Unité de Biochimie Microbienne, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Philippe Mazodier. Tel: +33 1 45 68 88 42. Fax: +33 1 45 68 89 38. e-mail: mazodier{at}pasteur.fr
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ABSTRACT |
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Keywords: heat regulation, repressor, RheA, hsp18
Abbreviations: HSP, heat-shock protein
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INTRODUCTION |
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Although the induction of HSPs is a universal response, organisms have diverse regulatory mechanisms for controlling HSP synthesis. The heat-shock response and its regulation were first studied in Escherichia coli, where two heat-shock regulons, positively controlled at the level of transcription by specific sigma factors (32 and
24), were identified (for reviews see Bukau, 1993
; Yura et al., 1993
). In Bacillus subtilis, at least four classes of heat-inducible genes are distinguished by their regulatory mechanisms (Derré et al., 1999b
; Hecker et al., 1996
). The thermoregulation of class I genes (groE, dnaK) depends on a repressor (HrcA) that interacts with an inverted repeat motif called CIRCE (for controlling inverted repeat of chaperone expression) (Schulz & Schumann, 1996
; Yuan & Wong, 1995
; Zuber & Schumann, 1994
). Class II heat-shock genes are positively regulated by the
B factor, the synthesis and activity of which are increased under stress conditions. Class III genes (clpP, clpC operon, clpE) are negatively regulated by CtsR (class three stress gene repressor), which recognizes a directly repeated heptanucleotide operator sequence (Derré et al., 1999a
, b
). Class IV comprises heat-shock genes of unknown regulation. Heat-shock regulation mediated by CIRCE and
32 coexists in some bacteria, for instance Agrobacterium tumefaciens, Caulobacter crescentus and Zymomonas mobilis (Avedissian & Gomes, 1996
; Barbosa et al., 1994
; Mantis & Winans, 1992
; Michel, 1993
; Reisenauer et al., 1996
; Roberts et al., 1996
; Segal & Ron, 1993
). In Bradyrhizobium japonicum, the heat-shock regulation system consists of mechanisms described previously (Babst et al., 1996
) as well as novel systems. Recently, Narberhaus et al. (1998
) showed that a conserved DNA element of approximately 100 bp called ROSE (for repression of heat-shock gene expression) confers heat inducibility upon a
70-type promoter by serving as a binding site for a putative regulatory protein. The ROSE system regulates genes that encode small HSPs (hspAhspH), the
32-like transcription factor (rpoH1) and degP (Münchbach et al., 1999
).
Streptomyces spp. are Gram-positive soil bacteria that undergo morphological differentiation and produce a wide variety of metabolites during growth. Their response to heat shock has been studied mostly in two species, Streptomyces coelicolor and Streptomyces albus. In S. albus, there are at least three different regulatory networks controlling the synthesis of different HSPs. Unlike in several Gram-positive bacteria, including Bac. subtilis, dnaK and groEL of Streptomyces are not regulated by the same mechanism: heat regulation of groEL involves a CIRCE motif and an HrcA repressor (Grandvalet et al., 1998 ), whereas the dnaK operon and clpB are regulated by the HspR repressor, which binds to an inverted repeat called HAIR (HspR-associated inverted repeat) (Bucca et al., 1995
, 1997
; Grandvalet et al., 1997
, 1999
). hspR is the last gene of the dnaK operon.
A third heat regulation mechanism, repressing hsp18, has been identified in S. albus. Transcription from a streptomycete vegetative promoter of hsp18, encoding a small HSP, is strongly induced following heat shock (Servant & Mazodier, 1995 ). An open reading frame (orfY), located 150 bp upstream and in the opposite orientation to hsp18, contributes to the transcriptional regulation of hsp18. Indeed, disruption of orfY generated mutants that synthesized a large amount of hsp18 mRNA at low temperature (Servant & Mazodier, 1996
). In addition, heat induction of the hsp18 gene is subject to post-transcriptional regulation, the mechanism of which is unknown. Here we report that the orfY gene, now referred to as rheA (repressor of hsp eighteen), encodes the repressor of hsp18. We also investigated the regulation of rheA in S. albus by in vivo transcriptional analysis. RheA appears to function as a negative autoregulator.
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METHODS |
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pDL was used as a source of bgaB (Yuan & Wong, 1995 ). ß-Galactosidase activities of bgaB, a thermostable ß-galactosidase from Bacillus stearothermophilus (Hirata et al., 1986
), were determined at 65 °C as described previously (Miller, 1972
; Msadek et al., 1998
), and are expressed as Miller units (mg protein)-1.
DNA manipulations.
Plasmid construction and transformation of E. coli were as described in Sambrook et al. (1989 ). Plasmid DNA was extracted and purified from E. coli with the Qiagen plasmid kit. Restriction enzymes were used as recommended by the manufacturers. DNA was sequenced by the dideoxy chain-termination method (Sanger et al., 1977
) with the Sequenase kit (Pharmacia).
Plasmid construction.
To overproduce RheA in E. coli, rheA (ATG to TGA) was inserted into the expression vector pET11a. A corresponding PCR fragment amplified from S. albus genomic DNA with oligonucleotides PS90 (5'-GTGGATCCTCAGGACCGCCCGGAC-3') and PS92 (5'-TGTGATCATATGACCACCGCCGACCGCCC-3') was digested with NdeI and BamHI and inserted into pET11a cut with NdeI/BamHI to obtain pPS310.
A DNA fragment corresponding to rheA and upstream sequence containing the hsp18 promoter was isolated from S. albus genomic DNA by PCR using oligonucleotides PS90 (5'-GTGGATCCCAGGACCGCCCGGAC-3') and PS97 (5'-GTGGATCCGAAGTCCAGGCAGTTGATGCC-3'). The fragment was digested with BamHI and inserted into the BamHI site of pUC19 to give pPS311.
A plasmid containing a transcriptional fusion between the hsp18 promoter and the Bac. stearothermophilus bgaB gene (Hirata et al., 1986 ), encoding a thermostable ß-galactosidase, was constructed from pPM1745. This plasmid carries the ampicillin-resistance gene and contains hsp18 and rheA from S. albus (Servant & Mazodier, 1995
). A BamHISacI fragment of pDL containing bgaB was ligated between the BamHI and SacI sites of pPM1745 to give pPM7001. The rheA gene was deleted from this plasmid by SalI digestion to yield pPM7002.
pPS310 was digested with XbaI and BamHI. The XbaI site is upstream from and adjacent to the NdeI site. The fragment containing rheA was inserted between the XbaI and BamHI sites of pSU2718 and pSU2719 to give pPM7003 and pPM7004, respectively. In pPM7004, rheA is expressed from the plac promoter, and in pPM7003 rheA is in the opposite orientation. pSU2718 and pSU2719 contain the chloramphenicol acetyltransferase gene (cat) and are based on the pACYC184 replicon (Martinez et al., 1988 ). They are compatible with ColE1-derived plasmids like pPM7002.
Double transformants of E. coli TG1 containing pPM7002 and either pPM7003 or pPM7004 were selected on ampicillin and chloramphenicol. The activity was assayed by measuring the level of ß-galactosidase in cells grown to mid-exponential phase at 37 °C. The results represent the mean of duplicate assays.
RNA manipulation.
Total RNA from S. albus was prepared and Northern blotting was carried out as described previously (Servant et al., 1994 ). Highly stringent conditions were used for hybridization, and the blots were washed with 0·5x SSC/0·1% SDS at 65 °C. The probe used to detect the rheA transcript was a 284 bp internal fragment of rheA obtained by PCR with the oligonucleotides XM73 (5'-GCCGTCGGGGCCGGACA-3') and XM87 (5'-GGCGAACAGCGCGTCCA-3').
The transcription start site upstream from rheA was determined by primer extension as previously described (Grandvalet et al., 1999 ). The rheA-specific oligonucleotide PS201 (5'-GGCCAGGAAGGAGGCCGGGTCGGGGGTGAA-3'), radiolabelled with [
-32P]dATP by polynucleotide kinase (Biolabs), was used with RNA preparations from heat-shocked and untreated S. albus cultures. Dideoxynucleotide chain-termination sequencing reactions performed with the same primer and an appropriate plasmid DNA template were electrophoresed alongside the samples.
Overproduction of RheA in E. coli.
pPS310 and pET11a were introduced into E. coli BL21DE3 in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter. Cultures of strains containing pET11a or pPS310 were induced by adding 1 mM IPTG. Five hours after induction, cells were harvested by centrifugation, resuspended in 50 mM NaCl/B buffer (B buffer: 10 mM Tris, pH 7·5; 10 mM MgCl2; 1 mM DTT; 0·1% Triton X-100; 1 mM EDTA; 10% glycerol) and subjected to sonication. The extract was clarified by centrifugation at 10000 g for 15 min at 4 °C and the supernatant was used directly as a crude extract in gel retardation assays.
DNA mobility shift assays.
A 300 bp BamHISalI fragment from pPS311 containing the hsp18 and rheA promoters was purified and used in gel retardation assays. This fragment was end-labelled with [-32P]dATP using the Klenow fragment of DNA polymerase I (Gibco-BRL). The labelled fragment was further purified using the Qiaquick PCR purification kit (Qiagen). Proteins were incubated for 10 min at 30 or 37 °C with the labelled DNA fragment (0·2 pmol) in a 20 µl reaction mixture containing 1 µg calf thymus DNA, 50 mM NaCl, 10 mM Tris (pH 7·5), 1 mM DTT, 10 mM MgCl2, 1 mM CaCl2 and 10% glycerol. Samples were then loaded directly on to a 6% polyacrylamide gel (50 mM Tris, pH 8·5; 400 mM glycine; 1·73 mM EDTA; 2·5% glycerol) for electrophoresis at 150 V (14 V cm-1). The gels were dried and autoradiographed. The DNA fragments used in competition assays were the hsp18 fragment (specific unlabelled competitor DNA) and the 305 bp groES gene of S. albus (Servant et al., 1993
) (non-specific competitor DNA).
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RESULTS |
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Overproduction of RheA in E. coli, and DNA-binding studies
Mobility shift assays were performed to ascertain whether the product of rheA interacts directly with the hsp18 promoter. RheA was overproduced in E. coli and crude extracts were prepared from both the control strain (E. coli BL21DE3 carrying pET11a) and the RheA production strain (E. coli BL21
DE3 carrying pPS310). Expression of rheA in this system was controlled by the IPTG-inducible T7 promoter. A protein of the expected size for RheA (23 kDa) was readily detected by SDS-PAGE of crude extracts in induced cells containing rheA (Fig. 1
, lane 2). In DNA-binding experiments, extracts of cells of the control strain, without RheA, did not affect the mobility of a 300 bp fragment containing the hsp18 promoter (Fig. 2
, lane 2). In the presence of 0·4 µg of a crude cell extract containing RheA, the labelled fragment was totally retarded (Fig. 2
, lane 3), demonstrating the high affinity of RheA for the hsp18 promoter fragment. In competition experiments to assess the specificity of the binding, the presence of excess unlabelled probe abolished the mobility shift of the labelled hsp18 promoter fragment (Fig. 2
, lane 5), whereas the control with excess S. albus groES DNA fragment had no effect (Fig. 2
, lane 6). These results confirm that RheA binds specifically to the hsp18 promoter region. Heating (37 °C) the E. coli extracts containing RheA before the gel retardation experiments did not affect the retardation pattern (data not shown).
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DISCUSSION |
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We have previously shown that disruption of rheA did not affect expression of genes encoding the main S. albus chaperones GroESL, DnaK and ClpB (Servant & Mazodier, 1996 ). Here we show that RheA is able to repress expression of the hsp18 gene in E. coli and to bind to the promoter region in vitro, indicating that RheA is the repressor of hsp18. To our knowledge, this is the first report of a repressor that specifically decreases expression of a small HSP in bacteria. In the databases there are currently no genes homologous to rheA, indicating that regulation by RheA is not a widespread regulatory mechanism. For example, rheA is absent from the Mycobacterium tuberculosis genome, although Streptomyces and mycobacteria are taxonomically related and both belong to the order of the Actinomycetales (Cole et al., 1998
). The rheA gene might be limited to Streptomyces or a sub-group of Streptomyces. Analysis of a panel of Streptomyces genomic DNA by Southern blotting with a rheA probe and subsequent cloning of the rheA homologues would resolve this issue and help identify a consensus target sequence for RheA. Genes highly similar to S. albus hsp18 appear to be present in Streptomyces pristinaespiralis, Streptomyces parvulus but not Streptomyces lividans, as detected by hybridization with the hsp18 probe (unpublished results). A perfect inverted repeat (TGTCATC 5N GATGACA) which overlaps -35 and -10 sequences of hsp18 could be the target of RheA. This hypothetical target of RheA also overlaps the -10 sequence of rheA (see Fig. 4
).
It would be interesting to determine how variation of temperature can modify RheA activity and to confirm that it is an autoregulatory protein. Thermoinduction of the synthesis of the repressor in heat-shock conditions, although at first sight paradoxical, appears to be a general feature of the heat-shock repressors. It is the case for HspR, HrcA and CtsR, which are autoregulatory repressor proteins. Induction of HSP after temperature increase is usually transient. The mechanisms which lead to the down-regulation of the transcriptional activity of heat-shock genes differ between species, but the main chaperones (GroEL or DnaK) are usually implicated. In E. coli, DnaK, DnaJ and GrpE negatively regulate heat-shock gene expression by controlling the synthesis and stability of 32 (Straus et al., 1990
). In Bac. subtilis, the GroE chaperonin machinery modulates the activity of the HrcA repressor (Mogk et al., 1997
). In the case of Hsp18, a constitutive high level of expression of hsp18 is observed at high temperature. This suggests that modulation of RheA activity is clearly different from the feedback mechanism previously described. The repressor might be degraded or might lose its binding capacity at high temperature. The reported experiments do not sustain the second hypothesis but clearly studies of this phenomenon will require a purified functional RheA protein.
In conclusion, in Streptomyces regulation of the heat-shock response involves a combination of negative regulators. HspR co-regulates expression of the dnaK operon and the clpB gene. HrcA represses expression of the groEL genes and RheA is the repressor of hsp18. However, other mechanisms will probably be discovered, as at least five groups of HSPs can be defined by the patterns of their induction kinetics after a temperature upshift in S. coelicolor (Puglia et al., 1995 ).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Babst, M., Hennecke, H. & Fischer, H. M. (1996). Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum. Mol Microbiol 19, 827-839.[Medline]
Barbosa, M. D. F. S., Yomano, L. P. & Ingram, L. O. (1994). Cloning, sequencing and expression of stress genes from the ethanol-producing bacterium Zymomonas mobilis: the groESL operon. Gene 148, 51-57.[Medline]
Bucca, G., Ferina, G., Puglia, A.-M. & Smith, C. P. (1995). The dnaK operon of Streptomyces coelicolor encodes a novel heat-shock protein which binds to the promoter region of the operon. Mol Microbiol 17, 663-674.[Medline]
Bucca, G., Hindle, Z. & Smith, C. P. (1997). Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein. J Bacteriol 179, 5999-6004.[Abstract]
Bukau, B. (1993). Regulation of the Escherichia coli heat-shock response. Mol Microbiol 9, 671-680.[Medline]
Chater, K. F. & Wilde, L. C. (1980). Streptomyces albus G mutants defective in the SalGI restrictionmodification system. J Gen Microbiol 116, 323-334.[Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[Medline]
Derré, I., Rapoport, G., Devine, K., Rose, M. & Msadek, T. (1999a). ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis. Mol Microbiol 32, 581-593.[Medline]
Derré, I., Rapoport, G. & Msadek, T. (1999b). CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram-positive bacteria. Mol Microbiol 31, 117-131.[Medline]
Georgopoulos, C. & Welch, W. J. (1993). Role of the major heat shock proteins as molecular chaperones. Annu Rev Mol Biol 9, 601-634.
Gibson, T. J. (1984). Studies on the EpsteinBarr virus genome. PhD thesis, Cambridge University.
Grandvalet, C., Servant, P. & Mazodier, P. (1997). Disruption of hspR, the repressor gene of the dnaK operon in Streptomyces albus G. Mol Microbiol 23, 77-84.[Medline]
Grandvalet, C., Rapoport, G. & Mazodier, P. (1998). hrcA encoding the repressor of the groEL genes in Streptomyces albus G is associated with a second dnaJ gene. J Bacteriol 180, 5129-5134.
Grandvalet, C., de Crécy-Lagard, V. & Mazodier, P. (1999). The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon. Mol Microbiol 31, 521-532.[Medline]
Hecker, M., Schumann, W. & Völker, U. (1996). Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol 19, 417-428.[Medline]
Hirata, H., Fukazawa, T., Negoro, S. & Okada, H. (1986). Structure of a ß-galactosidase gene of Bacillus stearothermophilus. J Bacteriol 166, 722-727.[Medline]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Jakob, U., Gaestel, M., Engel, K. & Buchner, J. (1993). Small heat shock proteins are molecular chaperones. J Biol Chem 268, 1517-1520.
Mantis, N. J. & Winans, S. C. (1992). Characterization of the Agrobacterium tumefaciens heat shock response: evidence for a 32-like sigma factor. J Bacteriol 174, 991-997.[Abstract]
Martinez, E., Bartolomé, B. & de la Cruz, F. (1988). pACYC184-derived cloning vectors containing the multiple cloning site and lacZ reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68, 159-162.[Medline]
Michel, G. P. F. (1993). Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis. J Bacteriol 175, 3228-3231.[Abstract]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F. X. & Schumann, W. (1997). The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J 16, 4579-4590.
Msadek, T., Dartois, V., Kunst, F., Herbaud, M. L., Denizot, F. & Rapoport, G. (1998). ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation. Mol Microbiol 27, 899-914.[Medline]
Münchbach, M., Nocker, A. & Naberhaus, F. (1999). Multiple small heat shock proteins in rhizobia. J Bacteriol 181, 83-90.
Naberhaus, F., Käser, R., Nocker, A. & Hennecke, H. (1998). A novel DNA element that controls bacterial heat shock gene expression. Mol Microbiol 28, 315-323.[Medline]
Puglia, A.-M., Vohradsky, J. & Thompson, C. J. (1995). Developmental control of the heat-shock stress regulon in Streptomyces coelicolor. Mol Microbiol 17, 737-746.[Medline]
Reisenauer, A., Mohr, C. D. & Shapiro, L. (1996). Regulation of a heat shock 32 homolog in Caulobacter crescentus. J Bacteriol 178, 1919-1927.[Abstract]
Roberts, R. C., Toochinda, C., Avedissian, M., Baldini, R. L., Gomes, S. L. & Shapiro, L. (1996). Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE. J Bacteriol 178, 1829-1841.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Schulz, A. & Schumann, W. (1996). hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J Bacteriol 178, 1088-1093.[Abstract]
Segal, G. & Ron, E. Z. (1993). Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J Bacteriol 175, 3083-3088.[Abstract]
Servant, P. & Mazodier, P. (1995). Characterization of Streptomyces albus 18-kilodalton heat shock responsive protein. J Bacteriol 177, 2998-3003.[Abstract]
Servant, P. & Mazodier, P. (1996). Heat induction of hsp18 gene expression in Streptomyces albus G: transcriptional and posttranscriptional regulation. J Bacteriol 178, 7031-7036.[Abstract]
Servant, P., Thompson, C. & Mazodier, P. (1993). Use of new Escherichia coli/Streptomyces conjugative vectors to probe the functions of the two groEL-like genes of Streptomyces albus G by gene disruption. Gene 134, 25-32.[Medline]
Servant, P., Thompson, C. & Mazodier, P. (1994). Post-transcriptional regulation of the groEL1 gene of Streptomyces albus. Mol Microbiol 12, 423-432.[Medline]
Straus, D., Walter, W. & Gross, C. A. (1990). DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of 32. Genes Dev 4, 2202-2209.[Abstract]
Strohl, W. R. (1992). Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res 20, 961-974.[Abstract]
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Yuan, G. & Wong, S.-L. (1995). Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK. J Bacteriol 177, 6462-6468.[Abstract]
Yura, T., Nagai, H. & Mori, H. (1993). Regulation of the heat-shock response in bacteria. Annu Rev Microbiol 47, 321-350.[Medline]
Zuber, U. & Schumann, W. (1994). CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J Bacteriol 176, 1359-1363.[Abstract]
Received 11 March 1999;
revised 28 May 1999;
accepted 7 June 1999.