Fachbereich Biologie, Abteilung Mikrobiologie, Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany
Correspondence
Bernhard Henrich
henrich{at}rhrk.uni-kl.de
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ABSTRACT |
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Present address: College of Agriculture and Life Sciences, Department of Food Science, PO Box 7624, North Carolina State University, Raleigh, NC 27695, USA.
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INTRODUCTION |
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Lb. gasseri ADH carries the inducible prophage adh, which has been described as a morphotype B1 siphovirus with double-stranded linear DNA (Raya et al., 1989
). Several other temperate phages of this group (FSW, A2, mv4,
g1e) have been isolated from Lactobacillus strains used in dairy fermentations (for a review see Brüssow, 2001
). So far,
adh is the only phage originating from a non-dairy Lactobacillus strain from which knowledge on distinct genetic features has been obtained. These include the integrase gene intG and the adjacent attP site (Fremaux et al., 1993
), which have been exploited to design a site-specific integration vector (Raya et al., 1992
), the lysis functions (Henrich et al., 1995
) and some regulatory elements of the lytic/lysogenic switch (Engel et al., 1998
). Recently, we established the complete 43·8 kb nucleotide sequence of
adh and verified some of its predicted genetic functions (Altermann et al., 1999b
).
Here, we report on the temporal transcription pattern of phage adh during its lytic life cycle. Detailed transcriptional studies have not previously been reported for Lactobacillus phages of either dairy or human origin. Molecular knowledge on the reproduction programme of such phages would aid our understanding of underlying regulatory mechanisms and also advance the development of food-grade molecular tools for future engineering of lactobacilli of dairy, probiotic or medical importance.
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METHODS |
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Isolation of RNA.
Prewarmed medium (500 ml) was inoculated with an overnight culture of strain NCK102 to give an OD600 of 0·1 and incubated at 37 °C. When an OD600 of 0·5 was reached, a 35 ml sample was removed and the rest of the culture was infected with adh (m.o.i. 10) and further incubated at 37 °C. Further samples (35 ml) were taken at intervals of 10 min up to 90 min after infection. Samples (in JA20 centrifuge tubes, Beckman) were chilled in liquid nitrogen for 15 s, and centrifuged for 2 s at 22 000 g and 4 °C. Supernatants were discarded and pelleted cells were frozen in liquid nitrogen and stored at -80 °C. Total RNA was prepared from the frozen cells as previously described (Altermann et al., 1999a
). RNA was dissolved in H2O and checked by A260/A280 measurement and 0·8 % agarose gel electrophoresis. RNase contamination of materials was minimized according to Sambrook et al. (1989)
.
Synthesis and labelling of probes.
Phage adh was propagated and purified, and phage DNA was isolated, as described by Raya et al. (1989)
. Using this DNA as template, probes for Northern hybridizations were amplified by PCR. Appropriate PCR primers (20- to 30-mers) were chosen from the nucleotide sequence of
adh (Altermann et al., 1999b
) and synthesized on an Applied Biosystems model 392 DNA/RNA synthesizer. Primer sequences can be requested from the authors. Endpositions of the amplified DNA fragments are given in Table 2
. PCR products were purified by agarose gel electrophoresis and subsequent extraction with the QIAquick kit (Qiagen). Unspecific labelling of both strands of purified probes was carried out with [
-32P]dCTP by using the High Prime DNA Labelling kit (Roche). For strand-specific labelling, PCR products were purified from remaining primers by at least two rounds of electrophoresis and QIAquick extraction. Single-strand labelling was then achieved by extending either the forward or the reverse primer for 60 min at 37 °C in separate reactions (24 µl), containing 0·1 µg of the DNA probe (denatured by boiling for 7·5 min), 100 pmol primer, 20 µCi [
-32P]dCTP (3000 Ci mmol-1, 111 TBq mmol-1) and 2 U Escherichia coli DNA polymerase I (Klenow fragment) in 37·5 mM Tris/HCl (pH 7·2), 7·5 mM MgCl2, 0·075 mM DTT, 0·15 mg BSA ml-1 and 18·75 µM each of dATP, dGTP and dTTP.
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Primer extension.
To determine the 5' ends of transcripts, nonradioactive primer extension (NAPE) analysis was used as previously published (Altermann et al., 1999a). Extension reactions were carried out with 9 pmol primer annealed to 10 µg RNA isolated from Lb. gasseri NCK102 at 40 and 60 min after
adh infection. Primers used in NAPE experiments carried a fluorescent label (IRD800) at their 5' ends.
Recombinant DNA techniques.
DNA from phage adh was prepared as described previously (Altermann et al., 1999b
). Standard procedures were used for DNA cloning (Sambrook et al., 1989
) and for the isolation of plasmids from Lc. lactis and Lb. gasseri (de Vos et al., 1989
). Restriction enzymes, T4 DNA ligase (New England Biolabs) and DNA polymerase (Stratagene) were used as recommended by the suppliers. Lc. lactis and Lb. gasseri were transformed by electroporation (Wells et al., 1993
; Luchansky et al., 1989
) with a Gene Pulser (Bio-Rad).
Promoter probing.
To probe the ori region for promoter activity, a 391 bp fragment corresponding to positions 1306013450 of the adh genome (GenBank entry AJ131519) was PCR-amplified from
adh DNA with PfuTurbo Hotstart DNA polymerase by using the primers ori-r (tailed with a KpnI restriction site) and ori-f (Table 1
). After cutting the PCR product with KpnI, it was inserted between the unique EcoRV and KpnI sites of the vector pEK1. The recombinant plasmid pEK1ori was detected after transformation of Lc. lactis MG1363. It contains the cloned DNA fragment in the orientation which would allow transcripts, originating from the ori region in the expected direction (left to right in Fig. 2
), to proceed to the downstream pepI gene (see Fig. 5e
).
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RESULTS |
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The lengths of 33 species of adh transcripts were calculated by averaging the sizes of corresponding hybridization bands obtained with overlapping probes. The map positions of individual transcripts were deduced from Northern hybridization data (Table 2
), taking into account the locations and extensions of genes and ORFs (Altermann et al., 1999b
), and the positions of experimentally verified or predicted promoters (see below). Directions of transcription were deduced from the orientation of the respective coding regions. Sets of overlapping transcripts which seemed to originate from common precursors were assigned to eight groups (Table 2
, Fig. 2
). Three temporal classes (early, middle, late) of
adh-specific mRNAs were distinguished. Examples of Northern hybridizations representative of individual transcript groups are shown in Fig. 3
. Due to nonspecific hybridization signals of the 16S and 23S rRNA species,
adh mRNAs of the corresponding lengths may have escaped detection.
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Middle transcripts
A second group of adh-specific mRNAs, represented by a large transcript of 8·4 kb (Fig. 2
), first appeared about 30 min after infection (Fig. 3
). This transcript spans the cos site, the putative terminase gene (ORF624) and a number of ORFs with unpredicted functions, which may also be related to DNA packaging. The location of the 8·4 kb transcript was determined with the larger probes 15, 16 and 17, and its 5' end was mapped to the ori region of the
adh genome. Three additional mRNA species (4·8, 0·8 and 0·6 kb), all of which are covered by the 8·4 kb transcript, were detected by using probes 44, 4 and 31.
Late transcripts
Late transcripts were first detected between 40 and 50 min after infection (Fig. 3) and fell into two divergently oriented sets, L1 and L2 (Fig. 2
). The L1 set covers a number of ORFs with predicted or established functions in capsid morphogenesis and host cell lysis (Altermann et al., 1999b
). It was divided into three subgroups of overlapping transcripts, most of which shared either common 3' ends (L1a, L1c) or common 5' ends (L1b). The largest mRNAs transcribed from the L1 region (5·3 kb L1a, 6·2 kb L1b, 10·8 kb L1c) were detected with probes 1824. The locations of three smaller L1a transcripts, five smaller L1b transcripts and two smaller L1c transcripts were deduced from hybridizations with probes 47, 6364, 34, 6, 46, 7, 36, 45, 8, 38, 41, 9, hol, lys and 37.
The second main group of late-appearing transcripts (L2) spans a 3·1 kb section of adh DNA, which starts in the region of the lytic/lysogenic switch and ends at the attP site. It includes the gene for a transcription regulator (rad), some ORFs with as yet unknown functions, one of which may specify an excisionase (Engel et al., 1998
), and the integrase gene intG (Fremaux et al., 1993
). The direction of L2 transcription was deduced from the orientation of these genes, which is opposite to that of the majority of ORFs in the
adh genome. The sizes and locations of five different transcripts belonging to the L2 group were determined by hybridizations with probes 11, int, 1 and 55 (Table 2
, Fig. 2
). L2 transcription was probably initiated at the previously identified promoter Prad (Engel et al., 1998
).
The L1 and L2 groups of late transcripts are separated by the attP site. No mRNAs spanning this site were detected.
Promoters
We used non-radioactive automated primer extension (NAPE) to precisely map the start sites of individual transcription units. NAPE was initially used to verify the transcription start points (positions 3322 and 3239) of the promoters Prad and Ptec (Altermann et al., 1999a), which had already been determined by Engel et al. (1998)
. In these studies Prad had been detected with RNA from
adh lysogens, and Ptec with RNA isolated 40 min after
adh infection of NCK102. This is in accordance with the presence of Ptec transcripts (group E1a) throughout the infection cycle (Fig. 3
).
In additional NAPE experiments, 11 different primers (Table 1), targeted to the predicted 5' ends of the E1b, E2 and M transcription units (Fig. 2
), were used with two RNA samples, extracted from Lb. gasseri NCK102 at 40 and 60 min after
adh infection. Specific extension products were only obtained with the primers 15, 16 and 18 (Fig. 5
). This allowed us to map four 5' ends of E1b and E2 transcripts within a 406 bp sequence, which covers the region between ORFs 127 and 65a (Fig. 4
). Three different extension products were obtained with primer 15, corresponding to 5' ends at the T in position 5439 (Fig. 5a
) and at two adjacent As in positions 5311/5312 (Fig. 5b
). Primer 18, oriented in the same direction as primer 15, and the divergently running primer 16 each generated single signals, mapping the 5' ends of the corresponding transcripts to a T in position 5562 (Fig. 5d
) and a T in position 5717 (Fig. 5c
), respectively.
Putative promoter sequences (P15, P16) with similarity to promoters of Lactobacillus delbrueckii subsp. lactis (Matern et al., 1994) were identified upstream of the mRNA 5' ends at positions 5311/5312 and 5717 (Fig. 4
). P15 is located within ORF127 and oriented in the opposite direction. The intensities of the two primer extension signals obtained for this site increased between 40 and 60 min after infection (Fig. 5b
), correlating with the overall increase of the amounts of middle and late transcripts in this time interval (Fig. 3
). Possible -10 and -35 regions of P15, deviating from the consensus hexamers in two and three positions, respectively, are separated by 19 bp, and the -10 sequence precedes the dual mRNA start site at a distance of 5 bp. Primer extension products ending at position 5439 were most abundant (Fig. 5a
), indicating that the E1b region was efficiently transcribed throughout the infection cycle. Since no obvious promoter elements are present upstream of the mRNA 5' ends detected at positions 5439 and 5562, the respective transcripts probably resulted from cleavage of a precursor which was initiated at the preceding P15 site.
The putative promoter P16 is located between the ORFs 127 and 65a, and its direction is opposite to that of P15. P16 has an extended -10 sequence (Ponting & Aravind, 1997) with a perfectly conserved 5'-TATAAT motif at a distance of 5 bp from the 5' end of the corresponding mRNA. The 5'-TATAAT motif is separated by 19 bp from a possible -35 hexamer which shares 4 bp with the consensus. The corresponding primer extension signal generated with mRNA extracted at 40 min after infection was readily visible, whereas no detectable extension products were obtained with mRNA isolated 20 min later (Fig. 5c
). This is in agreement with our assignment of P16 to the synthesis of E2 transcripts, which was found to be turned down between 20 and 50 min after infection (Fig. 3
).
The early and middle transcription units are separated by the origin of replication (ori) (Fig. 2), which is located in a 379 bp non-coding region between ORFs 771 and 68 (Altermann et al., 1999b
). This area contains at least two canditates of appropriately spaced (17±1 bp) -10 and -35 hexamers with similarity to Lactobacillus promoters (Matern et al., 1994
) (at least four matches with the consensus per hexamer). One of them has previously been identified 35 bp upstream of ORF68 (Altermann et al., 1999b
) and the other is located 88 bp downstream of ORF771. In primer extension experiments, however, no distinct mRNA 5' ends could be detected with any of seven different oligonucleotides (Fig. 2
) hybridizing to the ori region. To check the potential promoter activity of the ori region in Lb. gasseri, we used the new vector pEK1, which carries a promoterless pepI gene (Neu & Henrich, 2003
), as an effective reporter. A DNA fragment, covering the last 32 bp of ORF771 and 359 bp of the downstream intergenic sequence (including the two possible promoters, but excluding the putative ribosome-binding site of ORF68) was inserted 53 bp upstream of the initiation codon of pepI (Klein et al., 1994
), and transformants of Lb. gasseri with the resulting plasmid pEK1ori were assayed for peptidase I (PepI) activity. Considering the possibility that transcription from the ori region may require some supplementary phage factor(s) not specified by the cloned DNA fragment, we used transformants of two
adh-lysogenic strains. One of them (NCK102-adh) carried the unmodified prophage whereas the other (NCK102-adh
m-lys) had a large deletion of the
adh morphogenetic and lytic functions (ORFs 397 through lys) which enabled prophage induction without lytic production of infective progeny. PepI activities were measured in cell extracts of both transformant strains, grown under conditions of prophage repression and mitomycin C induction, respectively (Fig. 5e
). In the presence of pEK1ori, the specific PepI activities were about eightfold higher than the intrinsic PepI background in control transformants carrying the unmodified vector pEK1. This clearly showed that the cloned fragment from the ori region of
adh was capable of driving mRNA synthesis in the authentic host Lb. gasseri. Neither the simultaneous induction of the prophage nor the absence of morphogenetic and lytic prophage functions had significant effects on the expression of the reporter gene. It therefore appeared that, under the experimental conditions used, the activity of the respective promoter(s) was rather independent of auxiliary phage functions. The pEK1-based promoter probe system will be used to localize the promoter(s) in the ori region more precisely and to examine the middle and late regions of the
adh genome for the presence of additional transcription initiation sites.
Stemloop structures
The Terminator program of the Husar package (Brendel & Trifonov, 1984) was used to search both strands of the
adh DNA sequence for potential
-independent transcription terminators by applying the combined criteria of (i) high correlation to the distribution of dinucleotides in functional terminators (normalized correlation sum threshold 3·5) and (ii) presence of dyad symmetry adjacent to a poly(T) region (secondary structure threshold 0·0). Of the potential termination sites identified, 23 were located near the assigned 3' ends of 26 distinct mRNA species (Fig. 2
). They included a previously described terminator downstream of intG (T1) (Fremaux et al., 1993
), whereas a potential termination site after lys (Henrich et al., 1995
) was not detected by the computer program. Four of these structures (T1, 5, 8, 23) were identified on both DNA strands and therefore may function as bidirectional terminators.
The small number of confirmed or predicted promoters and the occurrence of transcript groups with staggered 3' ends (E1a, E2, L1b) suggest that some terminators must be leaky to ensure expression of distally located genes. Most of the potential terminators indicated in Fig. 2 have rather moderate normalized correlation sums (<5·0) and secondary structure values (<50) and therefore may be prone to transcriptional readthrough. The common 3' ends of E1b, L1a and L1c transcripts, in contrast, may be generated by efficient termination at T12 (ori), T17/T18 and T23/T1.
RNA secondary structures can also function as targets for endonucleases of the RNase III type. One structural feature of RNA substrates, probably required by all RNase III orthologues, is a double-stranded segment slightly longer than one -helical turn (for a review see Nicholson, 1999
). Searching the
adh sequence with the StemLoop program of the Husar package revealed a number of such hairpins. Five of them (H1, H4 and H5, H9 and H10) are located near the 3' ends of transcripts (0·8/1·9 kb L2, 0·6 and 0·8 kb M, 0·7 and 4·7 kb L1b) for which no potential terminators were predicted (Fig. 2
). They may be involved in processing rather than in termination of mRNA. From the 5'-staggering of E1b, M, L1a, L1c and L2 transcripts it appears that some of the respective 5' ends are generated by cleavage of larger precursors. Possible RNase III cleavage sites can be assigned to a number of these 5' ends (Fig. 2
).
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DISCUSSION |
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Initiation
Two experimentally verified (Prad, Ptec) and two predicted (P15, P16) promoters, clustered within 2·5 kb of the early genome region, were assigned to distinct 5' ends of L2, E1a, E1b and E2 transcripts (Fig. 2), determined by primer extension analysis. These (potential) promoters share similarity with the consensus of Lb. delbrueckii subsp. lactis promoters (Matern et al., 1994
) and therefore may be recognized by the unmodified host RNA polymerase.
As supported by promoter probe data (Fig. 5e), the bulk of middle/late transcription is probably initiated near the replication origin (between ORFs 771 and 68), which, due to extensive secondary structures (Altermann et al., 1999b
), seems unlikely to allow for productive readthrough from the preceding early promoters. Our initial efforts to relate putative promoters in this region to distinct mRNA 5' ends were inconclusive, probably because primer extensions were impeded by such secondary structures.
Whether the late (L1) region has separate promoters or is cotranscribed with the middle region remains unclear. The 187 bp sequence between ORFs 624 and 397, which covers the junction between the middle and L1 areas (Fig. 2), does not contain elements with obvious similarity to the Lactobacillus consensus promoter. If transcription initiation occurs at unorthodox sites, this may require a specificity change of RNA polymerase. Homology searches with the
adh DNA sequence, however, gave no indications of a phage-encoded RNA polymerase or
-like proteins capable of associating with the host RNA polymerase core.
Contrary to all other adh transcripts, which persisted throughout the replication cycle, the mRNAs which seemed to be initiated at P16 (group E2) disappeared between 20 and 50 min after infection (Fig. 3
). Database and motif searches with the only two protein products (gpORF127, gpORF82) specified by E2 transcripts gave no clues to their possible functions. One possibility for turning down the activity of P16 may be a drastic change in RNA polymerase specificity. This seems unlikely, since at least a fraction of the RNA polymerase molecules must retain their ability to support the observed continuous transcription from the other early promoters. Alternatively, P16 may be switched off either actively by a regulator or indirectly by enhanced countertranscription from Ptec. In both cases, some kind of DNA-binding protein may be involved which would either repress P16 or stimulate Ptec. The only obvious candidate for a
adh-encoded transcription regulator, Rad, represses transcription from Ptec rather than stimulating it (Engel et al., 1998
). In Northern hybridizations, transcripts covering the rad gene (L2 group), were not detected before 50 min after infection, and transcription from Ptec (group E1a) seemed unregulated (Fig. 3
). This indicated that, under the conditions of lytic phage reproduction, Rad is only weakly expressed and that a possible repression of P16 would rely on rather low amounts of the regulator which accumulate during the early stage. Two pairs of perfectly conserved direct repeats, one (14 bp) covering the -10 and -35 regions of P16, and the other (7 bp) located 6 bp downstream of the mRNA 5' end (Fig. 4
), may be involved in regulator binding. Similar sequences do not occur in any of the other identified
adh promoters.
Timing
Early synthesis of E1a and E2 transcripts by the host RNA polymerase most probably starts at Ptec and P16. A likely candidate for the initiation of the third group of early transcripts (E1b) is the putative promoter P15. Alternatively, both the E1b and E1a transcripts may arise from an undetected precursor of about 10 kb initiated at Ptec.
The distribution of potential transcription terminators suggests that the timing of adh transcription partly relies on polarity effects resulting from inefficient termination or antitermination. Although the bulk of middle/late transcription probably starts in the ori region, transcriptional readthrough from the preceding early region may contribute to unfolding secondary structures, making promoters accessible to RNA polymerase. In this respect, we suspect that, in the early stage of infection, countertranscription from the convergently oriented promoter P16 may interfere with mRNA synthesis from P15 and Ptec. This would result in low amounts of E1a and E1b transcripts, most of which may be terminated at the ori secondary structures. This control would be relieved by turning down P16-dependent E2 transcription between 20 and 50 min after infection (Fig. 3
), leading to more frequent readthrough and unfolding of the ori structures. Transcription from a plasmid-encoded fusion of the ori sequence to the pepI reporter was neither stimulated nor inhibited by simultaneous induction of the
adh prophage (Fig. 5e
). This suggests that temporal regulation of middle transcription may not depend on positive control by some additional phage protein(s). Whether negative control is involved, on the other hand, remains uncertain, since small amounts of a possible phage-encoded repressor may be titrated by multiple copies of the reporter plasmid.
Up to 50 min after infection, most of the transcripts probably do not proceed beyond the M region, due to efficient termination at the adjacent stemloop structures T14 and T15. As possible mechanisms for turning on subsequent expression of the downstream L1 region, transcription start at untypical promoters (by a modified RNA polymerase) or some kind of antitermination at T14/T15 may be considered. In addition, coupling of some middle/late transcription to DNA replication, as known from phage T4 (Brody et al., 1995), may be involved in transcriptional timing.
Transcripts of adh genes (rad, intG) which are most probably involved in the establishment and maintenance of the lysogenic state were detected rather late after infection (Fig. 3
) and therefore are described as the L2 group (Fig. 2
). This is in contrast to the expected role of the respective functions in the initial decision between the lytic and lysogenic reproduction pathways. Moreover, the similarity of the assigned L2 promoter Prad to vegetative Lactobacillus promoters (Engel et al., 1998
) indicates that it should be readily recognized by the unmodified host RNA polymerase. Our late detection of L2 transcripts is probably a consequence of analysing the phage transcripts under conditions of lytic phage reproduction, rather than an indication of delayed transcription initiation at Prad. In host cells which undergo lytic
adh reproduction Prad should be rather inactive due to repression of the lysogenic pathway, whereas in a small fraction of cells which are actually converting to the lysogenic state, the activity of Prad should adjust to a constitutively low level. From this it seems plausible that the overall amount of L2 transcripts in the infected culture continuously increased until it exceeded the detection threshold between 40 and 50 min after infection.
Phage adh shares a similar overall genome organization and even some amino acid sequence homologies with the S. thermophilus phage Sfi21 (Desiere et al., 2000
). Despite these structural similarities, the two phages apparently differ in the timing of their transcription programs. Based on quite short time intervals (about 5 min) between the successive detection of certain mRNA groups during lytic phage reproduction, Ventura et al. (2002)
presented a transcript map of Sfi21, in which two early transcribed genome regions are interrupted by an area of middle transcription which starts at the putative lytic/lysogenic switch. This surprising assignment of early and middle mRNAs is unprecedented in other phages of lactic acid bacteria and is also incongruous with the transcription profile of
adh. Early transcribed genes of
adh (E1b region) correspond to middle genes of Sfi21, while middle genes of
adh (between ori and cos) correspond to early genes of Sfi21. The transcript map of
adh, like those of many other temperate phages, has a rather conventional appearance, with all early transcripts starting in the predicted genetic switch region.
In conclusion, the complex pattern of distinct mRNAs indicates that expression control of adh genes is largely achieved by transcriptional and post-transcriptional mechanisms. They are probably based on promoter sequences and mRNA structures which have the potential to enable various regulatory tools such as RNA polymerase specificity, mRNA processing, and transcription retardation/termination. Further examination of such mechanisms in
adh gene expression will have to await the identification of the regulatory factors involved.
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ACKNOWLEDGEMENTS |
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Received 26 November 2002;
revised 4 July 2003;
accepted 11 July 2003.
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