Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA1
Author for correspondence: Julie A. Maupin-Furlow. Tel: +1 352 392 4095. Fax: +1 352 392 5922. e-mail: jmaupin{at}ufl.edu
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ABSTRACT |
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Keywords: pyruvate metabolism, thiamin pyrophosphate-dependent enzymes, alcohol dehydrogenase, ethanol fermentation, allosteric regulation
Abbreviations: ADH, alcohol dehydrogenase; E1, decarboxylase component of pyruvate dehydrogenase; IPD, indole-3-pyruvate decarboxylase; PDC, pyruvate decarboxylase; TPP, thiamin pyrophosphate; TK, transketolase.
The GenBank accession number for the sequence reported in this paper is AF354297.
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INTRODUCTION |
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PDC (EC 4 . 1 . 1 . 1) serves as the key enzyme in all homo-ethanol fermentations. This enzyme catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide using Mg2+ and thiamin pyrophosphate (TPP) as cofactors. Acetaldehyde is subsequently reduced to ethanol by alcohol dehydrogenase (ADH, EC 1 . 1 . 1 . 1) during the regeneration of NAD+. PDC is widespread among plants, absent in animals, and rare in prokaryotes. This is in contrast to ADH, which is widespread among eukaryotes and prokaryotes. Prior to this study, the only bacterial pdc gene described was from the Gram-negative -proteobacterium Zymomonas mobilis (Braü & Sahm, 1986
; Conway et al., 1987
; Neale et al., 1987a
; Reynen & Sahm, 1988
). Z. mobilis PDC has been purified to homogeneity, crystallized, and extensively characterized (Candy & Duggleby, 1998
). PDC has also been purified from one other bacterium, Sarcina ventriculi (Lowe & Zeikus, 1992
), an unusual Gram-positive organism.
S. ventriculi is an obligate anaerobe that grows from pH 2 to pH 10, fermenting hexose and pentose sugars to produce acetate, ethanol, formate, CO2 and H2 (Goodwin & Zeikus, 1987 ; Stephenson & Dawes, 1971
). In this organism, the relative production of ethanol and acetate varies with environmental pH. Under acidic conditions, where acetic acid is toxic to cells, ethanol is the primary product (Goodwin & Zeikus, 1987
). At neutral pH and above, a near-equimolar mixture of ethanol and acetate is produced, with low levels of formate (Lowe & Zeikus, 1991
). These changes in fermentation profiles have been attributed to changes in the levels of two enzymes that metabolize pyruvate: PDC and pyruvate dehydrogenase (Goodwin & Zeikus, 1987
; Lowe & Zeikus, 1991
).
The properties of the S. ventriculi PDC are very different from those of the Z. mobilis enzyme. Unlike the MichaelisMenten kinetics of Z. mobilis PDC (Hoppner & Doelle, 1983 ; Neale et al., 1987a
), the S. ventriculi enzyme was activated by pyruvate (Lowe & Zeikus, 1992
), similar to PDCs from yeast and higher plants. S. ventriculi PDC was reported to have an unusually high Km for pyruvate (13 mM), compared to Km values of 0·34·4 mM for other PDCs (Hoppner & Doelle, 1983
; Neale et al., 1987a
; Ullrich & Donner, 1970
). The phenylalanine content of purified S. ventriculi PDC was four- to fivefold greater than that of other PDCs, suggesting significant differences in primary structure (Lowe & Zeikus, 1992
).
To further examine the unusual nature of the S. ventriculi PDC, the pdc gene encoding it was cloned, sequenced, and expressed in recombinant E. coli. This approach provided a genetic determination of primary amino acid sequence and facilitated PDC purification for further kinetic and biophysical characterization.
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METHODS |
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Bacterial strains and media.
Table 1 lists the E. coli strains used in this study, including strains TB-1 and DH5
, which were used for routine recombinant DNA experiments. E. coli strain SE2309 was used to create a subgenomic DNA library in plasmid pBR322. E. coli strains ER1647, LE392 and BM25.8 were used in conjunction with
BlueSTAR for a subgenomic DNA library. E. coli strains BL21(DE3), BL21-CodonPlus-RIL and BL21-CodonPlus-RIL/pSJS1240 were used to examine the expression of the S. ventriculi pdc gene from plasmid pJAM419. E. coli strains were grown in LuriaBertani (LB) medium and supplemented with antibiotics as appropriate (30 mg chloramphenicol l-1, 100 mg carbenicillin l-1, 100 mg ampicillin l-1 and/or 50 mg spectinomycin l-1). S. ventriculi strain Goodsir was cultivated as described previously (Goodwin & Zeikus, 1987
).
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Cloning of the S. ventriculi pdc gene.
A degenerate oligonucleotide, 5'-AARGARGTNAAYGTNGARCAYATGTTYGGNGT-3' (R is A or G; N is A, C, G or T; Y is C or T), was synthesized based on the N-terminal amino acid sequence of PDC purified from S. ventriculi (Lowe & Zeikus, 1992 ). This oligonucleotide was labelled at the 3'-end using terminal transferase with digoxigenin-11-dUTP and dATP as recommended by the supplier (Roche Molecular Biochemicals) and was used to screen genomic DNA from S. ventriculi.
For Southern analysis, genomic DNA was digested with BglI, EcoRI or HincII, separated by 0·8% agarose electrophoresis, and transferred to positively charged nylon membranes (Southern, 1975 ). Membranes were equilibrated at 58 °C for 2 h in 5x SSC (1x SSC is 0·15 M NaCl plus 0·015 M sodium citrate) containing 1% blocking reagent (Roche Molecular Biochemicals), 0·1% N-lauroylsarcosine, and 0·02% SDS. After the probe (0·2 pmol ml-1) and poly(A) (0·01 mg ml-1) were added, membranes were incubated at 58 °C for 18·5 h. Membranes were washed twice with 2x SSC containing 0·1% SDS (5 min per wash) at 25 °C and twice with 0·5x SSC containing 0·1% SDS (15 min per wash) at 58 °C. Signals were visualized using colorimetric detection according to the supplier (Roche Molecular Biochemicals).
For generation of a subgenomic library in plasmid pBR322, S. ventriculi chromosomal DNA was digested with HincII and fractionated by electrophoresis. The 2·53·5 kb HincII DNA fragments were ligated into the EcoRV site of pBR322 and transformed into E. coli SE2309. Colonies were screened with the degenerate oligonucleotide by colorimetric detection. By this method, plasmid pJAM400, which carries a HincII fragment containing 1350 bp of the pdc gene was isolated.
The BlueSTAR Vector System (Novagen) was used to create an additional subgenomic library to facilitate isolation of the full-length pdc gene from S. ventriculi. Genomic DNA was digested with BclI, separated by electrophoresis in 0·8% agarose, and the 6·58·5 kb fragments were ligated with the
BlueSTAR BamHI arms. In vitro packaging and plating of phage was performed according to the supplier (Novagen). A DNA probe was generated using an 800 bp EcoRI fragment of the pdc gene from pJAM400 that was labelled with digoxigenin-11-dUTP using the random-primed method as recommended by the supplier (Roche Molecular Biochemicals). Plaques were screened using colorimetric detection. Cre-loxP-mediated subcloning was used to circularize the DNA of the positive plaques by plating
BlueSTAR phage with E. coli BM25.8, which expresses Cre recombinase (Novagen). The circularized plasmid pJAM410 was then purified and electroporated into E. coli DH5
.
For generation of a pdc expression vector, the promoterless pdc gene was subcloned into pET21d after amplification from pJAM413 (Table 1) by PCR. Primers were designed for directional insertion using BspHI (oligo 1) and XhoI (oligo 2) restriction sites. The resulting fragment was ligated into compatible NcoI and XhoI sites of pET21d (Novagen) to produce pJAM419 (Fig. 1
). The fidelity of the pdc gene was confirmed by DNA sequencing.
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Genepro 5.0 (Riverside Scientific), CLUSTAL W version 1.81 (Thompson et al., 1994 ), Treeview version 1.5 (Page, 1996
), and MultiAln (Corpet, 1988
) were used for DNA and/or protein sequence alignments and comparisons. Deduced amino acid sequences were compared to protein sequences available in the GenBank, EMBL and SWISS-PROT databases at the National Center for Biotechnology Information (Bethesda, MD) using the BLAST network server (Altschul et al., 1990
). The dense alignment surface (DAS) method was used for the prediction of transmembrane
-helices (Cserzo et al., 1997
).
Production of S. ventriculi PDC in recombinant E. coli.
Plasmid pJAM419 was transformed into E. coli BL21-CodonPlus-RIL containing plasmid pSJS1240 (Table 1). Expression of the pdc gene in this plasmid is regulated by the bacteriophage T7 RNA polymerase promoter system (Novagen). Freshly transformed cells were inoculated into LB medium containing ampicillin, spectinomycin and chloramphenicol and grown at 37 °C (200 r.p.m.) until cells reached an OD600 of 0·60·8. Transcription was then induced for 23 h by the addition of 1 mM IPTG. Cells were harvested by centrifugation at 5000 g (10 min, 4 °C) and stored at -70 °C or in liquid nitrogen.
Purification of the S. ventriculi PDC protein.
All purification buffers contained 1 mM TPP and 1 mM MgSO4 unless indicated otherwise. Recombinant E. coli cells (14·8 g wet wt) were thawed in 6 vols 50 mM sodium phosphate buffer at pH 6·5 (buffer A) and passed through a French pressure cell at 20000 p.s.i. (138 MPa). Cell debris was removed by centrifugation at 16000 g (20 min, 4 °C). Supernatant was removed and filtered through a 45 µm filter membrane. Filtrate (692 mg protein) was applied to a Q Sepharose Fast Flow 26/10 column (Pharmacia) that was equilibrated at 2 ml min-1 with buffer A containing 300 mM NaCl. Fractions containing PDC activity (326 mg protein) were precipitated with 80% saturation (NH4)2SO4. Protein was resuspended in buffer A, dialysed against buffer A (4 °C, 16 h), and filtered (45 µm membrane). The filtrate (287 mg) was applied to a Q Sepharose column equilibrated with buffer A and developed with a linear NaCl gradient (0400 mM NaCl in 220 ml buffer A) at 4 ml min-1. PDC active fractions eluted at 230300 mM NaCl and were pooled. The pooled sample (23 mg) was applied to a 5 ml Bio-scale hydroxyapatite type I column (Bio-Rad) that was equilibrated at 1 ml min-1 with 5 mM sodium phosphate buffer at pH 6·5 (buffer B). The column was washed with 15 ml buffer B and developed with a linear sodium phosphate gradient (5500 mM sodium phosphate at pH 6·5 in 75 ml). Protein fractions (11·4 mg) with PDC activity eluted at 200300 mM sodium phosphate and were pooled. For further purification, portions of this material (0·250·5 mg protein per 0·250·5 ml) were applied to a Superdex 200 HR 10/30 column (Pharmacia) equilibrated at 0·25 ml min-1 in 50 mM sodium phosphate at pH 6·5 with 150 mM NaCl and 10% (v/v) glycerol in the presence and absence of 1 mM MgSO4 and 1 mM TPP.
Activity assays and protein electrophoretic techniques.
PDC activity was assayed by monitoring the pyruvic-acid-dependent reduction of NAD+ with bakers yeast alcohol dehydrogenase (ADH) (Sigma) as a coupling enzyme at pH 6·5 as previously described (Conway et al., 1987 ). The assay also included the following modifications. Buffered enzyme (100 µl) was added to a final volume of 1 ml containing 0·15 mM NADH, 0·1 mM TPP, 025 mM pyruvate and 10 U ADH in 50 mM potassium MES or 100 mM sodium hydrogen maleate buffer with 5 mM MgCl2 at pH 6·5. Since this assay does not distinguish PDC from NADH-oxidizing enzymes such as lactate dehydrogenase, activity of cell lysate was estimated by correcting for control reactions performed in the absence of added ADH. One unit of enzyme activity is defined as amount of enzyme that generates 1 µmol acetaldehyde min-1. Thermostability was determined by incubating purified PDC in 50 mM sodium phosphate buffer at pH 6·5 with 1 mM TPP and 1 mM MgCl2 for 90 min and then assaying for activity with 10 mM pyruvate. Protein concentration was determined using Bradford protein reagent with bovine serum albumin as the standard (Bio-Rad).
Molecular mass and N-terminal amino acid sequence analyses.
Subunit molecular mass was estimated by reducing and denaturing SDS-PAGE using 12% polyacrylamide gels which were stained with Coomassie blue R-250. The molecular mass standards for SDS-PAGE were: phosphorylase b (97·4 kDa), bovine serum albumin (66·2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21·5 kDa) and lysozyme (14·4 kDa). For determination of native molecular mass, samples were applied to a Superdex 200 HR 10/30 column equilibrated in 50 mM sodium phosphate buffer at pH 6·5 with 150 mM NaCl, 10% glycerol, and no added cofactors. Molecular mass standards included: bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), -amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa).
The N-terminal sequence was determined for PDC protein purified from recombinant E. coli. The protein was separated by SDS-PAGE and electroblotted onto a PVDF membrane (Immobilon-P). The sequence was determined by automated Edman degradation at the protein chemistry core facility of the University of Florida Interdisciplinary Center for Biotechnology Research.
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RESULTS AND DISCUSSION |
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A partial ORF was identified 722 bp upstream of pdc which encodes a 177 amino acid protein fragment (ORF1*) (Fig. 1). ORF1* has identity (2829%) to several hypothetical membrane proteins (GenBank accession numbers CAC11620, CAC24018, CAA22902) and is predicted to form several membrane-spanning domains (data not shown).
S. ventriculi PDC protein sequence
The S. ventriculi pdc gene apparently encodes a protein of 552 amino acids (including the N-terminal methionine) with a calculated pI of 5·16 and anhydrous molecular mass of 61737 Da. Consistent with other Z. mobilis and fungal PDC proteins, the N-terminal extension of up to 47 amino acids that is common to plant PDC proteins is not conserved in the S. ventriculi PDC protein. Although the pI of the purified S. ventriculi PDC protein has not been experimentally determined, the calculated pI is consistent with the acidic pH optimum of 6·36·7 for stability and activity of this enzyme (Lowe & Zeikus, 1992 ). The amino acid composition of the protein deduced from the S. ventriculi pdc gene is similar to that determined for the Saccharomyces cerevisiae PDC1 (Kellermann et al., 1986
) and Z. mobilis pdc genes (Conway et al., 1987
; Neale et al., 1987b
). A notable exception is the alanine composition of Z. mobilis PDC, which is 1·8- to 2·2-fold higher than that of S. cerevisiae PDC1 and S. ventriculi PDC. Although the phenylalanine composition of the deduced S. ventriculi PDC protein is consistent with the other PDC proteins, it is almost 3·6-fold less than the composition previously reported for the purified S. ventriculi enzyme (Lowe & Zeikus, 1992
). The reason for this discrepancy remains to be determined.
The amino acid sequence of S. ventriculi PDC was aligned with the sequences of the yeast (Sce) PDC1 and Z. mobilis (Zmo) PDC proteins, both of which have been analysed by X-ray crystallography (Arjunan et al., 1996 ; Dobritzsch et al., 1998
; Dyda et al., 1993
; König, 1998
) (Fig. 2
). The conserved motif of TPP-dependent enzymes identified by Hawkins et al. (1989)
and known to be involved in Mg2+-TPP cofactor binding is highly conserved in all three PDC proteins. Amino acid residues located within 0·4 nm of the Mg2+- and TPP-binding site of the PDC proteins which have been crystallized from yeast and Z. mobilis are conserved in primary sequence with the S. ventriculi PDC protein. These include residues with similarity to the aspartate (SceD444, ZmoD440) and asparagine residues (SceN471, ZmoN467) that are involved in binding Mg2+. The S. ventriculi PDC appears to be more similar to the yeast PDC than to that of Z. mobilis in binding the diphosphates of TPP, where serine and threonine side chains (SceS446 and T390) as well as the main chain nitrogen of isoleucine (SceI476) are conserved. This contrasts with the Z. mobilis enzyme, which utilizes a main-chain nitrogen of aspartate (ZmoD390) instead of the threonine hydroxyl group (SceT390) for binding the ß-phosphate. S. ventriculi PDC residues are also similar to the aspartate, glutamate, threonine and histidine residues (SceD28, E477, T388, H114 and H115; ZmoD27, E473, T388, H113 and H114) which may potentially interact with intermediates during the decarboxylation reaction mediated by Z. mobilis and yeast PDCs. Furthermore, the isoleucine (Sce and Zmo I415) side chain, which appears to stabilize the V conformation of TPP through Van der Waals interactions, as well as the glutamate (SceE51, ZmoE50) which may donate a proton to the N1' atom of TPP, are conserved in the S. ventriculi PDC protein sequence.
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Cluster analysis was performed to compare the PDC proteins and other TPP-dependent enzymes, including indole-3-pyruvate decarboxylase (IPD), the E1 component of pyruvate dehydrogenase (E1), acetolactate synthase (ALS), and transketolase (TK) (Fig. 3). The comparison reveals that all of these proteins are related in primary sequence and that there is a significant clustering of the sequences into families based on specific enzyme function. Of these, the S. ventriculi PDC appears most closely related to eubacterial IPD proteins as well as the majority of fungal PDC proteins. In contrast, the Z. mobilis PDC protein is most closely related to plant PDCs in addition to a couple of outgrouping fungal PDCs. Thus, it appears that the IPD protein family has close evolutionary roots with the PDC family and specifically the S. ventriculi PDC protein. In addition, the distant relationship of the S. ventriculi and Z. mobilis PDC proteins is consistent with the biochemical differences between these two enzymes (Hoppner & Doelle, 1983
; Lowe & Zeikus, 1992
; Neale et al., 1987a
; Ullrich & Donner, 1970
). In contrast to Z. mobilis PDC, which may have originated by the horizontal transfer of a plant pdc gene, S. ventriculi PDC appears to have diverged quite early during evolution and last shared a common ancestor with most eubacterial IPD and fungal PDCs.
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Biochemical and biophysical properties of the S. ventriculi PDC protein from recombinant E. coli
The S. ventriculi PDC protein was purified over 136-fold from recombinant E. coli. The N-terminal amino acid sequence of this protein (MKITIAEYLLXR, where X is an unidentified amino acid) was identical to the sequence of PDC purified from S. ventriculi (Lowe & Zeikus, 1992 ). Both PDC proteins have an N-terminal methionine residue, which suggests that this residue is not accessible for cleavage by either the S. ventriculi or E. coli aminopeptidases.
The thermostability of the purified S. ventriculi PDC was examined in the presence of 1 mM cofactors TPP and Mg2+ at pH 6·5. Enzyme activity was stable up to 42 °C but was abolished after incubation for 6090 min at temperatures of 50 °C and above. This is consistent with the significant loss of PDC activity observed when a thermal treatment step (60 °C for 30 min) was included in the purification (data not shown). In contrast, methods used to purify Z. mobilis and other PDC proteins (König, 1998 ) typically incorporate thermal treatment to remove unwanted proteins. These results suggest that the recombinant S. ventriculi PDC protein is not as thermostable as other PDC proteins, including that of Z. mobilis.
PDC proteins have been shown to bind TPP and Mg2+ cofactors with high affinity at slightly acidic pH (König, 1998 ). Consistent with this, the recombinant S. ventriculi PDC retains full activity after incubation at 37 °C for 90 min in the presence of 25 mM EGTA or EDTA in pH 6·5 buffer without cofactors. This is similar to the PDC protein purified directly from S. ventriculi, which is fully active after similar treatment with metal chelators.
The recombinant S. ventriculi enzyme displays sigmoidal kinetics (Fig. 5), suggesting that the enzyme is substrate activated, similar to the fungal and plant PDC proteins (König, 1998
). This contrasts with the Z. mobilis PDC, which is the only PDC known to display non-sigmoidal, MichaelisMenten kinetics. Based on the kinetic data, it appears that the S. ventriculi PDC has two affinities for pyruvate (Km values of 2·8 and 10 mM) (Fig. 5
). The Km value for the low-affinity site is comparable to the Km value (13 mM) reported by Lowe & Zeikus (1992)
. The fivefold lower Km observed in this present study is apparently due to the difference in the buffer, since maleate buffer significantly inhibited the PDC activity at low pyruvate concentrations (Fig. 5
). This difference in activity of S. ventriculi in maleate buffer is similar to that observed with Z. mobilis PDC, with a Km of 4·4 mM for pyruvate in Tris/maleate buffer at pH 6 (Hoppner & Doelle, 1983
) compared to Km values of 0·30·4 mM in potassium MES buffer at pH 6 (Bringer-Meyer et al., 1986
) and sodium citrate buffer at pH 6·5 (Neale et al., 1987b
).
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At pH 6·5, the recombinant S. ventriculi PDC forms a 235 kDa homotetramer consisting of a 58 kDa protein, as determined by Superdex 200 gel filtration chromatography and SDS-12% PAGE (see Methods). Exclusion of the cofactors from the buffer during gel filtration chromatography at pH 6·5 did not alter the tetramer configuration or enzyme activity, suggesting that the cofactors are tightly bound. The configuration of the PDC complex is consistent with that purified from S. ventriculi as well as the majority of those isolated from fungi, plants and Z. mobilis. There are, however, plant PDCs which have been reported to form larger complexes, including the PDC from Neurospora crassa, which forms aggregated filaments of 810 nm (Alvarez et al., 1993 ) as well as the PDC from Pisum sativum, which forms up to 960 kDa complexes (Mücke et al., 1995
).
Conclusions
Based on this study, the S. ventriculi PDC protein appears to share similar primary sequence structure with TPP-dependent enzymes and is highly related to the fungal PDC and eubacterial IPD enzymes. The close relationship of the S. ventriculi and fungal PDC structures is consistent with the similar biochemical and biophysical properties of these enzymes. Both types of enzymes display substrate cooperativity with similar affinities for pyruvate. The structure and biochemistry of the S. ventriculi PDC, however, dramatically contrast with the only other bacterial PDC (Z. mobilis) which has been characterized. The Z. mobilis PDC is closely related to plants in primary structure and it is the only PDC known to display MichaelisMenten kinetics.
This study also demonstrates the synthesis of active, soluble S. ventriculi PDC protein in recombinant E. coli. Only two other genes, the Z. mobilis pdc and Saccharomyces cerevisiae PDC1 genes, have been reported to synthesize PDC protein in recombinant bacteria (Braü & Sahm, 1986 ; Candy et al., 1991
; Conway et al., 1987
). Of these, at least 50% of the S. cerevisiae PDC1 forms insoluble inclusions in E. coli and thus has not been useful in engineering bacteria for high-level ethanol production (Candy et al., 1991
). Due to codon bias, accessory tRNA is essential for efficient production of S. ventriculi PDC in recombinant E. coli. However, the low-G+C codon bias of the S. ventriculi pdc gene should broaden the spectrum of bacteria that can be engineered as hosts for high-level production of PDC protein and the engineering of homo-ethanol pathways (Ingram et al., 1999
).
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ACKNOWLEDGEMENTS |
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Received 8 March 2001;
revised 26 May 2001;
accepted 4 June 2001.