Department of Biological Sciences1 and Howard Hughes Medical Institute2, Stanford University, Stanford, CA 94305, USA
Department of Biological Sciences, A527A Langley Hall, University of Pittsburgh, Pittsburgh, PA 15260, USA3
Author for correspondence: Valerie Oke. Tel: +1 412 624 4635. Fax: +1 412 624 4759. e-mail: voke{at}pitt.edu
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ABSTRACT |
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Keywords: sigma-32, transcription factor, symbiosis, stress
The GenBank accession numbers for the sequences reported in this paper are AF128845 (rpoH1) and AF149031 (rpoH2).
a Present address: Department of Biology, Trinity University, 715 Stadium Drive, San Antonio, TX 78212, USA.
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INTRODUCTION |
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A specialized sigma factor, 32 (RpoH), has been characterized as a component of the heat-shock re sponse in Escherichia coli (reviewed by Bukau, 1993
; Georgopoulos et al., 1994
; Yura, 1996
). In response to a sudden increase in temperature or other stresses, the levels of
32 rise transiently because of increased synthesis and protein stabilization. The induction of synthesis is mainly mediated by relief of translational repression due to a secondary structure in the mRNA (Morita et al., 1999
; Nagai et al., 1991
; Yuzawa et al., 1993
), though the level of rpoH transcription also increases slightly (Erickson et al., 1987
; Tilly et al., 1986
). Stabilization occurs with the release of
32 from a DnaK/DnaJ/GrpE chaperone complex as DnaK binds denatured proteins generated under stress conditions (Gamer et al., 1996
). Free
32 associates with core RNA polymerase to direct the transcription of genes encoding heat-shock proteins, which include molecular chaperones, such as DnaK, and proteases, such as Clp (Gross, 1996
). Alternative mechanisms for the response to heat shock can be found in other bacteria. For example, in Bacillus subtilis, multiple mechanisms for heat induction have been characterized, including negative regulation of some genes via the action of a repressor at an inverted repeat (called the CIRCE element: controlling inverted repeat of chaperone expression) and control of other genes by the alternative stress sigma factor,
B (Hecker et al., 1996
).
The nitrogen-fixing symbionts Rhizobium, Bradyrhizobium, Sinorhizobium, and their relatives exist both as soil saprophytes (subject to the conditions of the soil environment) and as symbionts that carry out a series of coordinated behaviours with a host plant. Numerous genes involved in symbiosis have been identified, and regulatory circuits have been partly elucidated. The free-living state of Rhizobium and related bacteria has not been extensively characterized, but both metabolism and regulation in response to environmental change are likely to be important as factors in bacterial survival and in strain competitiveness. Sigma factors, as components of the regulatory apparatus that guides general and specific bacterial behaviour, are of interest in Rhizobium and allied symbiotic bacteria. Bradyrhizobium japonicum and Sinorhizobium meliloti (previously Rhizobium meliloti) have been the major targets of molecular and biochemical study.
In Br. japonicum, the gene for the housekeeping sigma factor SigA (Beck et al., 1997 ), and two copies of the gene for RpoN (NtrA) (Kullik et al., 1991
), have been identified. RpoN directs expression of nitrogen-fixation (nif and fix) genes, and the two rpoN loci are able to replace each other for symbiotic nitrogen fixation. However, their expression is regulated differently: one is regulated by oxygen tension, and the other is subject to negative autoregulation. In addition, Br. japonicum contains three genes encoding
32-type heat-shock sigma factors (Narberhaus et al., 1996
, 1997
). These rpoH loci also show differential regulation and only one can fully function in an E. coli rpoH mutant. Although RpoH controls the expression of some heat-inducible genes in Br. japonicum, others are controlled, as in B. subtilis, by negative regulation using CIRCE or ROSE (repression of heat-shock expression) cis-acting elements (Babst et al., 1996
; Narberhaus et al., 1998a
). The presence of several copies of genes for particular alternative sigma factors suggests that rhizobia may contain multigene sigma families in order to respond more specifically to changes faced in either their symbiotic or free-living state.
In S. meliloti, the genes for the SigA housekeeping sigma factor and for the single RpoN sigma factor for nitrogen-fixation gene expression have been characterized (Ronson et al., 1987 ; Rushing & Long, 1995
). A previous attempt to isolate an rpoH homologue from S. meliloti by complementation of an E. coli rpoH amber mutant yielded the gene suhR, whose product has no homology to sigma factors and was postulated to function by stabilizing or increasing translation of
32 (Bent & Signer, 1990
). We report, here, the identification, characterization, and initial expression studies of two genes, rpoH1 and rpoH2, encoding alternative rpoH-like sigma factors from S. meliloti. These genes were also recently identified by Ono et al. (2001)
.
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METHODS |
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Isolation of the rpoH1 gene.
The production of a 146 bp PCR fragment (PCR1), corresponding to a portion of the S. meliloti sigA gene, has been described previously (Rushing & Long, 1995 ). The PCR1 fragment weakly hybridized with several plaques from a
library of S. meliloti DNA, which suggests the presence of additional genes encoding sigma factors. We therefore obtained lysates of these
isolates and performed a second round of PCR as described previously (Rushing & Long, 1995
), except that 2 µl phage lysate was used as the template. We obtained a PCR product (PCR2) of 227 bp, corresponding to rpoH1, that was cloned as an XhoIHindIII fragment in pBluescript SK+, creating pBGR24 (Fig. 1
). Southern analysis with the PCR2 probe indicated that the rpoH1 gene was located on a 4·8 kb BamHI chromosomal DNA fragment and that this fragment was contained in the
BglII clones from which PCR2 was derived. The 4·8 kb BamHI fragment was therefore subcloned from
in both orientations in pBluescript SK+, resulting in pBGR70a and pBGR70b.
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DNA manipulation and sequencing.
Plasmid constructions in E. coli, and DNA isolation, were carried out essentially as described by Sambrook et al. (1989) . The nucleotide sequence on both strands was determined with the dideoxy chain termination method using the Sequenase 2.0 kit (US Biochemicals), the IsoTherm DNA sequencing kit (Epicentre Technologies) and fluorescent sequencing with an Applied Biosystems model 373A or Prism 310 machine.
Sequence analysis.
Sequence assembly was performed using the University of Wisconsin Genetics Computer Group program (Devereux et al., 1984 ) or SEQUENCHER 3.0 (Gene Codes Corporation). Database searches were performed through the NCBI Web page by using BLAST 2.0 (Altschul et al., 1997
).
Construction of an rpoH1 null mutation.
To disrupt the rpoH1 gene, a 1·4 kb XhoIEcoRI fragment from pVO121 (Barnett et al., 2000 ), containing the aadA gene encoding spectinomycin resistance, was inserted into the SacI site of the rpoH1 ORF in pBGR70b, creating pMMT52. The rpoH1::aadA construct was removed from pMMT52 as a BamHI fragment and was inserted into pJQ200SK digested with BamHI, creating pMMT53. This plasmid contains the sacB gene from B. subtilis, allowing negative selection in Gram-negative bacteria when grown on sucrose (Gay et al., 1985
; Quandt & Hynes, 1993
). To construct a strain carrying an rpoH1 mutation in the chromosome, we first introduced pMMT53 into the wild-type strain Rm1021 by triparental mating followed by selection for spectinomycin resistance; this produced MMT12 (rpoH1::pMMT53). Integration of the plasmid by single-reciprocal recombination generated a disrupted copy of rpoH1 (rpoH1::aadA) in tandem with a full-length copy of rpoH1. If the plasmid is removed from the chromosome by homologous recombination, a single copy of either rpoH1::aadA or the wild-type rpoH1 gene will remain in the chromosome, depending on the point of crossover. To select for cells containing only rpoH1::aadA in the chromosome, MMT12 was grown on LB medium containing 5% sucrose (to select for the loss of pMMT53) and spectinomycin (to select for the retention of rpoH1::aadA), resulting in VO3128 (rpoH1::aadA). All strains were confirmed by Southern analysis.
Construction of an rpoH2 null mutation.
To construct a disruption of rpoH2, a 475 bp BglIISacI DNA fragment internal to the rpoH2 ORF was removed from pBGR38a and ligated into pBR322 digested with PstI, creating plasmid pVO101 (Fig. 1). pVO101 was introduced into the wild-type strain Rm1021 by triparental mating followed by selection for tetracycline resistance. Since the plasmid is unable to replicate in S. meliloti, the resulting strain, VO2148, contained pVO101 integrated by single-reciprocal recombination at the rpoH2 locus, which was confirmed by Southern analysis. Integration of the plasmid generates two partial copies of the rpoH2 gene. The first copy is under the control of the rpoH2 promoter and is deleted for 63 codons at the 3' end of the gene encoding region 4.2, which is involved in recognition of the -35 region of promoters (Lonetto et al., 1992
). The second copy, which is probably not expressed because of the lack of a promoter, has a deletion of 67 codons at the 5' end of the gene encoding region 2.1 and part of region 2.2 (Lonetto et al., 1992
). Thus, we expect no functional RpoH2 protein to be produced in this strain.
Construction of an rpoH1gusA fusion.
To construct a transcriptional rpoH1gusA fusion, we started with pBGR47, which contains the 5' end of the rpoH1 ORF, ending at the SacI site. To make use of the XhoI site located in the rpoH1-coding region 84 bp from the start codon as the point of fusion to the reporter gene gusA (uidA) encoding ß-glucuronidase, we first had to destroy an upstream XhoI site. We accomplished this by digesting pBGR47 to completion with SacI and then partially with XhoI. The 1·4 kb XhoISacI fragment that contains 690 bp of upstream DNA and the start of rpoH1 with the desired internal XhoI site was then ligated into SalISacI-digested pUC119. The resulting plasmid, pBGR72, was digested with XhoI, and a 3·4 kb SalI DNA fragment containing a promoterless gusA gene and the aph gene encoding neomycin resistance from pGK19 was inserted, creating pBGR79 (rpoH1gusA). pBGR79 was digested fully with HindIII and partially with EcoRI to recover the 4·8 kb rpoH1gusA fusion for insertion into the broad-host-range plasmid pLAFR3, creating pBGR86 (Fig. 1).
Construction of an rpoH2gusA fusion.
To construct an rpoH2gusA transcriptional fusion, both pVO147 (containing rpoH2) and pVO155 (containing a promoterless copy of gusA and the aph gene encoding neomycin resistance to allow selection in S. meliloti; Oke & Long, 1999 ) were digested with XhoI, filled in with Klenow, and then digested with BsaI (which cuts in the bla gene of the vector backbones). The fragments were ligated together, resulting in pVO194. The plasmid essentially contains the BstXIXhoI fragment of rpoH2 in front of gusA in pVO155 such that the ShineDalgarno sequence for gusA is maintained and rpoH2 and gusA are in different reading frames (Fig. 1
). pVO194 was introduced into the chromosome of Rm1021 by single-reciprocal recombination, as confirmed by Southern analysis, creating strain VO2257.
Assay of ß-glucuronidase activity.
Cells collected for ß-glucuronidase assays were either assayed immediately or placed on ice or frozen at -80 °C until assayed for activity. ß-Glucuronidase activity was determined as described previously (Swanson et al., 1993 ), except that in some cases the cells were permeabilized with lysozyme (200 µg ml-1; 37 °C for 10 min).
Nodules were sectioned and stained for ß-glucuronidase activity as described previously (Swanson et al., 1993 ).
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RESULTS |
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Approximately 200 bp upstream of the rpoH1 locus in S. meliloti we identified part of an ORF with similarity to a gene known as rluD (sfhB) (42% identity) from E. coli which encodes an enzyme responsible for the insertion of pseudouridine residues in 23S rRNA (Raychaudhuri et al., 1998 ). rluD was identified as a suppressor of a temperature-sensitive mutant of ftsH (cited in Myler et al., 1994
), a gene encoding a protease that regulates the levels of
32 in E. coli (Herman et al., 1995
; Tomoyasu et al., 1995
). rluD in E. coli is located upstream of clpB, a
32-regulated gene encoding a protease (Kitagawa et al., 1991
; Squires et al., 1991
). A similar ORF is also located upstream of the Br. japonicum rpoH2 gene (Narberhaus et al., 1997
).
Southern analysis indicated that the rpoH1 DNA is not located on either of the S. meliloti megaplasmids (data not shown). Therefore, the gene is presumably located on the main chromosome. This result has been confirmed by the presence of the gene in the chromosome sequence data from the genome project (Galibert et al., 2001 ).
Isolation of a second gene in S. meliloti encoding an RpoH homologue
Hybridization of the DNA fragment from sigA to the BglII library also identified two
clones that shared a common 1·2 kb DNA fragment which did not correspond to either sigA or rpoH1 (Rushing, 1995
). Sequencing of the fragment revealed a partial ORF, and we isolated a 2·3 kb DNA fragment containing the intact gene by integrating a plasmid into the chromosome and then retrieving the plasmid along with flanking DNA, as described in Methods. Sequence analysis demonstrated that the DNA fragment (Fig. 1
) consists of a partial ORF encoding a protein with homology to a hypothetical protein in Mycobacterium tuberculosis and the CarD transcription factor in Myx. xanthus (Nicolas et al., 1996
), a full-length ORF encoding the protein with homology to the sigma factors, a repetitive DNA element of the Rhizobiaceae (Øster
s et al., 1995
), and a partial ORF in the opposite direction encoding a putative protein with no homology to proteins in the database. As with rpoH1, Southern analysis indicated that this region of DNA is not located on either of the S. meliloti megaplasmids (data not shown) and is therefore located on the chromosome, as confirmed by the genome sequencing project (Galibert et al., 2001
)
The predicted protein encoded by the intact ORF is 288 amino acids long and has homology, along its length, to the 70 class of sigma factors. We have named the gene rpoH2 because the predicted protein product shares greatest homology (
43% identity, 63% similarity) with the heat-shock
32 factors from the
-proteobacteria, then with the related family of developmental sigma factors SigB and SigC from Myx. xanthus and the
32 factors from the
-proteobacteria. The rpoH1 and rpoH2 nomenclature is consistent with the designation of these genes in the annotation of the S. meliloti genome (Galibert et al., 2001
). Ono et al. (2001)
show an alignment of RpoH2 with RpoH1 of S. meliloti and other RpoH proteins. The RpoH2 protein has one mismatch [QKALFFNLR] within the signature RpoH box [Q(R/K)(K/R)LFFNLR] (Nakahigashi et al., 1995
). RpoH2 is no more related to the S. meliloti RpoH1 protein (42% identity, 64% similarity) than to the RpoH proteins from other
-proteobacteria.
Function of S. meliloti rpoH-like genes in E. coli
As a direct test of whether S. meliloti RpoH1 and RpoH2 can function as heat-shock sigma factors, we investigated whether the S. meliloti rpoH1 or rpoH2 genes could complement the temperature-sensitive phenotype of an E. coli strain, KY1612, carrying an rpoH deletion (Zhou et al., 1988 ). Although KY1612 originally was unable to grow at temperatures above 20 °C (Zhou et al., 1988
), several laboratories, including ours, have found the strain to be capable of growing at room temperature; thus, 23 °C is the permissive temperature and 37 °C is the restrictive temperature. We introduced pBGR91, containing the S. meliloti rpoH1 gene downstream of the lac promoter, or pVO198, containing rpoH2 downstream of the lac promoter, into KY1612 cells. Transformation with either the parent vector or pVO198 (rpoH2) resulted in 50600 times more colonies at 23 °C than at 37 °C. In contrast, transformation with pBGR91 (rpoH1) resulted in equal numbers of colonies at 23 °C and at 37 °C. Therefore, the S. meliloti rpoH1 gene is capable of producing a protein that can functionally replace the heat-shock
32 in E. coli. Western analysis of extracts from KY1612 cells carrying pBGR91 or pVO198, using antibody to E. coli
32, detected S. meliloti RpoH1 but not RpoH2 (data not shown). Recently, Ono et al. (2001)
demonstrated, with a different construct, that the S. meliloti rpoH2 gene can complement the E. coli rpoH mutant. Therefore, it is most likely that the S. meliloti rpoH2 gene in our construct is not expressed in E. coli (for unknown reasons).
Expression of the rpoH1gusA fusion
To characterize rpoH1 in more detail, we determined the pattern of rpoH1 gene expression during free-living growth and under symbiotic conditions by using a plasmid-borne transcriptional rpoH1gusA fusion. We followed the expression of the rpoH1gusA fusion during growth of Rm1021/pBGR86 cells in rich medium. The fusion was expressed during growth, and the ß-glucuronidase activity markedly increased as the cells entered stationary phase (Fig. 2a).
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To test whether rpoH1gusA is expressed during symbiosis within the nodule, we inoculated alfalfa plants with bacteria containing the plasmid-borne fusion, and stained the resulting nodules for ß-glucuronidase activity. The rpoH1gusA fusion was expressed throughout the nodule, whereas a negative control fusion in which gusA is in the reverse orientation from rpoH1 showed little or no expression (Fig. 3a).
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Expression of the rpoH2gusA fusion
We determined the pattern of gene expression of a transcriptional chromosomal rpoH2gusA fusion under free-living and symbiotic conditions. The fusion was not expressed during growth or stationary phase in LB medium, but was expressed in M9 minimal medium after the cells reached the stationary phase of growth (Fig. 2b).
The transcriptional rpoH2gusA fusion was modestly induced (fivefold by 10 h) by a shift to 40 °C during growth in M9 minimal medium (data not shown); however, induction was correlated with the cessation of cell growth in response to the heat treatment, and therefore might not be specific to heat shock.
To test if rpoH2gusA was expressed during symbiosis, we inoculated plants with cells containing the fusion and stained the resulting nodules for ß-glucuronidase activity. The transcriptional rpoH2gusA fusion was expressed at a low level within the nodules. Although there was some variability in the staining pattern, a typical example is shown in Fig. 3(b): staining occurs at the tip of the nodule and then there is punctate staining at other locations. No expression was observed within the central region of the nodule (where the bacterial cells fix nitrogen). The basis of the pattern of expression is unknown but could reflect the response of bacterial cells to different microenvironments within the nodule.
Disruption of the rpoH2 gene and characterization of the mutant
We disrupted the rpoH2 gene by integrating a plasmid containing a fragment internal to the ORF (see Methods). Cells containing the rpoH2 mutation were viable at 30 °C, the optimal growing temperature for S. meliloti. We compared the growth and survival rates of wild-type and mutant cells at different temperatures. There was no difference in growth between mutant and wild-type cells at 30 °C or at 37 °C in TY rich medium (data not shown), and there was no difference at 30 °C or 34 °C in M9 minimal medium (Fig. 4b) (the higher temperatures approaching the upper limit for growth in the respective medium). In addition, rpoH2 mutant cells had the same survival rate (as measured by colony formation) as wild-type cells immediately after reaching stationary phase and with continued incubation for 1 week (data not shown). To test if rpoH2 might play a role during symbiosis, we tested the mutant cells for the ability to nodulate alfalfa successfully. rpoH2 was not required for nodulation or nitrogen fixation.
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DISCUSSION |
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Our studies show that rpoH1 and rpoH2 are not functionally equivalent. Only rpoH1 is required for growth in liquid at 37 °C and for successful symbiosis with alfalfa and the two genes are expressed differentially during growth in culture and during symbiosis. Additional support for the idea that these genes are not functionally equivalent comes from work of Ono et al. (2001) . These authors compared the pattern of protein synthesis in wild-type and rpoH1 and/or rpoH2 S. meliloti mutants after a temperature upshift. Nine putative heat-shock proteins were identified in wild-type cells. An rpoH1 mutant affected the production of a subset of these heat-shock proteins, whereas an rpoH2 mutant did not affect production of any of the heat-shock proteins. However, an rpoH1 rpoH2 double mutant showed further reduction of two heat-shock proteins from the levels seen with rpoH1 alone. In addition, Ono et al. (2001)
demonstrated that the rpoH1 rpoH2 double mutant is unable to nodulate alfalfa. Therefore, the combination of the results from this paper and from Ono et al. (2001)
suggests that rpoH1 and rpoH2 have distinct but overlapping functions. To understand the different roles of these proteins in S. meliloti, it will be revealing to analyse the responses of RpoH1 and RpoH2 during heat shock, and to identify and analyse promoters that are dependent on RpoH1 and/or RpoH2.
The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Br. japonicum (another nitrogen-fixing, root-nodule symbiont of legumes), which has three rpoH genes (Narberhaus et al., 1996 , 1997
). BjRpoH2 in Br. japonicum is capable of replacing
32 in E. coli at 37 °C, and BjRpoH1 and BjRpoH3 can replace
32 at lower temperatures. Like rpoH1 and rpoH2 in S. meliloti, BjrpoH1 and BjrpoH3 in Br. japonicum are dispensable for growth. In contrast, a disruption of BjrpoH2 in Br. japonicum was not obtained at either 18 °C or 30 °C.
A conserved, nine-residue sequence called the RpoH box has been identified in members of the RpoH family (Nakahigashi et al., 1995 ). The RpoH box is contained within a region that has been suggested to be involved in the DnaK/DnaJ-mediated control of the translation and stability of
32 (Nagai et al., 1994
), or, more recently, in the binding of
32 to core RNA polymerase (Arsène et al., 1999
; Joo et al., 1998
). Interestingly, as is the case for RpoH2 in S. meliloti, BjRpoH1 and BjRpoH3 each have a single mismatch in the RpoH box, albeit at a residue other than that of the mismatch in RpoH2. The other rpoH sequences in the database that encode confirmed RpoH-acting proteins (usually by complementation of an E. coli rpoH mutant) contain a perfect match for the conserved sequence. Therefore, the altered RpoH box may have functional significance for the action of these proteins in organisms with multiple rpoH genes.
The different functions of the three rpoH-like genes in Br. japonicum are unclear (Narberhaus et al., 1997 , 1998b
). The Br. japonicum BjrpoH2 cannot be disrupted under standard laboratory conditions. On the basis of data regarding differences in the affinity of each protein for the groESL1 and dnaK promoters and differences in the induction of the rpoH genes by heat shock, a model (Narberhaus et al., 1997
) has been presented in which BjRpoH2 provides a basal level of RpoH-directed gene expression during normal growth, while BjRpoH1 is responsible for the induction of genes after heat shock. The possible functions of BjRpoH3, however, remain unknown. Further work characterizing the regulon of genes specifically controlled by each RpoH protein in S. meliloti and Br. japonicum might help to determine why these soil bacteria, which are also endosymbionts of plants, make use of a family of rpoH genes.
In S. meliloti, both rpoH1gusA and rpoH2gusA fusions are induced during the stationary phase of growth: rpoH1gusA is expressed during exponential phase, and expression then increases late in exponential phase/early in stationary phase in rich medium; rpoH2gusA expression, however, increases later in stationary phase in minimal medium. A link between starvation and other stress responses, including heat shock, has been observed in many bacterial species. Entry of cells of several species, including Rhizobium leguminosarum bv. phaseoli, into stationary phase leads to multiple stress resistances, including protection against pH, heat, oxidants, and osmotic shock (Thorne & Williams, 1997 ). In E. coli, levels of
32 increase during stationary phase, and the protein is required for the induction of several heat-shock proteins that are induced during starvation (Jenkins et al., 1991
). However, although an E. coli rpoH mutant is impaired for survival at high temperatures, starved cells are more thermotolerant than growing cells; this implies that
32 is not required for the thermal cross-protection provided by starvation (Jenkins et al., 1991
). Since rpoH1 and rpoH2 in S. meliloti are induced during stationary phase, we speculate that the proteins they encode could play roles in the general stress tolerance that develops in starved cells.
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ACKNOWLEDGEMENTS |
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Received 5 January 2001;
revised 1 June 2001;
accepted 15 June 2001.