©D 1990 Oxford University Press
5556 Nucleic Acids Research, Vol. 18, No. 18
Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D Thomas Binz, Hisao Kurazono, Michel R.Popoff, Melvin W.Eklund2, Genji Sakaguchi3, Shunji Kozaki3, Kerstin Kriegistein4, Agnes Henschen4, D.Michael Gill5 and Heiner Niemann* Institut fur Mikrobiologie, BFAV, Posifach 1149, 74 TObingen, FRG, 1Unite des Anaerobies, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France, 2Northwest Fisheries Center, 2725 Montlake Blvd E, Seattle, WA 98112, USA, 30saka Prefecture University, College of Agriculture, 8 Mozu Ume-machi, 4-cho Sakai-shi, Osaka 591, Japan, 4Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 and 5Department of Molecular Biology and Microbiology, Tufts University Schools of Medicine, Boston, MA 0211 1, USA Submitted August 13, 1990
The seven serologically distinct types of Clostridium botulinum neurotoxins, designated BoNT/A to BoNT/G, are synthesized as single-chain polypeptides of about 150 kDa. Proteolytic activation yields N-terminal light (=50 kDa) and C-terminal heavy chains (= 100 kDa) connected by single disulfide bonds. BoNT/D, like BoNT/Cl (1), is encoded by a bacteriophage (2, 3). The sequence shown below was established from cloned bacteriophage fragments the sizes of which were smaller than 2.0 kb (experiments registered by ZKBS Az 1494/1). The initial clone was identified with a synthetic 23-mer oligonucleotide containing only A or T residues in the wobble positions. The sequence (underlined) was deduced from a peptide obtained from purified BoNT/D by cyanogen bromide fragmentation. The sequence of the BoNT/D gene contains a single open reading frame beginning at the ATG codon in position 47 and encodes a polypeptide of 1276 amino acid residues (Mr 146,872 Da). Cys437 and Cys450 are probably involved in the disulfide bond between the light and the heavy chain. BoNT/D shares 49.6% amino acid sequence identity with BoNT/C (46.3% within the light chain, 67% within the N-terminal part of the heavy chain, and merely 35 % within the putative fragment C). No sequence similarity with exoenzyme C3 was found which is also encoded by C. botulinum type C and D phages (4).
ACKNOWLEDGEMENTS We thank F.Poysky and L.Jordan for help in preparing clostridial phage DNA. This work was supported by grant Nie 175/5-2 from the Deutsche Forschungsgemeinschaft to H.N. and by fellowships from the Alexander von Humboldt Foundation to H.K. and from Graduiertenforderung of the University Giessen to T.B.
REFERENCES 1. Hauser,D., Eklund,M.W., Kurazono,H., Binz,T., Niemann,H., Gill,D.M., Boquet,P. and Popoff,M.R. (1990) Nucl. Acids Res. 18, 4924.
*
To whom
correspondence should be addressed
EMBL accession
no.
X54254
2. Eklund,M.W., Poysky,F.T., Reed,S.M. and Smith,C.A. (1971) Science 172, 480-482. 3. Eklund,M.W. and Poysky,F.T. (1989) in: Botulinwn Neurotoxins and Tetanus Toxin (Simpson,L.L. ed.) pp. 25-51, Academic Press, San Diego. 4. Popoff,M.R., Boquet,P., Gill,D.M. and Eklund,M.W. (1990) Nucd. Acids Res. 18, 1291.
( ( ( ( ( ( ( ( ( ( ( (
1)AAGGTGCACA CTTGTGGATA TTAGAAAGTT AGGAGATGTT AGTATTATGA CATGGCCAGT AMAGATTTT
71)AATTATAGTG ATCCTGTTAA TGACAATGAT ATATTATATT TAAGAATACC ACAAAATMG 141)CACCTGTAAA AGCTTTTATG ATTACTCAM ATATTTGGGT AATACCAGM AGATTTTCAT TTMTTACTA CAGATACTM 211)TCCAAGTTTA AGTAAACCGC CCAGACCTAC TTCAAAGTAT CAAAGTTATT ATGATCCTAG TTATTTATCT 281)ACTGATGAAC AAAMGATAC ATTTTTAMA GGGATTATAA AATTATTTAA MGAATTAAT GAAAGAGATA 351)TAGGAAAAAA ATTAATAAAT TATTTAGTAG TTGGTTCACC TTTTATGGGA GATTCAAGTA CGCCTGMGA 421)TACATTTGAT TTTACACGTC ATACTACTM TATTGCAGTT GMMGTTTG AAMTGGTAG TTGGMAGTA 491)ACAAATATTA TMCACCAAG TGTATTGATA TTTGGACCAC TTCCTAATAT ATTAGACTAT ACAGCATCCC 561)TTACATTGCA AGGACAACM TCAAATCCAT CATTTGAAGG GTTTGGAACA TTATCTATAC TMAAGTAGC 631)ACCTGAATTT TTGTTMCAT TTAGTGATGT AACATCTAAT CAAAGTTCAG CTGTATTAGG CAAATCTATA 701)TTTTGTATGG ATCCAGTMT AGCTTTAATG CATGAGTTAA CACATTCTTT GCATCAATTA TATGGMTM 771)ATATACCATC TGATAAAAGG ATTCGTCCAC MGTTAGCGA GGGATTTTTC TCTCAAGATG GACCCAACGT 841)ACAATTTGAG GAATTATATA CATTTGGAGG ATTAGATGTT GAAATAATAC CTCAMTTGA MGATCACAA 911)TTAAGAGAAA AAGCATTAGG TCACTATAAA GATATAGCGA AAAGACTTAA TMTATTAAT AMACTATTC 981)CTTCTAGTTG GATTAGTAAT ATAGATAAAT ATAAAAAAAT ATTTTCTGAA AAGTATAATT TTGATAAAGA (1051)TAATACAGGA AATTTTGTTG TAAATATTGA TMATTCMT AGCTTATATT CAGACTTGAC TAATGTTATG (1121)TCAGAAGTTG TTTATTCTTC GCAATATAAT GTTMAAACA GGACTCATTA TTTTTCAAGG CATTATCTAC (1191)CTGTATTTGC AAATATATTA GATGATAATA TTTATACTAT AAGAGATGGT TTTAATTTAA CAMTAAAGG (1261)TTTTAATATA
(1331)GTAGTAGATT (1401)AAGTTAAAAA (1471)AATTATTACA (1541)GGGCAAGTTC
(1611)TTCCAGGTGA
(1681)TTTGGAATCT (1751)GGTTATAGCA
(1821)GTTTATTCTT
(1891)GGATAAAATA (1961)TTAAGGGGAA (2031)TTACTATACC (2101)TATAGAAAAT (2171)TTGTCAAGAA (2241)ATGCAATCAA (2311)TCAAGTTGAA (2381)ATACGAGAAT (2451)TTGATTTAAG (2521)AGATAGATTA (2591)AATAATTCTT (2661)TACAAAACAA (2731)TCAACTTAAT (2801)AATAATAATA (2871)ATTTAACTAA (2941)TATTAGGAAT (3011)TATAGTGAAT (3081)TGGGGTATAT (3151)TAAGTTAGAT (3221)GATTTTAATA (3291)GAAATGTTAT
GAAAATTCGG TATTTACAAA TAATAGATTA GATGAGACTA CTATTAATCC AGAAATAGTA CAAAAATTAA ATAAGATATA AAATTGGGCG TCAGATGTAT ATTTTAATCA TGCACTCGGT TGTTTGGAAC TTACTACTCA AGCTAAAATA
AATTTAAAAA
GTTCTGTAAC AACTAAAACA AAAGCAAAAG TATTAAAAGA AAAAAATGCT
ACGATATATA
TTTTATATAG TTCTCATAAT GGCAATATAG CATTAAGTCA GAAACTTTAT AAAACCATAG TTTTTTCTAA TAAAGATTAT
(3361)TTATATAGAT AGGTATATAG (3431)TTATATACTG GAAATCCTAT
(3501)GAGATAATAT AATTCTTCAT (3571)ATATGCAACA CAAGGAGGAG (3641)AATTATGGTA TAGGTATATT (3711)CTAGTTTTAG GGAAAATACA
(3781)ATACACGCCA GTTGCAGTAA (3851)TCTAGGGATC CAGGATGGGT (3921)TTTAGAAATT TATCGTATAA
GTCAGAATAT AGTATGTTTA CCTTATGTAG ATGTACAAAA TGAMTAGTA
AGAAAGGAAT CCTGCACTAC AAMGCTTAG TTCAGAAAGT AGATTMCAA AAAATAGTAG AGATGATTCA ACATGTATTA
CTGATAAAGA TAGCATTTCA CAAGAAATAT TTGAAMTM TTATTCAGAT AAATTTTCAT TAGATGAATC TATTTTAGAT GATCCACTAT TACCCAATGT TMTATGGM CCTTTAATC TTTTATGATG ATATTACTAA ATATGTTGAT TATTTAAATT CTTATTATTA GTAATMTGT TGAAAATATT ACTCTTACAA CTTCAGTTGA AGMGCATTA CACATTTTTA CCTAGCTTAG CTGAAAAAGT GAATAAAGGT GTTCMGCAG
MTGMGTAG TTGAGGATTT TACTACAMT TCCATATATA GGACCTGCCT ACAGCTGGTG TAGCTTTTTT TTTATAGTTC TATTCAAGAA
ATTATGAAGA MGATACATT TAAATATAGG AAATTCAGCA ATTAGAGGGA TTTCCAGAGT AGAGAGAAM TTATTAAAAC GAGATGGAM GATTCATATC AATGGATGGT ATCAMTTGG ATAMTTATC AAATGTATGA TTCTTTAAGT TATCAGGCAG ATAAAAAATA CTCAGGAAGT GATMAGAAA ATATMAAAG ATAGTTTAGA TGTAAAAATT TCGGAAGCA& TGQTTAT AATAAATTT ATACTTATTT AAAAATATGC TCCCTAAAGT MTTGACGAA TTAAATAAGT GAATTAATTA ATCTTATAGA TAGTCATAAT ATTATTCTAG TTGGTGAAGT TAAATGACAG TTTTGAAAAT ACMTGCCTT TTMTATTTT TTCATATACT TATAATTMT GAATATTTCA ATAGTATTM TGATTCAAAA ATTTTGAGCT TTAGTGGATA CATCAGGATA TAATGCAGAA GTGAGGGTAG GAGATAATGT CAAATGACTT TAAATTAAGT AGTTCAGGAG ATAAAATTAT AGTAAATTTA CGCTATTTAT GAGAACTCTA GTGTTAGTTT TTGGATTAAG ATATCTAAAG GMTATACAA TAATTAACAG TATAGAACAA AATTCTGGGT GGAAATTATG AATGGATTTT ACAAGATGTT AATAGAAAGT ATAAAAGTTT AATTTTTGAT TACAGGATAT ACAAATAAAT GGTTTTTTGT TACTATAACT AATAATATM ATAAATGGAG AATTAAAGCA GAGTCAAAAA ATTGMGATT TAGATGAGGT TATTTGGAAT AGATGAGAAT ATAGATGAGA ATCAGATGCT TTGGATTAGA AGAATTAAGT AATGAAGATA TTAATATTGT ATATGAGGGA CAAATATTM TGGGGAAATC CTTTGAAGTT TGATACAGAA TATTATATTA TTAATGATM CACCTGAAAG TAATGTACTT GTACTTGTTC AGTATCCAGA TAGATCTAAA TACTATTAAA TCAGTATCTG ATMGAATCC TTATAGTAGA ATTTTAAATG ATGTTATATA ATAGTAGGAA ATATATGATA ATAAGAGATA CTGATACMT AGTGTTCACA AAATTGTGTA TATGCATTAA AATTACAGAG TAATTTAGGT TAGTATAAAA AATATTGTAT CTAAAAATAA ATATTGTAGT CAAATTTTCT ATGCTTCTAG CAGATATATA TAAACCTTGG AGATTTTCTT TTAAAAATGC CTAATTATGA AACAAAACTA TTATCAACTT CATCTTTTTG GAAATTTATT AGAGTMTAC AATAAAAATT TAATATAAAC TATTMATTA TATTACAACT AATGTTGMT TC(3952)
CAGTAATAAT AGCATTTGCA GTATTTACCT AAAGAGTTAA ATTTAATCAT GATTTAGAAT
5556 Nucleic Acids Research, Vol. 18, No. 18
Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D Thomas Binz, Hisao Kurazono, Michel R.Popoff, Melvin W.Eklund2, Genji Sakaguchi3, Shunji Kozaki3, Kerstin Kriegistein4, Agnes Henschen4, D.Michael Gill5 and Heiner Niemann* Institut fur Mikrobiologie, BFAV, Posifach 1149, 74 TObingen, FRG, 1Unite des Anaerobies, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France, 2Northwest Fisheries Center, 2725 Montlake Blvd E, Seattle, WA 98112, USA, 30saka Prefecture University, College of Agriculture, 8 Mozu Ume-machi, 4-cho Sakai-shi, Osaka 591, Japan, 4Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 and 5Department of Molecular Biology and Microbiology, Tufts University Schools of Medicine, Boston, MA 0211 1, USA Submitted August 13, 1990
The seven serologically distinct types of Clostridium botulinum neurotoxins, designated BoNT/A to BoNT/G, are synthesized as single-chain polypeptides of about 150 kDa. Proteolytic activation yields N-terminal light (=50 kDa) and C-terminal heavy chains (= 100 kDa) connected by single disulfide bonds. BoNT/D, like BoNT/Cl (1), is encoded by a bacteriophage (2, 3). The sequence shown below was established from cloned bacteriophage fragments the sizes of which were smaller than 2.0 kb (experiments registered by ZKBS Az 1494/1). The initial clone was identified with a synthetic 23-mer oligonucleotide containing only A or T residues in the wobble positions. The sequence (underlined) was deduced from a peptide obtained from purified BoNT/D by cyanogen bromide fragmentation. The sequence of the BoNT/D gene contains a single open reading frame beginning at the ATG codon in position 47 and encodes a polypeptide of 1276 amino acid residues (Mr 146,872 Da). Cys437 and Cys450 are probably involved in the disulfide bond between the light and the heavy chain. BoNT/D shares 49.6% amino acid sequence identity with BoNT/C (46.3% within the light chain, 67% within the N-terminal part of the heavy chain, and merely 35 % within the putative fragment C). No sequence similarity with exoenzyme C3 was found which is also encoded by C. botulinum type C and D phages (4).
ACKNOWLEDGEMENTS We thank F.Poysky and L.Jordan for help in preparing clostridial phage DNA. This work was supported by grant Nie 175/5-2 from the Deutsche Forschungsgemeinschaft to H.N. and by fellowships from the Alexander von Humboldt Foundation to H.K. and from Graduiertenforderung of the University Giessen to T.B.
REFERENCES 1. Hauser,D., Eklund,M.W., Kurazono,H., Binz,T., Niemann,H., Gill,D.M., Boquet,P. and Popoff,M.R. (1990) Nucl. Acids Res. 18, 4924.
*
To whom
correspondence should be addressed
EMBL accession
no.
X54254
2. Eklund,M.W., Poysky,F.T., Reed,S.M. and Smith,C.A. (1971) Science 172, 480-482. 3. Eklund,M.W. and Poysky,F.T. (1989) in: Botulinwn Neurotoxins and Tetanus Toxin (Simpson,L.L. ed.) pp. 25-51, Academic Press, San Diego. 4. Popoff,M.R., Boquet,P., Gill,D.M. and Eklund,M.W. (1990) Nucd. Acids Res. 18, 1291.
( ( ( ( ( ( ( ( ( ( ( (
1)AAGGTGCACA CTTGTGGATA TTAGAAAGTT AGGAGATGTT AGTATTATGA CATGGCCAGT AMAGATTTT
71)AATTATAGTG ATCCTGTTAA TGACAATGAT ATATTATATT TAAGAATACC ACAAAATMG 141)CACCTGTAAA AGCTTTTATG ATTACTCAM ATATTTGGGT AATACCAGM AGATTTTCAT TTMTTACTA CAGATACTM 211)TCCAAGTTTA AGTAAACCGC CCAGACCTAC TTCAAAGTAT CAAAGTTATT ATGATCCTAG TTATTTATCT 281)ACTGATGAAC AAAMGATAC ATTTTTAMA GGGATTATAA AATTATTTAA MGAATTAAT GAAAGAGATA 351)TAGGAAAAAA ATTAATAAAT TATTTAGTAG TTGGTTCACC TTTTATGGGA GATTCAAGTA CGCCTGMGA 421)TACATTTGAT TTTACACGTC ATACTACTM TATTGCAGTT GMMGTTTG AAMTGGTAG TTGGMAGTA 491)ACAAATATTA TMCACCAAG TGTATTGATA TTTGGACCAC TTCCTAATAT ATTAGACTAT ACAGCATCCC 561)TTACATTGCA AGGACAACM TCAAATCCAT CATTTGAAGG GTTTGGAACA TTATCTATAC TMAAGTAGC 631)ACCTGAATTT TTGTTMCAT TTAGTGATGT AACATCTAAT CAAAGTTCAG CTGTATTAGG CAAATCTATA 701)TTTTGTATGG ATCCAGTMT AGCTTTAATG CATGAGTTAA CACATTCTTT GCATCAATTA TATGGMTM 771)ATATACCATC TGATAAAAGG ATTCGTCCAC MGTTAGCGA GGGATTTTTC TCTCAAGATG GACCCAACGT 841)ACAATTTGAG GAATTATATA CATTTGGAGG ATTAGATGTT GAAATAATAC CTCAMTTGA MGATCACAA 911)TTAAGAGAAA AAGCATTAGG TCACTATAAA GATATAGCGA AAAGACTTAA TMTATTAAT AMACTATTC 981)CTTCTAGTTG GATTAGTAAT ATAGATAAAT ATAAAAAAAT ATTTTCTGAA AAGTATAATT TTGATAAAGA (1051)TAATACAGGA AATTTTGTTG TAAATATTGA TMATTCMT AGCTTATATT CAGACTTGAC TAATGTTATG (1121)TCAGAAGTTG TTTATTCTTC GCAATATAAT GTTMAAACA GGACTCATTA TTTTTCAAGG CATTATCTAC (1191)CTGTATTTGC AAATATATTA GATGATAATA TTTATACTAT AAGAGATGGT TTTAATTTAA CAMTAAAGG (1261)TTTTAATATA
(1331)GTAGTAGATT (1401)AAGTTAAAAA (1471)AATTATTACA (1541)GGGCAAGTTC
(1611)TTCCAGGTGA
(1681)TTTGGAATCT (1751)GGTTATAGCA
(1821)GTTTATTCTT
(1891)GGATAAAATA (1961)TTAAGGGGAA (2031)TTACTATACC (2101)TATAGAAAAT (2171)TTGTCAAGAA (2241)ATGCAATCAA (2311)TCAAGTTGAA (2381)ATACGAGAAT (2451)TTGATTTAAG (2521)AGATAGATTA (2591)AATAATTCTT (2661)TACAAAACAA (2731)TCAACTTAAT (2801)AATAATAATA (2871)ATTTAACTAA (2941)TATTAGGAAT (3011)TATAGTGAAT (3081)TGGGGTATAT (3151)TAAGTTAGAT (3221)GATTTTAATA (3291)GAAATGTTAT
GAAAATTCGG TATTTACAAA TAATAGATTA GATGAGACTA CTATTAATCC AGAAATAGTA CAAAAATTAA ATAAGATATA AAATTGGGCG TCAGATGTAT ATTTTAATCA TGCACTCGGT TGTTTGGAAC TTACTACTCA AGCTAAAATA
AATTTAAAAA
GTTCTGTAAC AACTAAAACA AAAGCAAAAG TATTAAAAGA AAAAAATGCT
ACGATATATA
TTTTATATAG TTCTCATAAT GGCAATATAG CATTAAGTCA GAAACTTTAT AAAACCATAG TTTTTTCTAA TAAAGATTAT
(3361)TTATATAGAT AGGTATATAG (3431)TTATATACTG GAAATCCTAT
(3501)GAGATAATAT AATTCTTCAT (3571)ATATGCAACA CAAGGAGGAG (3641)AATTATGGTA TAGGTATATT (3711)CTAGTTTTAG GGAAAATACA
(3781)ATACACGCCA GTTGCAGTAA (3851)TCTAGGGATC CAGGATGGGT (3921)TTTAGAAATT TATCGTATAA
GTCAGAATAT AGTATGTTTA CCTTATGTAG ATGTACAAAA TGAMTAGTA
AGAAAGGAAT CCTGCACTAC AAMGCTTAG TTCAGAAAGT AGATTMCAA AAAATAGTAG AGATGATTCA ACATGTATTA
CTGATAAAGA TAGCATTTCA CAAGAAATAT TTGAAMTM TTATTCAGAT AAATTTTCAT TAGATGAATC TATTTTAGAT GATCCACTAT TACCCAATGT TMTATGGM CCTTTAATC TTTTATGATG ATATTACTAA ATATGTTGAT TATTTAAATT CTTATTATTA GTAATMTGT TGAAAATATT ACTCTTACAA CTTCAGTTGA AGMGCATTA CACATTTTTA CCTAGCTTAG CTGAAAAAGT GAATAAAGGT GTTCMGCAG
MTGMGTAG TTGAGGATTT TACTACAMT TCCATATATA GGACCTGCCT ACAGCTGGTG TAGCTTTTTT TTTATAGTTC TATTCAAGAA
ATTATGAAGA MGATACATT TAAATATAGG AAATTCAGCA ATTAGAGGGA TTTCCAGAGT AGAGAGAAM TTATTAAAAC GAGATGGAM GATTCATATC AATGGATGGT ATCAMTTGG ATAMTTATC AAATGTATGA TTCTTTAAGT TATCAGGCAG ATAAAAAATA CTCAGGAAGT GATMAGAAA ATATMAAAG ATAGTTTAGA TGTAAAAATT TCGGAAGCA& TGQTTAT AATAAATTT ATACTTATTT AAAAATATGC TCCCTAAAGT MTTGACGAA TTAAATAAGT GAATTAATTA ATCTTATAGA TAGTCATAAT ATTATTCTAG TTGGTGAAGT TAAATGACAG TTTTGAAAAT ACMTGCCTT TTMTATTTT TTCATATACT TATAATTMT GAATATTTCA ATAGTATTM TGATTCAAAA ATTTTGAGCT TTAGTGGATA CATCAGGATA TAATGCAGAA GTGAGGGTAG GAGATAATGT CAAATGACTT TAAATTAAGT AGTTCAGGAG ATAAAATTAT AGTAAATTTA CGCTATTTAT GAGAACTCTA GTGTTAGTTT TTGGATTAAG ATATCTAAAG GMTATACAA TAATTAACAG TATAGAACAA AATTCTGGGT GGAAATTATG AATGGATTTT ACAAGATGTT AATAGAAAGT ATAAAAGTTT AATTTTTGAT TACAGGATAT ACAAATAAAT GGTTTTTTGT TACTATAACT AATAATATM ATAAATGGAG AATTAAAGCA GAGTCAAAAA ATTGMGATT TAGATGAGGT TATTTGGAAT AGATGAGAAT ATAGATGAGA ATCAGATGCT TTGGATTAGA AGAATTAAGT AATGAAGATA TTAATATTGT ATATGAGGGA CAAATATTM TGGGGAAATC CTTTGAAGTT TGATACAGAA TATTATATTA TTAATGATM CACCTGAAAG TAATGTACTT GTACTTGTTC AGTATCCAGA TAGATCTAAA TACTATTAAA TCAGTATCTG ATMGAATCC TTATAGTAGA ATTTTAAATG ATGTTATATA ATAGTAGGAA ATATATGATA ATAAGAGATA CTGATACMT AGTGTTCACA AAATTGTGTA TATGCATTAA AATTACAGAG TAATTTAGGT TAGTATAAAA AATATTGTAT CTAAAAATAA ATATTGTAGT CAAATTTTCT ATGCTTCTAG CAGATATATA TAAACCTTGG AGATTTTCTT TTAAAAATGC CTAATTATGA AACAAAACTA TTATCAACTT CATCTTTTTG GAAATTTATT AGAGTMTAC AATAAAAATT TAATATAAAC TATTMATTA TATTACAACT AATGTTGMT TC(3952)
CAGTAATAAT AGCATTTGCA GTATTTACCT AAAGAGTTAA ATTTAATCAT GATTTAGAAT
