Mol Gen Genet (1992) 234:325-328 © Springer-Verlag 1992

Short communications Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis M. Zagorec and P.W. Postma E.C. Slater Institute for BiochemicalResearch, Universityof Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands Received March 4, 1992

Summary. The ptsG gene of Bacillus subtilis encodes Enzyme II ale of the phosphoenolpyruvate: glucose phosphotransferase system. The 3' end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III a~ of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5' end. These results, combined with the sequence of the 3' end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II 6~° and II Nag of E. coli but which are present in the II6~¢-like protein encoded by the E. coli malX gene. Key words: Glucose permease - Bacillus subtilis Phosphotransferase system - p t s G - Enzyme II ~1c

In many bacteria several carbohydrates are transported in the cell and phosphorylated by the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The general enzymes, Enzyme I and HPr, are required for all the PTS carbohydrates while the Enzyme II/III complexes are carbohydrate-specific (for review see Postma and Lengeler 1985). In Bacillus subtilis, several Enzymes II have been characterized. Some of the genes encoding these membrane-bound proteins have been cloned and sequenced. In a previous study we have shown that the glucose permease of B. subtilis is a membrane-bound protein, containing II ~1c and III ~c domains. The B. subtilis glucose permease is encoded by the ptsG gene, which is located upstream from the ptsH, I cluster that encodes HPr and Enzyme I (Gonzy-Tr6boul et al. 1989, 1991). Approximately two-thirds of the B. subtilis ptsG gene was previously cloned and sequenced (Gonzy-Trdboul et al. 1989, 1991). There have been several unsuccessful Correspondence to : P.W. Postma

attempts, using classical techniques, to clone the entire ptsG gene of B. subtilis in Escherichia coll. These results suggested that the ptsG gene could not be stably maintained, probably because expression of the B. subtilis ptsG gene is lethal for E. coli (G. Gonzy-Tr6boul and M. Zagorec, unpublished results). Therefore, another method was devised to clone the complete ptsG gene. Cloning of the ptsG 9ene in B. subtilis A chromosomal mutant carrying a deletion of the Y end ofptsG was constructed by transformation of the B. subtills wild-type strain PY480 (Poth and Youngman 1988) with plasmid pTS102SB, obtained from M. Steinmetz. pTS102SB contains the 3' end of ptsG, but the BglIIHindIII fragment of this gene has been deleted and replaced by a DNA fragment carrying the phleomycin resistance gene and the spac promoter of pDG148 (Stragier et al. 1988). pTS102SB was introduced into the chromosome of PY480 by a double crossing-over event, leading to a chromosomal deletion of the 3' end ofptsG. The resulting mutant, MZ109, grew normally on most PTS substrates, but its doubling time on 10 mM glucose was twice that of the corresponding wild-type strain. The rate of uptake of 0.5 mM [14C]glucose in the mutant was 20 % of that of the wild-type strain and [l*C]methyl a-glucoside uptake (0.5 raM) was not detectable, as has been observed for other ptsG mutants (Gonzy-Tr6boul et al. 1991). The residual growth on glucose was slow enough to allow a positive screen for restoration of growth on minimal C medium containing glucose as the carbon source. A chromosomal DNA library was constructed in plasmid pCV2, as described by Poth and Youngman (1988) and Youngman et al. (1989), by partial Sau3A digestion of chromosomal DNA of the B. subtilis wild-type strain 168. PY480 was transformed with this library, selecting for chloramphenicol resistance (the resistance marker associated with pCV2). In order to obtain a library in phage SP[~ c2, del2, present in PY480 104 transformants were pooled, and a lysate was prepared and used to transduce MZ109 (ptsG), selecting for growth on 10 mM

326

glucose-minimal C medium in the presence of chloramphenicol. Five positive transductants were pooled. A lysate was prepared and again used to transduce MZ109 for resistance to chloramphenicol; 90% of the chloramphenicol-resistant transductants obtained were able to grow normally on glucose. One of those, MZ110, containing a mutated ptsG gone at the pts locus and a wildtypeptsG + copy at the SPI3 locus, was selected for further experiments. We attempted to clone the ptsG +-containing fragment in E. eoli. SP[3 DNA of MZ110 was extracted, cut with NotI, ligated, then used to transform E. eoli cells, selecting for resistance to ampicillin, as described by Poth and Youngman (1988). Two isogonic strains were used: TG1 and TG90; the latter carries a mutation which lowers the copy number of plasmids containing a ColE1 origin of replication (Lopilato et al. 1986; Gonzy-Trdboul etal. 1992). No transformants were obtained. Since direct cloning in E. eoli was unsuccessful, we tried to transfer the ptsG + gone to a shuttle vector. The DNA fragment of M Z l l 0 containing ptsG was transferred to pTV51 by in vivo recombination (Youngman 1987). For this purpose, the wild-type B. subtilis strain 168 was transformed with pTV51 (Tetr). The resulting strain was subsequently transformed with chromosomal DNA of MZ110, containing the ptsG + gone linked to the chloramphenicol resistance marker. Transformants were selected which showed resistance to chloramphenicol and tetracycline in addition to erythromycin (as a result of the Ermr marker associated with the SPI3 fragment). Plasmid DNA from one of those transformants was isolated. The corresponding plasmid, pTSG6, was used to transform MZ109, carrying theptsG mutation and transformants were selected for tetracycline resistance. All the transformants also acquired erythromycin and chloramphenicol resistance and showed growth on glucose. The presence of Enzyme II G1¢ was tested by Western blot experiments with antibodies that were raised against III ~c of Salmonella typhimurium and that can recognize the C-terminal part of B. subtilis II Glc (Gonzy-Tr6boul et al. 1991). The protein was not detected in MZ109 or in M Z l l 0 , cured of the recombinant phage, and was overproduced approximately tenfold in the membrane fraction of MZ109 transformed with pTSG6. In MZ110 it was present at the same level as in the wild-type strain (data not shown). This result showed thatptsG + and its promoter were indeed present in pTSG6. When pTSG6 was used to transform E. eoli TG1 and TG90, selecting for resistance to ampicillin, only a few transformants were obtained. All the transformants contained plasmids with very large deletions; thus apparently the ptsG gone is not stable or its gone product is lethal in E. eoli.

Cloning of a mutated ptsG gene in E. coli In order to be able to clone and sequence the missing part of ptsG, we cloned a mutated ptsG gone, in which the BglII-HindIII fragment at the 3' end was deleted. For this purpose, pTSG6 was used to transform MZ109, carrying this 3' end deletion, selecting for resistance to tetracy-

ptsG' r--

Bg[]]

Hind]]/ phteo ptsH ptsZ I ...............................

I

_

[I

_. hromosomo, ON.

pTSG6

p

BglII

t

~

Hind 131

ptsH

pTSG7 Fig. l. Plasmid pTSG7, containing a ptsG gone with a deletion of the BglII-HindIII fragment, was constructed by double crossingover. For this purpose, strain MZ109 containing this partially deletedptsG gone and plasmid pTSG6, which contains the wild-type ptsG + gone of Bacillus subtilis, were utilized, phleo, Phleomycin resistance marker

cline. We expected that some plasmids would recombine with the chromosome at the pts locus. Then, by double crossing-over, the mutated ptsG gone of the chromosome of MZ109, carrying a marker for resistance to phleomycin, might recombine with pTSG6 (Fig. 1). Two hundred Phl r transformants were pooled and plasmid DNA was used to transform wild-type B. subtilis strain 168, selecting for phleomycin resistance. Transformants were selected which were also resistant to tetracycline, erythromycin and chloramphenicol. The plasmid DNA was extracted and restriction analysis showed that the resulting plasmid, pTSG7, indeed corresponded to pTSG6 in which the p tsG + gone was replaced by the p tsG gone with a deletion of the BglII-HindIII fragment. The ptsH gone and the beginning of the ptsI gone were also present on this plasmid, pTSG7 could no longer transform ptsG mutants for growth on glucose, confirming that a mutated gone had been cloned. pTSG7 could be transformed in to E. coli strain TG90 but not other E. eoli strains. Thus, the plasmid could not be maintained at a high copy number, so presumably expression of even the first half of the gone was lethal for most E. eoli strains. TG90/pTSG7 was used for further experiments. A DNA fragment corresponding to the 5' end of ptsG and ending at an HpaI site located in the previously cloned part was subcloned in M13mpl 8 and sequenced.

Nucleotide sequence of the B. subtilis ptsG gone Sequence analysis of the 5' end of the cloned ptsG fragment showed that an extra DNA fragment of 1284 bp had been cloned, containing 452 bp upstream from the ATG start codon. As stated before, the fragment should include the promoter of ptsG because the longer fragment on pTSG6 was sufficient to restore growth of B. subtilis MZ109 on glucose and resulted in an immunedetectable II ~1cprotein. Putative - 10 and - 35 promoter sequences were observed 343 bp upstream from the ATG start codon. The sequenced fragment showed an overlap

327 GAT CAGGCATATCAGATTTACAATAGAAAGAATTAAAAAAGAAGAACCGACTAAAGAACCAGAAAAATTAATGTTGTTAT TGAAAAATGAATATCGCTATGCTACAATACAGCTTGGAAATTGATTAAAATC

80

T TG C A G C A A A C A T T G A A G A A A C C A G T T C

160

ATGAGG CGGAAG CGGTTTAT CTGACGCTTCATCTGATAC CGATTAACCAATAAAATTTCATAAATTCAGTTTAT CCTTAT AACGTGTTACTGATTCGATCAGGCATCAGTGATTGAGGGAAAAA

CGGGAAGTTCATTCTCGTTCTTTTGCGCACCCAA

240 320

TTTTG CTCATGCCTTTTTGTTGTG TAAAAGGG CAAATG TAAACGGTTAAACTGGAAGACTTACGCCTGTGAATTCGTTG T

400

CATGATTTTTAGCTGTAAGGTCAGACTAGTAAAAAGAGGAGGTCAATTCTTATG TTTAAAGCATTATTCGGCGTT CTTCA M F K A L F G V L Q

480 i0

AAAAATTGGGcGTGcGcTTATGcTTccAGTTGCGATCCTTCCGGCTGCGGGTATTTTGCTTGCGATCGGGAATGCGATGC K I G R A L M L P V A I L P A A G I L L A I G N A M

560 36

AAAATAAGGACATGATTCAGGTCCTGCATTTCTTGAGCAATGACAATGTTCAGCTTGTAGCAGGTGTGATGGAAAGTGCT Q N K D M I Q V L H F L S N D N V Q L V A G V M E SA

640 63

GGGCAGATTGTTTTcGATAACcTTccGCTTcTTTTCGcAGTAGGTGTAGccATcGGGcTTGccAATGGTGATGGAGTTGc GQ I V F D NL PL L F A V G V A I G L A N G D G V A

720 90

AGGGATTGcAGCAATTATcGGTTATCTTGTAATGAATGTATCCATGAGTGcGGTTcTTCTTGCAAAcGGAACCATTCcTT G I A A I I G Y L V M N V S M S A V L L A N G T I P

800 116

cGGATTCAGTTGAAAGAGCCAAGTTcTTTAcGGAAAAccATcCTGcATATGTAAACATGcTTGGTATACcTAccTTGGCG S D S VE RA K F F T E N H P AY V N M LG I P T L A

880 143

AcAGGGGTGTTCGGcGGTATTATcGTcGGTGTGTTAGcTGcATTATTGTTTAAcAGATTTTAcAcAATTGAAcTGccGCA TG V F GG I I VG V L A A L L F N R F Y T I E L P

Q

960 170

ATAccTTGGTTTCTTTGcGGGTAAACGTTTCGTTccAATTGTTAcGTcAATTTcTGcAcTGATTCTGGGTcTTATTATGT Y L G F F A G K R F V P I V T S I S A L I L G L I M

1040 196

TAGTGATcTGGCcTCCAATccAGcATGGATTGAATGCcTTTTcAACAGGATTAGTGGAAGcGAATCcAAcccTTGcTGcA LV I W P P I QH G L NA F S TG L V E AN P T L A A

1120 223

TTTATcTTcGGGGTGATTGAAcGTTcGCTTATCCCATTCGGATTGCACCATATTTTcTATTCACcGTTCTGGTATGAATT F I F G V I E RS L I P FG L H H I FY S P F W Y E

F

1200 250

e cTTcAGCTATAAGAGTGCAGCAGGAGAAATcAT•CGCGGGGATCAGCGTATCTTTATGGcGcAGATTAAAGACGGCGTAc F S Y K S A A G E I I R G D Q R I F M A Q I K D G V

1280 276

AGTTAA•GGCAGGTAcGTTCATGAcAGGTAAATATCCATTTATGATGTTCGGTCTGccTG•TGcGGcGcTTGCcATTTAT Q L T A G T F MT G K Y P F MM F G L P A A A LA I Y

1360 303

CATGAAGcAAAAC•GCAAAAcAAAAAAcTcGTTGCAGGTATTATGGGTTCAGCGGCcTTGAcATCTTT•TTAAcGGGGAT H E A K P Q N K K L V A G I MG S A A L T S F L T G

I

CACAGAGCcATTGGAATTTTcTTTcTTATTcGTTGCTC•AGTCcTGTTTGCGATTCAcTGTTTGTTTGcGGGAcTTTcAT T E P L E F S F L F V A P V L F A I H C L F A G L S

1440 330 1520 356

TCATGGTCATGcAGCTGTTGAATGTTAAGATTGGTATGACATTCTCCGGCGGTTTAATTGAcTAcTTccTATTCGGTATT F M V M Q L L N V K I G M T F S GG L I D Y F L F G

I

1600 383

TTAccAAACCGGACGGCATGGTGGcTTGTcATCCCTGTCGGCTTAGGGTTAGCGGTCATTTACTAcTTTGGATTCcGATT L P N R T A W W LV I P VG LG L A V I Y Y FG F R

F

1680 410

TGCCATccGCAAATTTAATCTGAAAACAcCTGGAcGCGAGGATGCTGCGGAAGAAACAGCAGCAcCTGGGAAAAcAGGTG A I R K F N L K T P G R E D A A E E T A A P G K T G

1760 436

AAGCAGGAGATcTTcCTTATGAGATTcTGCAGGCAATGGGTGAccAGGAAAACATCAAACAcCTTGATGcTTGTATcAcT E A G D L PY E I L Q A M G D QE N I KH L D A C I T

1840 463

cGTcTGCGTGTGACTGTAAACGATcAGAAAAAGGTTGATAAAGAccGTCTGAAACAGCTTGGcGcTTccGGAGTGCTGGA R L R V T V N D Q K KV D K D R L KQ L G A S GV LE

1920 490

AGTCGGCAACAACATTCAGGCTATTTTCGGACCGCGTTCTGACGGGTTAAAAACACA~TGCAAGACATTATTGCGGGAC V G N N I Q A I F G P R S D G L K T Q M Q D I I A G

2000 516

GCAAGC CTAGACCTGAGC CGAAAACATCTGCT CAAGAGGAAGTAGGCCAGCAGG TTGAGGAAGTGATTG CAGAACCGCTG RK P R P E P KT S A Q E E VG Q Q V E E V I AE p L

2080 543

CAAAATGAAATAGGCGAGGAAGTTTTCGTTTCTCCGATTACCGGGGAAATTCACCCAATTACGGATGTTCCTGACCAAGT Q N E I G E E V F V S P I T G E I H P I TD V p D Q V

2160 570

CTTCTCAGGGAAAATGATGGGTGACGGTTTTG CGATTCTCCCTT CTGAAGGAATTGTCGTATCACCGGTTCGCGGAAAAA F S G K M M G D G F A I L P S E G I V V S P V R G K

2240 596

TTCTCAATGTGTTCcCGACAAAACATGCGATCGGCCTGCAATCCGACGGCGGAAGAGAAATTTTAATCCACTTTGGTATT I L N V F P T KH A I G LQ S D G G R E I L I H F G I

2320 623

GATACCGTcAGcCTGAAGGGCGAAGGATTTACGTcTTTCGTATCAGAAGGAGACcGCGTTGAGCcTGGACAAAAACTTcT D T V S L KG E G F T S F V S E G D RV E P G Q KL

2400 L 650

TGAAGTTGATCTGGATGCAGTcAAACCGAATGTA•CATCTCTcATGACAcCGATTGTATTTACAAACCTTGCTGAAGGAG E V D L D A V K P N V P S L M T P I V F T N L A E G

2480 676

AAACAGTCAGCATTAAAGCAAGCGGTTCAGTCAACAGAGAACAAGAAGATATTGTGAAGATTGAAAAATAAGGGTGTTAG E T V S I K A S G S V N R E Q E D I V K I E K *

2560 699

TACGCCGTGCTTGTCAGATGACAAGTACGGTTGTATGATATAATATTGTGAAGTAATAAAGCTT

2624

Fig. 2. Nucleotide sequence of the B. subtilis ptsGgene and upstream region. Putative promoter sequences at - 1 0 and - 3 5 are indicated. The sequence has been deposited in the EMBL nucleotide sequence data library under the accession number Z11744

328

of 92 bp with the previously published sequence of the Y end of the gene (Fig. 2; Gonzy-Tr6boul et al. 1991; Sutrina et al. 1990). We observed an open reading frame, starting with an ATG codon at position 452 (Fig. 2) and preceded by a potential Shine-Dalgarno sequence. The first 30 amino acids encoded by this open reading frame shared 66.7 % identical amino acids with the N-terminal part of Enzyme II ~c of E. coli. The homology strongly suggested that the D N A sequence was the start o f p t s G (Erni and Zanolari 1986). After combining the sequence of our 1284 bp fragment with the previously determined sequence of the Y end of the ptsG gene, a single open reading frame of 699 amino acids was observed, encoding a protein with a molecular weight of 75 521 Da. This agrees well with the apparent molecular weight that was previously estimated (Gonzy-Tr6boul et al. 1991 ; Sutrina et al. 1990). The N-terminal part of the protein encoded by ptsG shared strong identity with several Enzymes II:39.6% of the 571 amino acids of Enzyme II Nag of E. coli (Rogers et al. 1988) and 44.1% of the 465 amino acid residues of Enzyme II c~c ofE. eoli (Erni and Zanolari 1986) were identical with those of B. subtilis II G~°. The B. subtilis II GI° domain was 31 amino acids longer than that of E. coli II a~¢, due to extra amino acids between positions 35 and 55 (12 amino acids) and 108 and 144 (16 amino acids). The length of the B. subtilis II ~1~ domain was more similar to that of the IIGlc-like protein encoded by the malXgene ofE. coli (Reidl and Boos 1991). These two proteins were also highly similar, sharing 36.9% identical amino acids. Small internal similarities were also observed between B. subtilis II a~° (residues 444-485) and several Enzymes IIS": 40 % identical amino acids in II set of Vibrio aloinolytitus (residues 9-48; Blatch et al. 1990), 31% in II s°r encoded by the sacP gene of B. subtilis (residues 10-50; Fouet et al. 1987), and 42% in the negative regulator, IIS% encoded by the saeX gene of B. subtilis (residues 8-40; Zukowski et al. 1990). These similarities corresponded to the same region in the three proteins and also corresponding regions in Enzymes II Nag, II Bgl and IIS% This domain is thought to be involved in the binding of III c~° or III~*-like domains (Lengeler et al. 1990). The cloning is reported of the ptsG gene of B. subtilis. It appears that this gene cannot be stably maintained in E. toll; the 3' end of the gene was previously cloned in E. coli (Gonzy-Tr6boul et al. 1989). We succeeded in cloning the 5' end ofptsG ÷ in E. coli, but only in a strain carrying a mutation that decreases the copy number of plasmids. It is thus possible that the expression of (part of) the B. subtilis ptsG gene is lethal in E. coli; however, such effects were not observed in B. subtilis, ptsG + appears to encode a membrane-bound protein; possibly the presence of elevated amounts of this (truncated) protein leads to lysis of E. coli cells.

Acknowledgements. We thank Michel Steinmetz for the gift of plasmid pTS102SB, and Genevieve Gonzy-Tr6boul for the gift of strain TG90. This work was supported by EEC BRIDGE and EMBO fellowships.

References Blatch GL, Scholle RR, Woods DR (1990) Nueleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region. Gene 95:17-23 Erni B, Zanolari B (1986) Glucose permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme II ~lc. J Biol Chem 262:16398-16403 Fouet A, Arnaud M, Klier A, Rapoport G (1987) Bacillus subtilis sucrose-specific enzyme II of the phosphotransferase system: Expression in Escherichia coli and homology to enzymes II from enteric bacteria. Proe Natl Acad Sci USA 84:8773-8777 Gonzy-Tr6boul G, Zagorec M, Rain-Guion M-C, Steinmetz M (1989) Phosphoenolpyruvate: sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5' end ofptsI and evidence for a ptsHI operon. Mol Microbiol 3 : 103-112 Gonzy-Tr6boul G, de Waard JH, Zagorec M, Postma PW (1991) The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for II Gle and III Glc domains. Mol Microbiol 5 : 1241-1249 Gonzy-Tr6boul G, Karmazyn-Campelli C, Stragier P (1992) Developmental regulation of transcription of the Bacillus subtilis f t s A Z operon. J Mol Biol 224:967-979 Lengeler JW, Titgemeyer F, Vogler AP, W6hrl BM (1990) Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins. Phil Trans R Soc Lond [B] 326:489-504 Lopilato J, Bortner S, Beckwith J (1986) Mutation of a new chromosomal gene of Escherichia coli K12, penB, reduces plasmid copy number of pBR322 and its derivatives. Mol Gen Genet 205: 285-290 Postma PW, Lengeler JW (1985) Phosphoenolpyruvate: carbohydrate phosphotransferase system of bacteria. Microbiol Rev 49: 232-269 Poth H, Youngman P (1988) A new system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors. Gene 73: 215-226 Reidl J, Boos W (1991) The malX malY operon of Escherichia coli encodes a novel Enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system. J Bacteriol 173 : 4862M-876 Rogers MJ, Ohgi T, Plumbridge J, $611 D (1988) Nucleotide sequences of the Escherichia coli nagE and nagB genes: the structural genes for the N-acetylglucosaminetransport protein of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and for glucosamine-6-phosphate deaminase. Gene 62:197-207 Stragier P, Bonamy C, Karmazyn-Campelli C (1988) Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression. Cell 52:697-704 Sutrina SL, Reddy P, Saier MH, Reizer J (1990) The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease. J Biol Chem 265:18581-18589 Youngman P (1987) Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus subtilis and other Grampositive bacteria. In: Hardy K (ed) Plasmids: a practical approach. IRL Press, Oxford, pp 79-103 Youngman P, Poth H, Green B, York K, Olmedo G, Smith K (1989) Methods for genetic manipulation, cloning and functional analysis of sporulation genes in Bacillus subtilis. In : Smith I, Slepecky R, Setlow P (eds) Regulation of prokaryotic development. American Society for Microbiology, Washington, pp 65-87 Zukowski MM, Miller L, Cogswell P, Chen K, Aymerich A, Steinmetz M (1990) Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes. Gene 90:153-155

Communicated by J. Lengeler

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis.

The ptsG gene of Bacillus subtilis encodes Enzyme IIGlc of the phosphoenolpyruvate: glucose phosphotransferase system. The 3' end of the gene was prev...
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