Cloning and expression of the inorganic pyrophosphatase gene fromthe amino acid producer Brevibacterium lactofermentum

ATCC 13869

Angelina Ramos, Sirin A.I. Adham, Jose¤ A. Gil �

Area de Microbiolog|¤a, Departamento de Ecolog|¤a, Gene¤tica y Microbiolog|¤a, Facultad de Biolog|¤a, Universidad de Leo¤n, 24071 Leo¤n, Spain

Received 6 June 2003; accepted 10 June 2003

First published online 5 July 2003

Abstract

A 20-kDa Brevibacterium lactofermentum protein was detected when purifying the His-tagged FtsZBL. The protein was identified bymatrix-assisted laser desorption/ionisation time of flight as the inorganic pyrophosphatase encoded by the ppa gene, which is present as asingle copy in the genome of Corynebacterium glutamicum. The ppa gene was cloned from B. lactofermentum chromosomal DNA bypolymerase chain reaction; it seemed to be an essential gene and it might represent an attractive target for drug discovery. The cloned ppagene complemented a ppa3 Escherichia coli mutant and a ppa-gfp gene fusion revealed that the gene product mainly accumulated at thecell poles in both E. coli and B. lactofermentum.5 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Inorganic pyrophosphatase; FtsZ; DivIVA; Corynebacterium; Cell division; Green £uorescence protein

1. Introduction

Inorganic pyrophosphatases catalyse the hydrolysis ofpyrophosphate into phosphate (PPi to 2Pi). This is anessential process, since pyrophosphate represents a by-product in many important anabolic pathways such asRNA, DNA, and oligosaccharide biosynthesis, and thecharging of tRNAs. PPi hydrolysis is important for pullingthe above anabolic pathways in the direction of biosyn-thesis and replenishes the orthophosphate (Pi) required forphosphorylation [1].

There are two major types of inorganic pyrophospha-tases : soluble (sPPases) and membrane-embedded (Hþ-PPases). sPPases are ubiquitous proteins whose functionis to remove the PPi produced by anabolic reactions, andthey have been found in bacteria, fungi and animal cells.Membrane-embedded pyrophosphatases are protonpumps that utilise PPi as the driving force for Hþ transferacross biological membranes [2,3]. Hþ-PPases have been

identi¢ed in higher plants, parasitic protists, archaea, bac-teria and photosynthetic bacteria, but they are absent inanimals, fungi, and some bacteria, including enterobacte-ria.

Prokaryotic inorganic pyrophosphatase genes have beencloned from eubacteria (Escherichia coli [4], Bacillus sub-tilis [5], Bartonella [6], Rhodospirillum [7]) and from Ar-chaea (Sulfolobus [8], Aquifex [9], Methanococcus [10],Thermus [11] and Thermoplasma [12]). When we attemptedto characterise the proteins that interact with the essentialcell division protein FtsZ from Brevibacterium lactofer-mentum, the PPase from this microorganism was puri¢ed.Because PPases are considered to be excellent targets forantibacterial/antifungal drug development [13], we wereprompted to characterise the PPase from B. lactofermen-tum, which is used in our laboratory as a model to studythe cell division process in this pleomorphic and non-spor-ulating actinomycete.

2. Materials and methods

2.1. Bacterial strains, plasmids, and culture conditions

The bacterial strains and plasmids used in this work are

0378-1097 / 03 / $22.00 5 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.doi :10.1016/S0378-1097(03)00485-3

* Corresponding author. Tel. : +34 (987) 291503;Fax: +34 (987) 291479.

E-mail address: degjgs@unileon.es (J.A. Gil).

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www.fems-microbiology.org

described in Table 1. E. coli cells were grown on Luriabroth or Luria agar [14] at 37‡C with aeration. Comple-mentation of the E. coli ppa mutant was performed in2UYT medium [15]. When necessary, kanamycin (Km),chloramphenicol (Cm) and ampicillin (Ap) were addedto a ¢nal concentration of 50 Wg ml31. B. lactofermentumcells were grown in TSB (trypticase soy broth, Difco) orTSA (TSB containing 2% agar) at 30‡C.

2.2. Nucleic acid isolation and manipulation

Plasmid DNA was isolated from E. coli according to themethod of Holmes and Quigley [16]. E. coli cells weretransformed by the method of Hanahan [14].

The plasmids to be transferred by conjugation fromE. coli to B. lactofermentum were introduced by transfor-mation into the donor strain E. coli S17-1. B. lactofermen-tum R31 was used as recipient strain. The protocol forconjugation was a slight modi¢cation of the method de-veloped by Schafer et al. [17].

Puri¢cation of DNA fragments was carried out usingthe Gene Clean kit (Bio101, La Jolla, CA, USA). Restric-tion enzymes were purchased from Promega and Biolabs.

Total DNA from corynebacteria was isolated using themethod described for Streptomyces [18], but the cells weretreated with lysozyme for 4 h at 30‡C.

2.3. Cloning the ppa gene by PCR

The ppa gene from B. lactofermentum was cloned bypolymerase chain reaction (PCR), using primers designedfrom the DNA sequence of Corynebacterium glutamicum(NCBI accession number NC-003450) to amplify a 781-bp

fragment carrying the whole ppa gene and upstream se-quence (196 bp):

Ppa-1: 5P-CGCGGATCCGCTCCTTCTCCTGCAAA-TTTTCTTG-3P

Ppa-2: 5P-CGCGGATCCGGACACCCCTCCCAGCC-CAAG-3P.

Because of the presence of a BamHI site (GGATCC) inthe upper and lower primers, the ampli¢ed fragment wasdigested with BamHI and cloned into the conjugative vec-tor pECM2 (Table 1) digested with BglII, or in pBlue-script SKþ digested with BamHI, a¡ording plasmidspECPPA1 and pCPA respectively (Table 1).

2.4. Plasmid constructions

To disrupt the ppa gene by single recombination, aninternal 200-bp DNA fragment was cloned into the con-jugative suicide plasmid pK18mob (Table 1). The internalfragment of ppa was ampli¢ed by PCR using the followingprimers:

Ppa-3: 5P-CCGGAATTCATGGCATACCCACTGGA-CTACGG-3P

Ppa-4: 5P-CTAGTCTAGACTTGAGGAAATCGGA-CACGTCGG-3P.

Because of the presence of EcoRI (GAATTC) and XbaI(TCTAGA) in the primers, the ampli¢ed fragment wasdigested with those enzymes and cloned into the EcoRI-XbaI-digested pK18mob plasmid. The new plasmid wasnamed pKPPA (Table 1). If a single recombination eventtakes place, two interrupted ORF8s will be created, onelacking 51 amino acids from its C-terminus and the otherlacking 43 amino acids from its N-terminus.

A ppa-gfp translational fusion was made by PCR. The

Table 1Bacterial strains and plasmids

Strain or plasmid Relevant genotype or description Source or reference

StrainsE. coli DH5K r3m3 used for general cloning [14]E. coli K31 EKTRP (pEP) ppa3 temperature-sensitive mutant Dr P. PlateauE. coli JM109 (DE3) JM109 derivative containing a chromosomal copy of the gene for T7 RNA polymerase PromegaB. lactofermentum 13869 wt ATCCB. lactofermentum R31 13869 derivative used as host for transformation, electroporation or conjugation [29]PlasmidspBS KS/SK E. coli vector containing bla, lacZ, orif1 StratagenepECM2 Mobilisable E. coli/B. lactofermentum bifunctional plasmid containing kan and cat [26]pECPPA1 pECM2 derivative containing ppa from B. lactofermentum and upstream sequences Fig. 2pCPPA pBluescript SKþ derivative containing ppa from B. lactofermentum and upstream sequences Fig. 2pK18mob Mobilisable plasmid containing an E. coli origin of replication and kan [30]pKPPA pK18mob derivative containing an internal 200 bp from B. lactofermentum ppa gene

ampli¢ed by PCRFig. 2

pKMM3 pK18mob derivative containing an internal 200-bp HaeIII fragment from the gluconatekinase (gntK) gene from C. glutamicum

Michal Letek and Luis M.Mateos, unpublished

pECPPA2 pECM2 derivative containing the whole ppa gene and upstream sequences fused to gfp Fig. 2pET-28a(+) E. coli vector containing kan, lacI, orif1, N-terminal and C-terminal His tag NovagenpETPPA pET28a derivative containing ppa-6UHis under Plac Fig. 2

ATCC: American Type Culture Collection.

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3P end of ppa was ampli¢ed by PCR using primer Ppa-1and the following primer:

Ppa-5: 5P-GGGAATTCCATATGTGCCTTGTAGCG-GTCG-3P.

These primers were designed to replace the stop codon(TAA) of ppa by CAT (His) (ATG, in the lower primer),which after NdeI digestion and ligation with gfp is imme-diately followed by the ATG start of gfp. Because of thepresence of restriction sites in the primers [BamHI(GGATCC) and NdeI (CATATG)], the ampli¢ed frag-ment was digested with these enzymes and the BamHI-NdeI-ampli¢ed fragment was cloned together with gfp(as a NdeI-XbaI fragment) in plasmid pECM2, generatingpECPPA2. The gfp gene used was egfp from Clontech,which includes the mutations V163A and S175G intro-duced by Siemering et al. [19]. The ppa-gfp fusion wascon¢rmed to be correct by sequencing, using the dideoxynucleotide chain termination method of Sanger.

Plasmid pETPPA was constructed for the expression ofHis-tagged PPase in E. coli by PCR ampli¢cation, usingthe following primers and subcloning into pET-28a(+).The primers used were Ppa-2 and:

Ppa-6: 5P-GGGAATTCCATATGAGCATCGAAGT-AACCGTCG-3P.

The ampli¢ed fragment was digested with NdeI (CAT-ATG)+BamHI (GGATCC) and cloned downstream ofPlac into pET-28a(+) digested with the same enzymes.

2.5. PPase activity

To determine PPase activity, cell-free extracts of B. lac-tofermentum R31 and B. lactofermentum [pECPPA1] weredisrupted by sonication in PPase bu¡er (50 mM Tris^HCl,3 mM MgCl2, 0.2 mM EGTA, pH 7.5) containing 0.2%bovine serum albumin. Sonication was carried out overperiods of 30 s at 1-min intervals in an ice-cooled tubeusing a Branson sonicator (model B-12) at 75^100 W untilthe cells had been completely disrupted, as observed mi-croscopically. Cell debris was removed by centrifugation,and supernatants were dialysed against the same bu¡er toeliminate the inorganic phosphate present in the extracts.The enzymatic assay was performed as described previ-ously [20], using malachite green reagent (3 parts of0.045% malachite green+5 ml l31 of concentrated HCland 1 part of 4.2% ammonium molybdate in 4 M HCl)to quantify the inorganic phosphate released. Samplescontaining phosphate developed a green colour, while neg-ative controls remained yellow. One unit of activity wasde¢ned as the amount of enzyme that liberated 1 Wmol ofinorganic pyrophosphate in 30 min at 25‡C.

2.6. Puri¢cation of His-tagged PPase from E. coli

Puri¢cation of the His-tagged protein from isopropyl-L-D-thiogalactose-induced E. coli JM109 DE3 [pETPPA]cells, grown at 37‡C in LB medium supplemented with

100 Wg ml31 ampicillin, was accomplished according tothe standard procedures of the manufacturer Novagen(http://www.novagen.com/docs/NDIS/C281-000.pdf)

2.7. Protein^protein interactions, PAGE and MALDI-TOF

Protein interactions were performed using eitherFtsZBL-6UHis or PPaseBL-6UHis as the primary protein,attached to Ni-NTA magnetic agarose beads, and a cell-free extract of B. lactofermentum R31 was used as thesolution containing potentially interacting macromole-cules. Protocol 3 from Qiagen was used (http://www.qiagen.com/literature/Handbooks/PDF/Protein/INT/NiNTA_Magnetic_Agarose_Beads/1018847HBNiNTAMB_prot03.pdf).

Eluted proteins were analysed by sodium dodecyl sul-fate^polyacrylamide gel electrophoresis (SDS^PAGE) es-sentially as described by Laemmli [21]. Electrophoresiswas performed at room temperature in a vertical slab gel(170U130U1.5 mm), using 12% (w/v) polyacrylamide at100 V and 60 mA. After electrophoresis, the proteins weresilver-stained (Bio-Rad) and the protein bands of interestwere cut out from the gel, and analysed by matrix-assistedlaser desorption/ionisation time of £ight (MALDI-TOF)at the Proteomic Service, National Centre of Biotechnol-ogy (Madrid, Spain).

2.8. Microscopic techniques

B. lactofermentum and E. coli cells containing construc-tions carrying green £uorescent protein (GFP) were ob-

1 2 3 4 5

FtsZ+6His

PPA

97.4

45

31

21.5

14.4

66.2

Fig. 1. Isolation of PPase from B. lactofermentum. Ni-NTA magneticagarose beads were loaded with His-tagged FtsZBL (in Protein BindingBu¡er, Qiagen) that had been expressed in E. coli, and washed severaltimes with Wash bu¡er (Qiagen) to remove unwanted E. coli proteins(lane 2). Then, a B. lactofermentum R31 cell-free extract (lane 3) (in In-teraction Bu¡er, Qiagen) was added to the His-tagged FtsZBL-Ni-NTAmagnetic agarose beads, washed several times with Interaction Bu¡er(Qiagen) to remove any B. lactofermentum protein non-speci¢callybound to FtsZ, and potential FtsZ-associated proteins were eluted usingElution Bu¡er (lane 4). Lane 1, molecular mass markers. Lane 5, E. coliextract expressing His-tagged FtsZBL.

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served under a Nikon E400 £uorescence microscope. Pic-tures were taken with a DN100 Nikon digital camera andassembled using Corel Draw.

3. Results and discussion

3.1. Identi¢cation of the inorganic pyrophosphatase fromB. lactofermentum/C. glutamicum

A protein of c. 20 kDa from B. lactofermentum co-pu-ri¢ed with FtsZBL-6UHis when we isolated B. lactofer-mentum proteins that interact with the essential cell divi-sion FtsZBL protein (Fig. 1). This 20-kDa protein wasidenti¢ed by MALDI-TOF as the inorganic pyrophospha-tase from C. glutamicum (NCBI accession number:NP_601896) (Fig. 2). Since inorganic pyrophosphatasesare essential enzymes [22,23] and, at least in E. coli, theirsynthesis is stimulated when E. coli cells are cultured in thepresence of penicillin, which inhibits cell division andcauses ¢lamentation [24], we attempted to establish a pos-sible relationship between FtsZ and PPase and hence de-cided to undertake the cloning of the ppaBL gene.

In C. glutamicum, the open reading frame (CGL2607)for ppa contains 477 bp and encodes a putative protein of158 amino acids with a predicted molecular mass of 17.9kDa. The ppa gene is located, in opposite direction, 201 bpdownstream of the open reading frame CGL2606 whichencodes a D-alanyl-D-alanine carboxypeptidase (PBP4).

500 bp downstream of ppa, and in the opposite direction,the speE gene, encoding spermidine synthase (CGL2608),is found (Fig. 2).

There are 38 sPPase sequences currently available inGenBank: prokaryotic PPases have 158^233 amino acidresidues per subunit, whereas membrane-bound PPases aremuch larger (660^770 amino acid residues per monomer)and do not have any sequence similarity to sPPases [5].Accordingly, the C. glutamicum PPase is clearly a sPPase,belonging to family I and showing the typical PrositeDGDPLD signature (three conserved aspartates that areinvolved in the binding of divalent cations).

Most prokaryotes do have Mg2þ-dependent sPPasesand no PPase activity can be observed in the absence ofbivalent cations. However, this absence can be compen-sated by the presence of spermine and spermidine. It hasbeen deduced that polyamines are able to form a complexwith PPi that serves as a substrate for PPase [25]. There-fore, the singular presence of both genes (ppa and speE) inthe genome of C. glutamicum might be of as yet unknownevolutionary or ecological signi¢cance.

3.2. Cloning of the ppaBL gene and its expression inB. lactofermentum

The ppaBL gene and upstream sequences were ampli¢edby PCR and cloned into the conjugative plasmid pECM2[26], a¡ording pECPPA1 (Fig. 2). This plasmid was intro-duced into B. lactofermentum by conjugation, and the

Fig. 2. Map of the ppa region on the C. glutamicum genome (accession number NC-003450). The ppa gene (CGL2607) is preceded by the open readingframe (CGL2606) annotated as ‘D-alanyl-D-alanine carboxypeptidase’. The open reading frame CGL2608 has the opposite orientation, and is annotatedas ‘spermidine synthase’. Gene orientations are indicated by thin arrows. The inserts in relevant plasmids are shown and the name of the vector DNAis indicated in parentheses. Squares represent PCR primers and parentheses the name of the restriction enzyme used for digestion after PCR ampli¢ca-tion.

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transconjugants behaved exactly like the untransformedstrain.

Crude protein extracts of exponentially growing B. lac-tofermentum R31 and B. lactofermentum [pECPPA1] wereprepared and assayed for PPase activity. B. lactofermen-tum R31 showed a pyrophosphatase activity of 0.028 WmolPi Wg31 of protein, whereas B. lactofermentum [pECPPA1]released 0.045 Wmol Pi Wg31 of protein. This indicated thatoverexpression of PPase is not toxic in B. lactofermentum,as has been described for E. coli [4].

Our attempts to disrupt ppaBL using plasmid pKPPA(Table 1; Fig. 2) were consistently unsuccessful. It couldbe argued that 200 bp is a small fragment of DNA forrecombination, but control experiments using plasmidpKMM3 (containing a 200-bp internal fragment ofgnkP) always a¡orded kanamycin-resistant transconju-gants unable to use gluconic acid as the sole carbonsource. The observations that PPase is essential for theviability of E. coli [22], Legionella pneumophila [27] andyeasts [23], together with our unsuccessful attempts toconstruct a mutation in ppa, suggested that this gene isprobably essential for the viability of B. lactofermentum.

3.3. Identi¢cation of the ppa gene product

In order to con¢rm the biological activity of the clonedppa gene from B. lactofermentum, we attempted to com-plement an E. coli ppa temperature-sensitive mutant [E.coli K31 EKTRP (pEP)] with the whole ppa present inplasmid pCPPA (Fig. 2). This E. coli mutant was con-structed by Chen et al. [22] and contained the chromosom-al copy of ppa disrupted by the insertion of kan and atemperature-sensitive plasmid (pEP) carrying cat and withthe wild-type ppa. As can be observed in Fig. 3, the E. colimutant was able to grow at 37‡C but unable to grow at42‡C, whereas the mutant transformed with pCPPA wasable to grow at both temperatures. To con¢rm the loss ofplasmid pEP at 42‡C, and therefore the real complementa-tion of the mutant by PPaseBL, cells from E. coli K31EKTRP [pEP+pCPPA] grown at 42‡C were inoculated at37‡C on chloramphenicol-containing plates and no growthwas detected. By contrast, they were able to grow at both37‡C and 42‡C on ampicillin+kanamycin plates. There-fore, the cloned gene must encode a pyrophosphataseable to complement the E. coli mutant.

37ºC 42ºC

2x YT+Km

2x YT+Km+Cm

E. coli K37 EKTRP (pE')

37ºC

42ºC

37ºC

2x YT+Ap+Km

2x YT+Ap+Km

2x YT+Km+Cm

37ºC 42ºC

2x YT+Ap+Km

E. coli K37 EKTRP (pE' + pCPPA)

Fig. 3. Complementation of the E. coli ppa mutant with ppa from B. lactofermentum. For details, see text.

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3.4. Construction of ppaBL-gfp fusions

To the best of our knowledge, there are no reportsabout the cellular location of sPPase in bacteria, althoughit may be assumed that it should be located throughoutthe cell. There is only a report addressing the quanti¢ca-tion of ppa expression in the intracellular pathogenL. pneumophila, in which its PPase is induced during intra-cellular infection [27].

The bifunctional plasmid pECPPA2 (containing ppaBL-gfp under the control of Pppa) (Fig. 2) was introducedinto both E. coli DH5K and B. lactofermentum R31. Mi-croscopic observation of both strains revealed that PPase-GFP was located mainly at one of the cell poles (Fig. 4).Similar results were obtained when we studied the cellularlocation of the B. lactofermentum DivIVABL-GFP in E. coliand B. lactofermentum and FtsZ-GFP in B. lactofermen-tum (Ramos et al., unpublished results). The size and in-tensity of the £uorescent region of PPase-GFP at the endof the cells indicated that a large number of molecules isconcentrated there, and this suggests either that a highPPase activity must be taking place there or that this local-isation could be an artefact due to the presence of GFPfused to PPase. We therefore suggest that PPaseBL is anessential protein with possible function at the growing cellpoles of B. lactofermentum.

3.5. PPase-His puri¢cation and interaction studies

Because PPaseBL co-puri¢es with FtsZBL-6UHis, we de-cided to obtain a large amount of PPase to perform the

reverse experiment, and thus purify FtsZBL by interactionwith PPaseBL-6UHis. Puri¢cation of PPase 6UHis-taggedenzyme was accomplished in a single step by chromatog-raphy of the crude extract from induced E. coli [pETPPA]cells on Ni-NTA agarose (Novagen). 1.3 mg of PPase-6UHis protein was obtained from only 50 ml of culture,which seemed to be at least 90% pure as judged by SDS^PAGE (Fig. 5). The speci¢c pyrophosphatase activity ofthe puri¢ed enzyme was 20 Wmol Wg31 of protein. Noenzymatic activity was detected in the absence of Mg2þ

Fig. 4. Subcellular localisation of PPaseBL-GFP. The strains used were B. lactofermentum [pECPPA2] (A, AP) and E. coli [pECPPA2] (B, BP). Phasecontrast microscopy and £uorescence microscopy pictures were exposed for 1/15 and 1 s respectively. Pictures A and B were taken at the same magni¢-cation (U400), whereas pictures AP and BP were enlarged digitally.

1 2 3 4 5 6 7 8

97.4

31

45

66.2

21.5

14.4

Fig. 5. Puri¢cation of the PPase-6UHis. An E. coli cell-free extract (ex-pressing His-tagged PPaseBL) was passed through a Ni-NTA column,and washed with Wash bu¡er (Novagen) to remove unwanted E. coliproteins. Then, proteins bound to the column were eluted using Elutebu¡er (Novagen) (di¡erent fractions, lanes 2^8). Lane 1, molecular massmarkers.

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in the reaction mixture or in the presence of 1 mM EDTA,as occurs with the E. coli enzyme [28].

Interaction assays was performed by binding PPaseBL-6UHis to Ni-NTA magnetic agarose beads and adding acell-free extract of B. lactofermentum as a source ofFtsZBL. Under di¡erent experimental conditions, FtsZBL

failed to co-purify with PPaseBL-6UHis. However, in re-peated experiments PPase consistently co-puri¢ed whenFtsZBL-6UHis was bound to Ni-NTA agarose beads.Moreover, untagged PPase was not retained by the Ni-NTA agarose beads. It may therefore be concluded thatthe co-puri¢cation of PPaseBL with FtsZBL-6UHis mustbe due to some non-speci¢c and hitherto unknown inter-actions between FtsZ bound to Ni-NTA agarose beadsand PPaseBL and not to the binding of PPase to the Ni-NTA agarose beads.

In conclusion, PPase seems to be an essential enzyme inB. lactofermentum with a strict requirement for Mg2þ thatwas detected because it co-puri¢ed with FtsZ in interac-tion experiments. PPase-GFP localised to the cell poles ofB. lactofermentum and E. coli, suggesting a still unknownrole in cell morphology or cell growth at the cell poles.

Acknowledgements

This work was supported by the Junta de Castilla yLeo¤n (LE 24/01) and the Ministerio de Ciencia y Tecno-log|¤a (BIO 2002-03223) grants. A.R. and S.A.I.A. wererecipients of fellowships from the Junta de Castilla yLeo¤n and from the Agencia Espan‹ola de Cooperacio¤nInternacional (AECI) respectively. We thank Dr PierrePlateau (Laboratoire de Biochimie, Centre National dela Recherche Scienti¢que, Ecole Polytechnique, Palaiseau,France) for E. coli K31 EKTRP (pEP). Thanks are alsodue to N. Skinner for supervising the English version ofthe manuscript.

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