., 1992
; Hood et al., 1996
). We anticipate that organisms expressing lex2, facilitating the extension of a second digalactoside on the LPS, may be selected for during systemic infection. The digalactoside can mimic antigens found on the surface of a number of host cell types. Thus, the conserved presence of lex2 in serotype b strains may contribute to the virulence of these strains, those most frequently causing systemic and meningeal infection (Turk & May, 1967
). Organisms contained in the cerebrospinal fluid of newly diagnosed cases of meningitis showed a majority (>99 %) of organisms binding mAb 4C4 compared to only <0·1 % when organisms were cultured in vitro (Weiser et al., 1989b
). The importance to the organism of the digalactoside in the globoside extension from HepI might be indicated by the emphasis placed on the switching of its expression. Its assembly evidently requires the expression of three independent phase-variable loci, lex2, lic2A and lgtC, each adding sequential sugar residues.
Lex2B may also compete with alternative steps in LPS biosynthesis. Recently, a novel sialylated lacto-N-neotetraose structure has been reported in H. influenzae strains that contain the lex2 locus (Cox et al., 2002). The terminal four sugars of this structure [
-Neu5Ac-(2-3)-
-D-gal-(1-4)-
-D-glcnac-(1-3)-
-D-Gal] are an alternative addition to the same
-glucose attached to HepI.
What is unclear about the lex2 locus is the relationship between the glycosyltransferase, Lex2B, and its phase-variable expression apparently mediated by changes in the repeat numbers within lex2A. It had been previously postulated that the function of lex2B depends upon the successful translation of lex2A (Jarosik & Hansen, 1994). A functional relationship between the two genes has been confirmed by evidence that they are co-transcribed. It has been shown in Helicobacter pylori that expression of adjacent co-transcribed genes can co-phase vary through transcription/translational coupling (de Vries et al., 2002
). However in our study, the separate in-frame fusions of the lacZ gene to lex2A and lex2B in strain RM7004 indicated that these two genes are translated independently of each other. The LPS from strain RM7004lex2AlacZ appeared different from wild-type and equivalent to that examined in detail from strain RM7004lex2B. This alteration in LPS structure is unlikely to be due only to a polar effect of the lex2A mutation on lex2B as introduction of a non-polar mutation in lex2A resulted in the same LPS phenotype as observed for strain RM7004lex2B. It remains a possibility that translation of lex2B is influenced by translation of lex2A through reinitiation of translation at the initiation codon of lex2B by ribosomes that have completed translation of lex2A, or that message stability is altered through failure to translate the first reading frame. However, the uniform colouration of blue colonies of strain RM7004lex2BlacZ grown on medium containing X-Gal indicated a consistent and constitutive translation of this gene. No clue to Lex2A function can be discerned through database searches; its contribution to the function of the lex2 locus requires further investigation.
In summary, lex2B is required for the addition of the second -glucose in the oligosaccharide extension from the first heptose of the LPS of H. influenzae strain RM7004. lex2 has a role in the phase-variable expression of H. influenzae LPS and its activity is key to the extension of a digalactoside that may be associated with enhanced survival of the organism during systemic infection.
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ACKNOWLEDGEMENTS |
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Received 2 April 2003;
revised 18 July 2003;
accepted 29 July 2003.
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