1 Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Correspondence
Gennady Kholodii
kholodii{at}img.ras.ru
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ABSTRACT |
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This work is dedicated to the memory of Roman B. Khesin.
The accession numbers for the nucleotide sequences reported in this work are: AF213017 (pKLH2); AJ487050 and AJ251126 (pKLH204); AJ251306 and AJ251307 (pKLH201); AJ486855 (pKLH203); AJ486857 (pKLH202); AJ250171 and AJ486856 (pKLH207); AJ459234 (pKLH205); AJ251272 (pKLH208).
A table showing genetic similarities for the Acinetobacter determinants identified within the regions flanking the mercury-resistant loci is available as supplementary data with the online version of this paper (at http://mic.sgmjournals.org).
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INTRODUCTION |
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A considerable fraction of Tn3 family mercury-resistance transposons found in environmental bacteria appeared to be transposition-deficient (Pearson et al., 1996; Yurieva et al., 1997
; Hobman et al., 1994
; Martinez et al., 2001
; Mindlin et al., 2001
; Kholodii et al., 2002
). The inability of at least some of these elements to transpose has not apparently prevented their further spread, since nearly identical copies of certain defective transposons have been found on different plasmids (pKLH272 and pKLH247) in bacteria from different taxa, which were isolated from geographically separated populations (Yurieva et al., 1997
; G. Kholodii, unpublished). If this apparent widespread dissemination of elements has not been associated with real transposition events, what mechanisms could ensure the translocation of defective transposons? To our knowledge, no single study on this question is found in the literature. One of the defective transposons we were interested in was a derivative of a Tn21-subgroup (Tn3 family) mercury-resistance transposon, which was found in plasmid pKLH2 from an Acinetobacter strain (Kholodii et al., 1993
; Osbourn et al., 1995
). This transposon, named here TndPKLH2, was found to be interrrupted at an incomplete hybrid res site (RS1) consisting of only one subsite, resI. From these data, it has been proposed that the participation of an aberrant resolution event, resulting in the loss of accessory resolution subsites (resII and resIII) of the original RS1 site, and abutted transposition genes (tnpR, A), occurred during the evolution of TndPKLH2 (Kholodii et al., 1993
).
Previously, short DNA sequences, identical or nearly identical to those of TndPKLH2, have been found in a number of Acinetobacter strains from geographically remote areas (Lomovskaya & Nikiforov, 1988; Pearson et al., 1996
; Kholodii, 2001
), raising the possibility that TndPKLH2-like elements are widespread. Where tested, these sequences were found to be located on plasmids (Mindlin et al., 1986
; S. Mindlin & Zh. Gorlenko, unpublished). In two plasmids, pKLH204 and 205, the termini of the presumptive TndPKLH2-like elements were sequenced and found to be identical to the termini of TndPKLH2; the location of these elements on different plasmids was inferred from differences in the flanking DNAs (Kholodii, 2001
). From these data, TndPKLH2-like elements appeared to be a good model for epidemiological studies, and elucidation of the mechanisms of transposition of defective transposons. One of the less obvious' mechanisms for transposition was tested previously (Kholodii, 2001
). In that work, a powerful co-integrative function of typical DNA resolution systems such as cinH-RS2 (identified in pKLH2, 204 and 205 near the mer locus) and tnpR-res (from Tn1721) was identified. These systems (especially cinH-RS2) were able to act on heterogeneous res sites with a range of identity from 35 to 100 %. What was inferred from the data obtained (Kholodii, 2001
) is that movement of DNA fragments by typical resolvases is possible. In the present work, to understand the modes of inter-replicon movement of defective transposons, extended epidemiological and molecular studies on environmental Acinetobacter HgR loci were undertaken. The results obtained have allowed us to propose additional mechanisms for translocation of DNA fragments.
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METHODS |
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Genetically modified bacterial strains, plasmids and matings.
The strains and plasmids used are listed in Table 1. Intra- and interspecies matings between Acinetobacter sp. and Escherichia coli strains were performed on LB plates as described previously (Kholodii et al., 1995
).
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The permafrost strains were isolated from ice core samples removed from a geological layer (the Edoma suite) in the Arctic (152153°E, 69·570·5°N; the Khomus Yuryakh river region, Kolyma-Indigirka lowland, North-Eastern Siberia). In this region, the late Pleistocene layer the so-called Icy Complex or Edoma suite is represented by syngenetically frozen sediments with ice veins, which indicates that they have not thawed since first frozen. On the basis of 14C studies of the Kolyma-Indigirka lowland permafrost grounds (Sher & Plakht, 1988) and the stratigraphic characterization of the sediment cores recovered from drill-holes 6X-Yu and 4/89-34E (from which the Edoma samples used in this work originated), the age of permafrost bacteria within these samples has been estimated to be between 15 000 and 40 000 years old. The drilling/sampling techniques, storage/transportation of permafrost samples, and the precautions against and tests for exogenous contamination were similar to those described previously (Vorobyova et al., 1997
; Shi et al., 1997
; Petrova et al., 2002
).
HgS and HgR bacterial strains were isolated on LB plates without any drug and on LB plates supplemented with HgCl2, respectively, or by using an enrichment culture method (Mindlin et al., 1986). The strains were identified according to morphological, cultural and biochemical characteristics of the genus Acinetobacter (Juni, 1978
), and the results of genetic transformation of strain BD413 ilv (performed as described previously: Mindlin et al., 1990
), which is extremely specific for bacteria from the genus Acinetobacter (Juni, 1978
).
Screening for active HgR transposons resident in environmental Acinetobacter strains.
To test for transposition of HgR determinants, a broad-host-range plasmid (RP1) was introduced into Acinetobacter isolates by mating with E. coli K-12 HB101(RP1). HgR Acinetobacter transconjugants containing RP1 were propagated for 90120 generations on selective LB plates and mated with E. coli K-12 HB101. HgR StrR transconjugants expected to carry an RP1-containing transposable HgR determinant (RP1 : : HgR) were selected on LB plates with 46 µg HgCl2 ml1 and 100 µg streptomycin ml1. Genetic linkage between RP1 and the mercury-resistance determinant was verified in crosses with E. coli strain K-12 JF238, and further analysis was performed if genetic linkage was found. The transposition frequencies were assessed by dividing the fraction of RP1 : : HgR transconjugants by the RP1 transfer rate.
Plasmid size estimation and localization of HgR determinants.
Plasmids were identified in crude cell extracts according to the method of Eckhardt (1978). Plasmid size was assessed by using known plasmids as size standards, and by restriction enzyme analysis of isolated DNA. Plasmid DNA was tested for the presence of HgR determinant(s) by Southern blotting with a 32P-labelled 0·512 kb EcoRIHindIII fragment from the mer operon of pKLH2 (Fig. 1
, top). The presence of an HgR determinant in a conjugative plasmid was determined from transmission of mercury resistance in crosses with a StrR derivative of BD413 ilv, followed by identification of the characteristic pKLH2 fragment in Southern hybridization using transconjugant total DNA and the probe described above.
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RESULTS |
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Southern hybridization showed that the majority of the HgR strains contained a 2·7 kb EcoRI fragment of a mer operon characteristic of TndPKLH2 (Table 2, columns 3 and 4). In the transposition assay, which was similar to those applied to bacteria from genera other than Acinetobacter (Kholodii et al., 1993
, 1997
, 2000
, 2002
; Mindlin et al., 2001
), no active HgR transposons were identified in the collection (Table 2
, last two columns), although some of the strains carried Tn21-related tnpA sequences (column 5). Several strains, including KHW14 detailed below, were suspected to contain an active HgR transposon, as each of them was found in the transposition assay (described in Methods) to produce transconjugants with genetic linkage between the target plasmid RP1 and the HgR determinant. Detailed molecular and genetic analyses showed, however, that genetic linkage was caused by formation of a fusion between RP1 and the full HgR-containing plasmid. The mechanism of fusion formation was not related to transposition of an HgR determinant, but was probably due to IS element activity (data not shown). Most of the HgR, but not the HgS, isolates hybridized with an IS26 probe (Table 2
, column 6), suggesting physical linkage between the HgR determinants and IS elements. To confirm this linkage, HgR plasmids from arbitrarily chosen strains which contained the 2·7 kb EcoRI fragment were taken for further study. Of these, four strains (KHW14, TC108, NC13-1 and BW3; Table 3
) hybridized with the IS26 probe, and one (ED23-35) did not. Additionally, HgR plasmids that had previously been partially sequenced were studied. These were the reference plasmid pKLH2 (Kholodii et al., 1993
; Kholodii, 2001
), which was subjected to further sequencing 3' to the merR gene and 5' to the cinH gene; and pKLH204 and pKLH205, previously sequenced in the regions responsible for the function of the CinH recombinase (Kholodii, 2001
).
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Determination of fuctional activity of the merB gene from pKLH208
pKLH208.5 and pKLH2.41, containing respectively the mer operons from pKLH208 and pKLH2 (cloned in pACYC184), and pACYC184 were used to test the resistance of the transformed E. coli strain K-12 JM83 to an organomercurial drug, phenylmercuric acetate (PMA). PMA resistance, associated in E. coli with MerB activity (Ogawa et al., 1984; Hobman & Brown, 1997
), was determined by bacterial culture titration on LB plates containing different concentrations of the drug. Inhibitory concentrations of PMA were determined according to complete growth inhibition. JM83(pKLH208.5), which was resistant to PMA, grew at 7·5 µg PMA ml1, whereas the susceptible strains, JM83(pKLH2.41) and JM83(pAYC184), grew at 2·5 µg PMA ml1. Each experiment was repeated four times.
Sequences surrounding the TndPKLH2-like elements
The adjacent regions, sequenced from the cloned fragments (Table 3, last column), contained a large number of recombinogenic elements (20 IS elements, 15 resolution system determinants, and two transposons from the Tn21 subgroup), five putative cation efflux system genes, and other determinants. DNA sequence data described below, and restriction enzyme analysis of unsequenced regions of the cloned fragments (data not shown), demonstrated that, except for the common regions clearly inherited from the last common ancestor of these plasmids (named parental plasmid DNA fragments, PPFs), the remainder of the DNAs varied greatly in molecular structure. These data were clearly indicative of the location of PPFs identified on different plasmids.
Characterization of plasmids bearing the cinH-RS2 system
The high sequence identity between the plasmids (>99·8 %), shown by the hatched bars in Fig. 2(a), continued in regions flanking the TndPKLH2-like elements in pKLH2, 204, 205 and 208. On the right (Fig. 2a
), the resolution site RS1 of these plasmids was followed by nearly identical 1451 bp regions, containing a 5'-truncated IS17-related element (IS1011.D1), and the cinH-RS2 DNA resolution system. The identity was interrupted at the centre of the resolution site RS2 crossover subsite, resI (Fig. 2b
), suggesting involvement of independent site-specific recombination in the generation of each particular breakpoint. On the left (Fig. 2a
), the identity was interrupted either because of the insertion of an IS26-related element (IS1007.1) in pKLH204, a transposon (Tn5049) in pKLH208, or an unknown recombination event in pKLH205. The exact breakpoint of identity with pKLH208 was not determined, but fine restriction analysis showed that pKLH2 did not contain Tn5049 5' to orf900, suggesting that Tn5049 or an ancestor of it had interrupted the original (common) DNA sequence in pKLH208. In the partially sequenced Tn5049, the 38 bp TIR and adjacent resolution system (tnpR-RS3) were most similar to those of Tn501 (AC Z00027), showing respectively 79 % and 93 % identity at the DNA level.
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DNA insertion sequences: molecular characterization
Most of the IS elements flanking the TndPKLH2 variant mer operons (Figs 2a and 3) were highly similar to either the 820 bp IS26 from Proteus mirabilis (Mollet et al., 1983
, 1985
) belonging to the IS6 family (Mahillon & Chandler, 1998
), or the 1040 bp IS17 recently found in Acinetobacter haemolyticus BM2714 (AC U95013). Nine out of a total of twenty IS elements identified in this study were truncated.
IS1011.D1 and IS17 shared 82 % identical nucleotides. In both elements, the longest ORF encoded a polypeptide that exhibited the highest similarity (60 % identical residues) to ORFL1, a putative transposase from Janthinobacterium sp. J3 (AC AB095952), and was 39 % identical to the transposase of a well-characterized element, IS903 (Weinert et al., 1983; Mahillon & Chandler, 1998
). The predicted transposases from elements IS1011.D1 and IS17 were characterized by a catalytic motif, D(73)D(67)E, closely related to the IS903 element's transposase motif, D(71)D(67)E (Polard & Chandler, 1995
; Mahillon & Chandler, 1998
). In accordance with these data, phylogenetic analysis (Fig. 4
a, bottom) placed IS1011.D1 and IS17 into the IS903 group that is part of the IS5 family (Mahillon & Chandler, 1998
).
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In the Acinetobacter plasmids studied, no target site duplication characteristic of transposition events of IS26 and other IS elements (Iida et al., 1984; Mahillon & Chandler, 1998
) was found in the insertion site of either of the individual ACI subgroup IS elements or potential transposons bracketed by these IS elements. DNA insertion sequences from other families (IS1, IS3 and IS21) were also identified (Figs 2a and 3
; see also the table published as supplementary data with the online version of this paper at http://mic.sgmjournals.org).
IS26 relatives: a functional study
Since the Acinetobacter IS elements were closely related to IS26, a functional study was performed on them similar to that described for IS26 (Iida et al., 1984), which is believed to transpose via a replicative mechanism. pUC19 (ApR) derivatives containing a cloned copy of IS1006 (pKLH203.L14), IS1007 (pKLH201.L1), IS1007.1 (pKLH204.27), IS1008 (pKLH201.5) or an IS26 variant (pKLH272.R1) were examined for IS-mediated fusion with the conjugative plasmid pOX38gen (GmR). After each pKLH plasmid was co-transformed with pOX38gen into E. coli K-12 HB101 (recA), GmR ApR clones were selected, replated three or four times on selective LB agar, mated with JF238 (recA+ NalR), and the frequencies of mobilization of the pUC19 derivatives measured by the ratio of ApR GmR NalR/GmR NalR transconjugants. In all but one case these were less than the lowest detectable (2·5x108). With pKLH201.5, the frequency was 401000 times higher. After the genetic linkage between the ApR and GmR markers was established, four JF238 ApR GmR NalR recA+ transconjugant clones of independent origin were replated three or four times to allow dissociation of the cointegrates which were expected to contain the direct copies of IS1008 at the junctions of pOX38gen and pKLH201.5 and then mated with a recA recipient, UB5201 RifR. In these matings,
1 % of GmR RifR transconjugants were ApS. Southern hybridization and terminal sequencing showed that these ApS segregants carried, in each case, a pOX38gen : : IS1008, probably resulting from a homologous recombination-mediated resolution event between the direct copies of IS1008 in the cointegrate. Where tested, a target site duplication of 8 bp flanking IS1008, was identified. These data were similar to those obtained in the case of IS26 (Iida et al., 1982
, 1984
).
Time elapsed from the last common ancestor of the PPFs
The isolation of TndPKLH2-type transposons and corresponding PPFs from the permafrost samples, and the divergence observed between them (Fig. 1, pKLH205 and 208; and data not shown), provided evidence that these entities evolved before they were frozen (15 00040 000 years ago). Given that the mean evolutionary synonymous distance (Ds) and the rate of synonymous site evolution (µ) are available, the divergence time elapsed since the last common ancestor of these entities may be computed by dividing Ds by µ (Rich et al., 1998
; Achtman et al., 1999
). Ds was computed from the polymorphisms observed within the contemporary TndPKLH2-like transposons (from pKLH2, 201204, 207; Fig. 1
) with the help of several models embedded in the MEGA2 software program (Kumar et al., 2001
). These models (NeiGojobori, modified NeiGojobori, LiWuLuo, PamiloBianchiLi, and Kumar's) described elsewhere (Nei & Kumar, 2000
), produced close estimates of Ds ranging from 0·00141±0·00072 to 0·00181±0·00101. A number of µ estimates were available: for the genus Pseudomonas, 2·3x109 mutations per synonymous site per year (Ochman et al., 1999
); for mammals, the same value or similar (Kumar & Subramanian, 2002
); for E. coli/Salmonella enterica, 4·5x109 (Ochman & Wilson, 1987
); and for other entities (Ochman et al., 1999
). The similar µ values observed amongst disparate life forms such as bacteria and mammals further confirm the data of Kumar & Subramanian (2002)
that neither the generation time, nor the particular features of the organism's physiology, nor the population size, provide factors determining µ. One more inference from these data seems to be that µ values for chromosomal and plasmid genes can hardly differ significantly. Taking this consideration into account, and using the µ value from the genus Pseudomonas [as, of bacteria for which a µ estimate was available (Ochman et al., 1999), Acinetobacter spp. were most closely related to Pseudomonas spp. (Olsen et al., 1994
)], we have calculated the age of the hypothetical plasmid that was the source of the Acinetobacter PPFs identified in this study as being between 300 000 and 830 000 years.
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DISCUSSION |
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Why is the inactive transposon widespread and how has it appeared in various plasmids? The fact that the transposon is often located on a transmissible plasmid (Table 3) answers the first of the questions. To answer the second question, we have sequenced the regions flanking TndPKLH2-like transposons in different plasmids. These regions appeared to contain multiple tracks of previous recombination events,
36 over
70 kb sequenced. Such events included: independent breakpoints of PPFs in RS2 (Fig. 2b
); insertions/deletions of different DNA elements and regions adjacent to them (Figs 2a and 3
); and homologous recombination events in mer operons and IS elements, producing genetic mosaics (Figs 1 and 4b
). Some older tracks, e.g. insertion of IS3 and IS17.D1 ancestors (Fig. 2
, pKLH204), were covered by later recombinations, so the whole list and succession of events cannot be reconstructed accurately. However, it is clear that certain events in the evolution of the Acinetobacter mer loci occurred before the formation of the Arctic permafrost. These events included: (i) recombination at the res site of the active transposon to produce the hybrid site RS1; (ii) recombination events underlying the formation of both the IS1011.D1-RS2 conglomerate and versions of RS2 in pKLH205 and 208 (Fig. 2
); (iii) recombination events that formed the left flank in pKLH205 and 208; and (iv) allelic exchanges that resulted in the mosaic mer loci (Fig. 1
).
An unexpected finding in this study was the high sequence identity between different Acinetobacter plasmids in the regions that flanked the TndPKLH2-like elements. The length of additional identity varied from 5 to 1451 bp (Figs 2a and 3, hatched bars) and, as a rule, this identity was interrupted by a mobile element (IS26- or Tn21-related), or ended at the recombination point of the cinH-RS2 DNA resolution system (Fig. 2b
). The very closely related and apparently transferable HgR units we have identified and named PPFs, which were flanked by recombinogenic elements, do not fit into any known categories of bacterial mobile elements, such as pathogenicity islands (Hacker & Kaper, 2000
), introns (Martínez-Abarca & Toro, 2000
; Dai & Zimmerly, 2002
), class II transposons (Sherratt, 1989
; Grinsted et al., 1990
; Craig, 1996
) (even including the possibility for a one-ended transposition mechanism: Mötsch et al., 1985
), IS elements (Mahillon & Chandler, 1998
; Mendiola et al., 1994
) and gene cassettes (Recchia & Hall, 1995
; Hall & Collis, 1998
).
The most reasonable view from the data we obtained is that PPFs are relics of an ancient (300 000800 000 years old) plasmid that has passed through numerous rounds of fusions with other plasmids followed by deletions (resolutions) stabilizing the resulting novel HgR plasmids. The hypothesis for the stabilizing events stems from the necessity to consider the cinH-RS2 DNA resolution system as a factor implicated in transposition of TndPKLH2-like elements, taking into account that its co-integrative and dissociative functions (and those of other typical resolution systems) are reversible (Bliska et al., 1991; Kholodii, 2001
). In support of this hypothesis, it seems to be no accident that multiple events of damage to the resolution determinants were seen in the plasmids we studied. These events included: the insertion of an IS1011.D1, substituting for res subsites II and III of the ancestral RS1 site (Figs 1 and 2a
, top); the impingement of IS1006.1 elements into RS1 in pKLH202 and 207 (shown by the vertical wavy lines in Figs 1 and 3
); deletions of the tnpR genes seen with the tnpR.D1 and tnpR.D2 relics (Fig. 3
, pKLH202, 207 and 204); and, in the case of pKLH201, 204, 202 and 207, partial deletions of the 44 bp element (shown by the diamond in Figs 2a and 3
), the most probable cis-acting factor stimulating the formation of replicon fusions by CinH (Kholodii, 2001
).
In contrast to true transposition, which is related to acquisition of a DNA segment, the models we propose, which take into account and explain why no target duplications associated with mobile elements were identified, are related to redistribution of genetic material between replicons. Thus based on the co-integrative capacity of typical resolvases (Kholodii, 2001), transposition of the pKLH208 fragment, delimited by RS3 and RS2 (Fig. 2a
), may be proposed by the mechanism described previously (Kholodii, 2001
), where both reactions producing transposition are resolvase-mediated. For the origin of pKLH208, insertion of a class II transposon (Tn5049 ancestor) into the common parental plasmid is sufficient to give pKLH208. Transposition by typical resolvases is also applicable for the movement of the pKLH2 PPF, if the second res site exists in the unsequenced region. Other scenarios, applicable for the movement of the pKLH204 PPF (Fig. 5
a) and the formation of pKLH205 (Fig. 5b
), differ from the mechanism discussed above. In these proposed mechanisms, the cointegrate could dissociate either by intramolecular duplicative transposition initiated by a relative of IS26 (possibly similar to the mechanism detailed in the IS1 system: Turlan & Chandler, 1995
), or by illegitimate recombination, which is suggested at the left flank of pKLH205. Considering the co-integrative activity of the IS26-related element as an initiating event, the succession of reactions shown in Fig. 5(c)
, which is the reverse of that shown in Fig. 5(a)
, is very feasible for the formation of pKLH204. The co-integrative function is also characteristic of other IS elements, but is best expressed in IS26 (Mahillon & Chandler, 1998
; Iida et al., 1984
). Our transposition models, explaining the origin of pKLH204 and 202/207 (Fig. 5c, d
), are based on this feature of transposition, as well as the fact that in the case of IS26, the cointegrates, which are believed to form by a replicative mechanism, show considerable stability, i.e. these are not resolved for a long time by homologous recombination to give the transposition products (Iida et al., 1982
). This latter feature allows rearrangement of these cointegrates to be produced by other recombination events. Apparently, precisely because of the deferred resolution and therefore the possibility of non-transposition type resolutions, i.e. those not involving homologous recombination between the two IS elements, the target site duplication produced by IS26 (Iida et al., 1984
) is rarely detectable near IS26 elements (database search results). Since the data we have obtained in this work for the IS26-related elements from the genus Acinetobacter [which form a new (ACI) subgroup (Fig. 4a
, top)] demonstrate or suggest all of the above properties characteristic of IS26, this gives a basis for the models proposed. The fact that an IS26 variant and most ACI subgroup elements tested in this work have not exhibited mobility might be explained by a particular nucleic acid environment, because there is evidence that the transposition activity of IS26 is significantly increased when the element is placed downstream of a strong promoter (K. Vögel, cited by Mahillon & Chandler, 1998
).
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ACKNOWLEDGEMENTS |
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Received 16 October 2003;
revised 4 December 2003;
accepted 4 December 2003.
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