Plant Molecular Biology 20: 997-1001, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
997
Update section
Short communication
Identification of a second psaC gene in the cyanobacterium Synechocystis sp. PCC6803 Klaus Steinm/iller
Institutf~irEntwicklungs-und Molekularbiologieder Pflanzen, Heinrich-Heine-Universittit, Universitiitsstrasse 1, 4000 Diisseldorf i, Germany Received 28 January 1992; accepted in revised form 12 July 1992
Key words:Synechocystis sp. PCC6803, photosystem I, iron-sulfur protein, psaC Abstract The psaC gene encodes the 9 kDa protein subunit of photosystem I that carries the iron-sulfur centers F A and FB. The paper describes the sequence and transcription analysis of a second psaC gene (psaC2) from Synechocystissp. PCC6803. The psaC2 gene is transcribed into an abundant monocistronic mRNA. The deduced amino acid sequence of PSI-C2 is very similar to the protein sequences from two other cyanobacteria (Synechococcus sp. PCC7002 and Nostoc sp. PCC8009). In contrast, the recently isolated psaC1 gene is not transcribed and the PSI-C1 amino acid sequence shows the highest similarity to the proteins from higher plants.
The PSI-C protein is the iron-sulfur protein of photosystem I that harbors the iron-sulfur clusters F A and FB [9, 11, 14, 25]. The nucleotide and the protein sequences of several psaC genes and PSI-C proteins, respectively, of cyanobacteria, algae and plants are known [7, 18]. The psaC sequence of the cyanobacterium Synechocystis 6803 is of special interest, because this organism is frequently used in studies of the function of proteins of the photosynthetic apparatus [3, 4, 6, 24]. Recently, the nucleotide sequence of a psaC gene of Synechocystis6803 was published [1]; however the deduced amino acid sequence of the
polypeptide resembles more closely the amino acid sequences of the PSI-C proteins of higher plants than those of cyanobacteria. This paper describes the isolation of a second psaC gene from Synechocystis 6803, whose deduced protein sequence is more closely related to the PSI-C proteins of other cyanobacteria. The 3369 bp Bgl II/Xba I fragment (nucleotides 116171-119540) of the tobacco plastid DNA subclone pNtcPsl (PstI fragment, nucleotides 99983-123672 [20,21]) was inserted into the Barn HI/Xba I sites of the vector Bluescript KS, yielding the subclone pD3369BX. From this clone
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X65170, Synechocystis sp. PCC6803, psaC gene.
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Fig. 1. Southern and northern btot analysis of the psaC2 gene of Synechocystis 6803. A, B, C. Southern blots of genomic D N A isolated from Synechocystis 6803 and tested with the tobacco probe (A) and the Synechocystis probe (B, C) as described in the text. The following restriction enzymes were used: lane 1: Hind III; lane 2: Eco RI; lane 3: Pst I; lane 4: Bgl II; lane 5: Bst EII; lane 6: Hinc II; lane 7: Ssp I; lane 8: Xba I; lane 9: Eco RI and SalI. Lane O: undigested DNA. Blot B was preincubated with 0.25 N HC1 as recommended [23] to improve the transfer of undigested D N A and the large Pst I fragment. Each lane contains 4 #g DNA. D. Northern blot analysis with RNA (5 #g) isolated from mixotrophically grown cells.
a 629 bp Hinc II/Xba I fragment, containing the psaC reading frame and the first 47 nucleotides of the ndhD gene, was isolated. The fragment was used to test a Southern blot of genomic D N A of Synechocystis 6803. The hybridization was performed at reduced stringency (51 ° C) in 250 m M sodium phosphate pH 7.2, 7~o SDS and 2.5 m M EDTA and the membranes were washed five times at 51 °C with 1.5x SSC, 0.1~o SDS. Under these conditions specific hybridization signals were obtained, which demonstrated that the tobacco probe can be used to identify the cyanobacterial psaC gene. (Fig. 1A). A library of genomic D N A of Synechocystis 6803 in the bacteriophage 2 dash was screened with the tobacco probe and one phage (261) was isolated, which contained the psaC gene on a 3.5 kb SaII fragment. The fragment was subcloned in the vector pSP65 (clone p61S3.5I) and mapped with restriction enzymes (Fig. 2). The
psaC reading frame was located around the Xba I site by hybridization with the tobacco probe (data not shown). Sequencing of this region identified the psaC reading frame (Fig. 3). It codes for 81 amino acids and specifies a polypeptide of the calculated molecular mass of 8829 Da. The gene was designated psaC2, to distinguish it from the psaC gene identified by Anderson and McIntosh
(psa Cl [ 11). F---
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Fig. 2. Restriction map of the 3.5kb SalI fragment of p61S3.5I. The left SalI and the left Eco RI site are derived from the multiple cloning site of 2 dash. The open box represents the psaC2 reading frame and the arrow indicates the direction of transcription.
999
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Fig. 3. Nucleotide and deduced amino acid sequence of the psaC2 gene of Synechocystis 6803. The nucleotide sequence is shown from the Ssp I to the Hinc II site. A possible ribosome binding site (rbs) is underlined.
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Fig. 4. Amino acid sequence comparison between the P S I - C polypeptides from various organisms. The sequences are derived from the following publications: Syneehococcus sp. PCC7002 [2, 15], Nostoc sp. PCC8009 [2], Synechococcus vulcanus [ 19], Cyanophora paradoxa [2, 15], Chlarnydornonasreinhardtii [22], liverwort [ 12] Synechocystis sp PCC6803 [ 1 ], tobacco [9], spinach [ 14], pea [5], barley [17], rice [10], wheat [5] and maize [ 16]. Points indicate identical amino acids•
1000 Southern hybridization was carried out with a homologous D N A fragment (nucleotides 4-366 of the sequence in Fig. 3). Fig. 1B shows that the same bands seen with the tobacco probe are labelled. In a second control experiment, restriction enzymes that cut within the 3.5 kb Sal I fragment were used. The labelled bands correspond well to the fragments predicted from the map of the 3.5 kb Sal I fragment (Fig. 1C). In these experiments, the blots were hybridized and washed at 61 °C and the salt concentration in the last three washing steps was lowered to 0.1 x SSC, 0.1~o SDS. Amino acid sequence comparison revealed that PSI-C2 is very similar to the PSI-C proteins of other cyanobacteria (Fig. 4). The amino acid sequence differs in only one position from the Synechococcus 7002 sequence, while there are eight to ten amino acid substitutions compared to the proteins from higher plants. In contrast, the amino acid sequence of PSI-C1 is identical to that of tobacco and is therefore closely related to the sequences from higher plants. As was pointed out by Anderson and McIntosh [1], this similarity is also reflected at the nucleotide level. For example, the psaC1 nucleotide sequence is 93 ~o identical to the tobacco sequence, but only 75 ~o to psaC2. In the light of this finding, it remains unclear why, under our hybridization conditions, the tobacco psaC probe did not detect the very similar psaC1 gene of Synechocystis 6803 but did detect the more divergent psaC2 gene (Fig. 1A). Interestingly, the nucleotide sequence of the ndhE gene, which flanks the psaC1 gene, is also more similar to the ndhE gene of tobacco (86 ~o identity) than to a recently identified second ndhE gene in Synechocystis 6803 (62~o identity; U. Ellersiek and K. Steinm~iller, in preparation). Moreover, this second ndhE gene is located at a different genomic position (i.e. more than 5 kb away). One may speculate, therefore, that the psaC1 gene and its flanking regions were derived from a transformation of Synechocystis 6803 with higher-plant chloroplast DNA. This process might have taken place in nature because Synechocystis 6803 can be transformed by naked D N A [8] and D N A is stabilized by adsorption to soil particles [ 13].
The expression of the psaC2 gene was studied in a northern blot experiment with the homologous psaC2 probe. The psaC2 gene is transcribed into an abundant m R N A of about 450 nucleotides (Fig. 1D). Monocistronic transcription and high m R N A levels have also been reported for the psaC genes from Synechococcus7002 and Nostoc 8009 [2]. In contrast, Anderson and Mclntosh did not detect specific transcripts for psaC1 [1]. In higher plants, psaC is cotranscribed with the downstream located ndhD gene [9, 16], while in the cyanobacterium Synechococcus7002, no adjacent reading frame for ndhD was found [15]. Preliminary hybridization and sequencing data for Synechocystis 6803 indicate that a ndhD gene is located on the opposite strand, upstream of psaC2. Thus the situation in cyanobacteria differs from that in chloroplasts and it remains to be seen what the regulatory implications for this gene arrangement are.
Acknowledgements I would like to thank Ulrike Ellersiek for excellent technical assistance and Alan Blowers for a gift of the tobacco plastid D N A subclone pNtcPsl. Many thanks to Don Bryant for helpful discussions and unpublished data and to Peter Westhoff for critical reading of the manuscript. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 189).
References 1. Anderson SL, McIntosh L: Partial conservation of the 5' ndhE-psaC-ndhD 3' gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts. Plant Mol Biol 16:497-499 (1991). 2. Bryant DA, Rhiel E, de Lorimier R, Zhou J, Stirewalt VL, Gasparich GE, Dubbs JM, Snyder W: Analysis of phycobilisome and photosystem I complexes of cyanobacteria. In: Baltscheffsky M (ed) Current Research in Photosynthesis, vol. II, pp, 1-9. Kluwer Academic Publishers, Dordrecht 1990. 3. Chitnis PR, Reilly PA, Miedel MC, Nelson N: Structure
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and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterinm Synechocystis sp. PCC6803. J Biol Chem 264:18374-18380 (1989). Debus RJ, Barry BA, Babcock GT, McIntosh L: Sitedirected mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 85:427-430 (1988). Dunn PPJ, Gray JC: Localization and nucleotide sequence of the gene for the 8 kDa subunit of photosystern I in pea and wheat chloroplast DNA. Plant Mol Biol 11:311-319 (1988). Dzelzkalns VA, Bogorad L: Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure. EMBO J 7:333-338 (1988). Golbeck JH, Bryant DA: Photosystem I. Curr Topics Bioenerget 16:83-177 (1991). Grigorieva G, Shestakov S: Transformation in the cyanobacterium Synechoeystis sp. 6803. FEMS Microbiol Lett 13:367-370 (1982). Hayashida N, Matsubayshi T, Shinozaki K, Sugiura M, Inoue K, Hiyama T: The gene for the 9 kd polypeptide, a possible apoprotein for the iron-sulfur centers A and B of the photosystem I complex, in tobacco chloroplast DNA. Curr Genet 12:247-250 (1987). Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun CR, Meng BY, Li YQ, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M: The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217:185-194 (1989). Hoj PB, Svendsen I, Scheller HV, Moller BL: Identification of a chloroplast-encoded 9-kDa polypeptide as a 214Fe-4S] protein carrying centers A and B of photosystern I. J Biol Chem 262:12676-12684 (1987). Kohchi T, Shirai H, Fukuzawa H, Sano T, Komano T, Umesono K, Inokuchi H, Ozeki H, Ohyama K: Structure and organization of Marchantia polymorpha chloroplast genome IV. Inverted repeat and small single copy regions. J Mol Biol 203:353-372 (1988). Lorenz MG, Wackernagel W: Impact of mineral surfaces on gene transfer by transformation in natural bacterial environments. In: KlingmOllerW (ed) Risk Assessment for Deliberate Releases, pp. 111-119. Springer-Verlag, Berlin/Heidelberg (1988). Oh-oka H, Takahashi Y, Kuriyama K, Saeki K, Matsubara H: The protein responsible for center A/B in spinach
photosystem I: Isolation wkh iron-sulfur cluster(s) and complete sequence analysis. J Biochem 103:962-968 (1988). 15. Rhiel E, Stirewalt VL, Gasparich GE, Bryant DA: The psaC genes of Synechococcus sp PCC7002 and Cyanophora paradoxa: cloning and sequence analysis. Gene (in press). 16. Schantz R, Bogorad L: Maize chloroplast genes ndhD, ndhE and psaC. Sequences, transcripts and transcript pools. Plant Mol Biol 11:239-247 (1988). 17. Scheller HV, Svendsen I, Moller BL: Amino acid sequence of the 9-kDa iron-sulfur protein of photosystem I in barley. Carlsberg Res Commun 54:11-15 (1989). 1.8. Scheller HV, Moller BL: Photosystem I polypeptides. Physiol Plant 78:484-494 (1990). 19. Sbimizu T, Hiyama T, Ikeuchi M, Koike H, Inoue Y: Nucleotide sequence of the psaC gene of the cyanobacterium Synechococcus vulcanus. Nucl Acids Res 18:3644 (1990). 20. Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chungwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazama K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M: The complete nncleotide sequence of the tobacco chloroplast genome. Plant Mol Biol Rep 4:111-147 (1986). 21. Sugiura M, Shinozaki K, Zaita N, Kusuda M, Kumano M: Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal protein genes. Plant Sci 44:211-216 (1986). 22. Takahashi Y, Goldschmidt-Clermont, Soen SY, Franz6n LG, Rochaix JD: Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I. EMBO J 10:2033-2040 (1991). 23. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683-3687 (1979). 24. Williams JGK: Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167:766-778 (1988). 25. Wynn RM, Malkin R: Characterization of an isolated chloroplast membrane Fe-S protein and its identification as the photosystem I Ee-SA/Fe-S Bbindingprotein. FEB S Lett 229:293-297 (1988).
997
Update section
Short communication
Identification of a second psaC gene in the cyanobacterium Synechocystis sp. PCC6803 Klaus Steinm/iller
Institutf~irEntwicklungs-und Molekularbiologieder Pflanzen, Heinrich-Heine-Universittit, Universitiitsstrasse 1, 4000 Diisseldorf i, Germany Received 28 January 1992; accepted in revised form 12 July 1992
Key words:Synechocystis sp. PCC6803, photosystem I, iron-sulfur protein, psaC Abstract The psaC gene encodes the 9 kDa protein subunit of photosystem I that carries the iron-sulfur centers F A and FB. The paper describes the sequence and transcription analysis of a second psaC gene (psaC2) from Synechocystissp. PCC6803. The psaC2 gene is transcribed into an abundant monocistronic mRNA. The deduced amino acid sequence of PSI-C2 is very similar to the protein sequences from two other cyanobacteria (Synechococcus sp. PCC7002 and Nostoc sp. PCC8009). In contrast, the recently isolated psaC1 gene is not transcribed and the PSI-C1 amino acid sequence shows the highest similarity to the proteins from higher plants.
The PSI-C protein is the iron-sulfur protein of photosystem I that harbors the iron-sulfur clusters F A and FB [9, 11, 14, 25]. The nucleotide and the protein sequences of several psaC genes and PSI-C proteins, respectively, of cyanobacteria, algae and plants are known [7, 18]. The psaC sequence of the cyanobacterium Synechocystis 6803 is of special interest, because this organism is frequently used in studies of the function of proteins of the photosynthetic apparatus [3, 4, 6, 24]. Recently, the nucleotide sequence of a psaC gene of Synechocystis6803 was published [1]; however the deduced amino acid sequence of the
polypeptide resembles more closely the amino acid sequences of the PSI-C proteins of higher plants than those of cyanobacteria. This paper describes the isolation of a second psaC gene from Synechocystis 6803, whose deduced protein sequence is more closely related to the PSI-C proteins of other cyanobacteria. The 3369 bp Bgl II/Xba I fragment (nucleotides 116171-119540) of the tobacco plastid DNA subclone pNtcPsl (PstI fragment, nucleotides 99983-123672 [20,21]) was inserted into the Barn HI/Xba I sites of the vector Bluescript KS, yielding the subclone pD3369BX. From this clone
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X65170, Synechocystis sp. PCC6803, psaC gene.
998
Fig. 1. Southern and northern btot analysis of the psaC2 gene of Synechocystis 6803. A, B, C. Southern blots of genomic D N A isolated from Synechocystis 6803 and tested with the tobacco probe (A) and the Synechocystis probe (B, C) as described in the text. The following restriction enzymes were used: lane 1: Hind III; lane 2: Eco RI; lane 3: Pst I; lane 4: Bgl II; lane 5: Bst EII; lane 6: Hinc II; lane 7: Ssp I; lane 8: Xba I; lane 9: Eco RI and SalI. Lane O: undigested DNA. Blot B was preincubated with 0.25 N HC1 as recommended [23] to improve the transfer of undigested D N A and the large Pst I fragment. Each lane contains 4 #g DNA. D. Northern blot analysis with RNA (5 #g) isolated from mixotrophically grown cells.
a 629 bp Hinc II/Xba I fragment, containing the psaC reading frame and the first 47 nucleotides of the ndhD gene, was isolated. The fragment was used to test a Southern blot of genomic D N A of Synechocystis 6803. The hybridization was performed at reduced stringency (51 ° C) in 250 m M sodium phosphate pH 7.2, 7~o SDS and 2.5 m M EDTA and the membranes were washed five times at 51 °C with 1.5x SSC, 0.1~o SDS. Under these conditions specific hybridization signals were obtained, which demonstrated that the tobacco probe can be used to identify the cyanobacterial psaC gene. (Fig. 1A). A library of genomic D N A of Synechocystis 6803 in the bacteriophage 2 dash was screened with the tobacco probe and one phage (261) was isolated, which contained the psaC gene on a 3.5 kb SaII fragment. The fragment was subcloned in the vector pSP65 (clone p61S3.5I) and mapped with restriction enzymes (Fig. 2). The
psaC reading frame was located around the Xba I site by hybridization with the tobacco probe (data not shown). Sequencing of this region identified the psaC reading frame (Fig. 3). It codes for 81 amino acids and specifies a polypeptide of the calculated molecular mass of 8829 Da. The gene was designated psaC2, to distinguish it from the psaC gene identified by Anderson and McIntosh
(psa Cl [ 11). F---
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Fig. 2. Restriction map of the 3.5kb SalI fragment of p61S3.5I. The left SalI and the left Eco RI site are derived from the multiple cloning site of 2 dash. The open box represents the psaC2 reading frame and the arrow indicates the direction of transcription.
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Fig. 3. Nucleotide and deduced amino acid sequence of the psaC2 gene of Synechocystis 6803. The nucleotide sequence is shown from the Ssp I to the Hinc II site. A possible ribosome binding site (rbs) is underlined.
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Spinach Barley/Rice/Wheat Maize
Fig. 4. Amino acid sequence comparison between the P S I - C polypeptides from various organisms. The sequences are derived from the following publications: Syneehococcus sp. PCC7002 [2, 15], Nostoc sp. PCC8009 [2], Synechococcus vulcanus [ 19], Cyanophora paradoxa [2, 15], Chlarnydornonasreinhardtii [22], liverwort [ 12] Synechocystis sp PCC6803 [ 1 ], tobacco [9], spinach [ 14], pea [5], barley [17], rice [10], wheat [5] and maize [ 16]. Points indicate identical amino acids•
1000 Southern hybridization was carried out with a homologous D N A fragment (nucleotides 4-366 of the sequence in Fig. 3). Fig. 1B shows that the same bands seen with the tobacco probe are labelled. In a second control experiment, restriction enzymes that cut within the 3.5 kb Sal I fragment were used. The labelled bands correspond well to the fragments predicted from the map of the 3.5 kb Sal I fragment (Fig. 1C). In these experiments, the blots were hybridized and washed at 61 °C and the salt concentration in the last three washing steps was lowered to 0.1 x SSC, 0.1~o SDS. Amino acid sequence comparison revealed that PSI-C2 is very similar to the PSI-C proteins of other cyanobacteria (Fig. 4). The amino acid sequence differs in only one position from the Synechococcus 7002 sequence, while there are eight to ten amino acid substitutions compared to the proteins from higher plants. In contrast, the amino acid sequence of PSI-C1 is identical to that of tobacco and is therefore closely related to the sequences from higher plants. As was pointed out by Anderson and McIntosh [1], this similarity is also reflected at the nucleotide level. For example, the psaC1 nucleotide sequence is 93 ~o identical to the tobacco sequence, but only 75 ~o to psaC2. In the light of this finding, it remains unclear why, under our hybridization conditions, the tobacco psaC probe did not detect the very similar psaC1 gene of Synechocystis 6803 but did detect the more divergent psaC2 gene (Fig. 1A). Interestingly, the nucleotide sequence of the ndhE gene, which flanks the psaC1 gene, is also more similar to the ndhE gene of tobacco (86 ~o identity) than to a recently identified second ndhE gene in Synechocystis 6803 (62~o identity; U. Ellersiek and K. Steinm~iller, in preparation). Moreover, this second ndhE gene is located at a different genomic position (i.e. more than 5 kb away). One may speculate, therefore, that the psaC1 gene and its flanking regions were derived from a transformation of Synechocystis 6803 with higher-plant chloroplast DNA. This process might have taken place in nature because Synechocystis 6803 can be transformed by naked D N A [8] and D N A is stabilized by adsorption to soil particles [ 13].
The expression of the psaC2 gene was studied in a northern blot experiment with the homologous psaC2 probe. The psaC2 gene is transcribed into an abundant m R N A of about 450 nucleotides (Fig. 1D). Monocistronic transcription and high m R N A levels have also been reported for the psaC genes from Synechococcus7002 and Nostoc 8009 [2]. In contrast, Anderson and Mclntosh did not detect specific transcripts for psaC1 [1]. In higher plants, psaC is cotranscribed with the downstream located ndhD gene [9, 16], while in the cyanobacterium Synechococcus7002, no adjacent reading frame for ndhD was found [15]. Preliminary hybridization and sequencing data for Synechocystis 6803 indicate that a ndhD gene is located on the opposite strand, upstream of psaC2. Thus the situation in cyanobacteria differs from that in chloroplasts and it remains to be seen what the regulatory implications for this gene arrangement are.
Acknowledgements I would like to thank Ulrike Ellersiek for excellent technical assistance and Alan Blowers for a gift of the tobacco plastid D N A subclone pNtcPsl. Many thanks to Don Bryant for helpful discussions and unpublished data and to Peter Westhoff for critical reading of the manuscript. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 189).
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