ORGANIZATION AND NUCLEOTIDE SEQUENCE OF GENES FOR HEMAGGLUTININ COMPONENTS OF CLOSTRIDIUMBOTULINUM TYPE B PROGENITOR TOXIN

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1 Vol. 39, No. 6, August 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages ORGANIZATION AND NUCLEOTIDE SEQUENCE OF GENES FOR HEMAGGLUTININ COMPONENTS OF CLOSTRIDIUMBOTULINUM TYPE B PROGENITOR TOXIN Gi-Hyeok Yang, Sang-Dal Rhee, Hyun-Ho Jung 1, and Kyu-Hwan Yang Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, , Korea and ~Department of Microbiology, Sunmoon University, Asan, , Korea Received May 22, 1996 SUMMARY: The genes for hemagglutinin components (33 kd, 17 kd, and 21.5 kd) of Clostlqdium botulinum progenitor toxin were cloned and sequenced. Analysis of the nucleotide sequence showed that the 33 kd, 17 kd, and 21.5 kd hemagglutinin genes were organized into an operon in the 5'upstream region of the toxin gene and their ORF orientation were opposite to that of the toxin gene. A comparison of amino acid sequences between the hemagglutinin components in and progenitor toxin showed significant homology. Northern blot analysis also revealed that all of the genes for the hemagglutinin components were transcribed as a polycistronic RNA. INTRODUCTION Clostridium botubnum is a Gram positive, anaerobic, endospore forming bacteria. The neurotoxins which are produced by the bacteria are classified into seven groups (type A to G) based on their antigenic characteristics (1). The neurotoxins are produced as a single chain and are separated into two fragments, designated as the heavy chain (100 kd) and the light chain (50 kd), by a reduction of a disulfide bond. Type B toxin exists in large molecular sizes of kd in the culture supernatant. These progenitor toxins are formed by the association of the 7S neurotoxin with a nontoxic fraction(s); a hemmaglutinin subunit(s) (HA) and a nontoxic-nonhemmaglutinin component (2). HAs have been thought to protect the neurotoxin from the gauntlet of acidity and proteases (3-5). To date, structural genes of the seven neurotoxin have been cloned and sequenced (6-13). However, in case of the genes for hemagglutinin components, only that of the strain was /96/ ~) /0 Copyright 1996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL reported (14-15). In this study, genes for hemagglutinin components in progenitor toxin were cloned and sequenced. METHODS Cloning of the Upstream Region of the Toxin Gene. Clostridium botulinum strain, Lamanna was grown in the culture medium as described by DasGupta et al. (16). Chromosomal DNA was prepared as described by Jung et al. and digested with XbaI, and southern blot analysis was performed with 0.4 kb XbaI-HindIII fragment from pu509 containing toxin gene previously cloned (17). The fragment was labeled with [32p]-dCTP using random primed DNA labeling kit (Promega, USA). Based on southern blot analysis, genomic library was prepared, and positive clones were screened by the colony hybridization technique. Restriction map was determined by using several six-cut restriction enzymes. Nucleotide Sequencin,~. After the subcloning of specific DNA fragments in pbluescript vectors, the sequencing reactions were performed by primer extension dideoxy termination method with alkali-denatured double stranded plasmid templates according to the procedure supplied with the Sequenase (version 2.0) kit (United States Biochemical Corp.,USA). [35S]-dATP (> 1,000Ci/mmol; Amersham Corp. UK) was used in labeling reaction. Forward and reverse M13/pUC universal primers (United States Biochem Corp.,USA) as well as custom oligonucleotide primers for sequencing internal regions were used as sequencing primers. Northern Blot Analysis. Total RNAs in C. botulinum were isolated using strong denaturant guanidine thicyanate method (18). A 0.3 kb DNA involving hem33/b gene was prepared from the clone pu921 by digestion with HindIII-XbaI(Fig.1.) These probes were labeled with [32p]-dCTP. Following gel electrophoresis of the prepared RNA (30 ~tg) and blotting onto Hybond-N + Nylon membrane (Amersham, UK), hybridization with the probes was performed. KEY : 1 Kb E EcoRI H Hindlll X Xbal xe X H H E H H H H X 438bp 873bp hem21.5/bhem17/b hem33/b Toxin Fig. 1. Organization of the hemagglutinin (HA) and neurotoxin genes from Clostridium botulinum. The chromosomal DNA were partially digested with XbaI. The fragments were cloned into puc19 plasmids to give pu921. The structural genes for progenitor toxin components and the orientation of those open reading frames indicated by the open arrows. 1142

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Analysis of the Sequences. The DNA sequence data was analyzed using computer programs, DNASIS and PCGENE, and the amino acid sequence homology search was carried out by the software program CLAUSTAL V. RESULTS AND DISCUSSION Nucleotide sequencing revealed that there are ORFs in the upstream flanking region of the toxin gene, which were named hem33/b, hem17/b, and hem21.5/b, respectively (Fig. 2). The three ORFs were exactly same as the N-terminal amino acids sequence of hemagglutinin components in a progenitor toxin previously reported (2). The sequence of hem33/b was 873bp in length which corresponded with the 293 amino acid residues with a deduced relative molecular mass of Da. Also, the hem17/b was 438 bp which corresponded with the 146 amino acid residues of Da. These three genes were transcribed in the opposite orientation to that of the toxin gene. The primary structures of the proteins from the three ORFs showed no characteristic signal peptide. As with all other clostridial botulinum genes characterized to date, the high A+T content of the DNA resulted in extreme bias towards the use of codons ending in A or T. The translational start codon was preceded by ribosome binding sites (Fig.2). A comparison of the amino acid sequence of hemagglutinin components of the type B progenitor toxin with that of the is shown in Fig.3. Hem33/B was found to share a 34.1% identity and 58 % similarity with the 33 kd hemagglutinin component in the progenitor toxin. 17 kd hemagglutinin components of both type progenitor toxin showed extreme identity (64.3%) and similarity (84.2 %). Such a high evolutionary conservation of the amino acid sequences suggested that the hemagglutinin components might play an impotant role in the toxicity of the botulinum toxin. The non-toxic components of the progenitor toxin have been reported to protect toxin from extremely low ph levels and proteolytic cleavage in the digestive tract (3-5). The hemagglutinin genes were shown to be closely oriented to each other. Heml7/B were located 65 bp apart from hem33/b, and hem21.5/b were located 16 bp apart from heml7/b (Fig2). To conform the possibility these genes are co-transcribed as a polycistronic transcript, northern blot hybridization was carried out by using a 0.3 kb HmdlII-XbaI hem33/b gene fragment as a probe. As shown in Fig. 4, a single transcript with a length of about 3.5 kb was obtained. This result agreed with the length of the 1143

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL ATTGCAAATATCTAAGCAATATCriT~rAAAGTCCTACTAATATATCTCTCTAAATCATITTGTGTA~ITGAAATTGCTCAAATCAA 86 N C I D L C Y R K L T R S I Y R E L D N Q T N F N S L D V Crr F f r [AAGTGTATACCATAAATGGTACAAAATATCATTGTAATTATCATATATATrATATFF~.CTAGTAAATATATT~`ATAGTr 192 K K L T Y W L H Y L I D N Y N D Y I N Y K R T F I N I T Tr HCAAAATCCYrAAAAAT~CITGAAACTCCTCATTGTCAC~IT~AACAT~rCAA~TGTAAAAACAA~U~AT~CATATFrAC 258 K E F H K F I E Q F E E N D S K L M E I Q L F L K N H CACTATCCCTCCATAATATACATATTATTGTTTTATTATACTATAAATGAACTAATTGCCTTTAAAT~TriAATAAGCCATrr~TC 344 SD CGAAATATGGATAATCAATrAT6TAAACATACTAATAAAAATTrGATATATTrAAATTTrAGGTITACAAAAAATAATr~GA~rAT > hem33/b TTATAT TI ATATGTAA ATrAATrATTAAATAAAGGAAT ATAATGGAACACTATTCAACAATCCAAAATrCA'UrAAATGACAA 516 SD M E H Y S T I O N S L N D K AATCGTrA~CATCTCCrGTAAGCCFAATACAGATTrA~ATCAAGTTCCCGGTAACGGTAACGTrAGCTTATTTCAACAAA 602 I V T I S C K A N T D L F F Y Q V P G N G N V S L F Q Q CTAGAAATTACCTGGAAAGATGGAGAATTATATATGATrCTAATAAAGCTG ITATAAAATAAAAAGTATGAATATCTATAATACT 688 T R N Y L E R W R I I Y D S N K A A Y K I K S M N I Y N T AATrTAurr r raacatggaatgcaccaacacataatatatcagcocttcaagattcaaatgcagataatcaata]t~atta]~r 774 N L V L T W N A P T H N I S A L Q D S N A D N Q Y W L L L AAAAGACATTGGTAACAATTCATITATTATTGCAA~TFATAAAAACCCTAACT~AGTATTATAT~CTGATACC~TAC~TCGTAA~ 860 K D I G N N S F I I A S Y K N P N L V L Y A D T V A R N TGAACLT~`AGCACACITAATAATrCAAGTrATATAAAATTTAT~ATAGAAGATrATGTAATATCAGATT~AAAAATTTCACATGT 946 L K L S T L N N S S Y I K F I I E D Y V I S D F K N F T C AGAATAAGTCCAATATTAGCCGGTGGTAAAG~ITGTACAACAAGTGTCTATGACAAATCTAGCTG'~TAATF~ATATAYITGGAACAA 1032 R 1S P I L A G G K V V Q O V S M T N L A V N L Y i W N N TGATCTCAATCAAAAATGGACAATTATATATAATGAAGAAAAAGCAGCATACCAGIT r r l raataaaatacittcaaacggag'itc lll8 D L N O K W T I I Y N E E K A A Y Q F F N K I L S N G V TAACATGGAT H-ITrCAGATGGTAATACTGTAAGAGTTTCTTCTAGTGCGCAAAATGATGCCCAATATTGCLTI'ATAAATCCTGTT 1204 L T W I F S D G N T V R V S S S A O N D A O Y W L I N P V TCAGATAATTATGACAGATATACAATTACTAATCTACGCTATAAAACTAAAGT~CTAGATTTATATGGCGGCCAAACAGCAGACGG 1290 s D N Y D R Y T I T N L R Y K T K V L D L Y G G 0 T A D G AACTACTATTCAAGTATITAATTCTAATGGAGGTGATAATCAGATATGGACTAT~AGTAACCCATAAAATrAATATCAACAAAGAT 1376 T T I Q V F N S N G G D N Q I W T ~I S N P... >heml7/b TFAATATGAATACTATCATCTAATTTATAAAGAGGTGAATTATATGTCAGCTGAAAGAAu f r r rctacctaatggtaattacaata 1462 SD M S A E R T F L P N G N Y N TAAAATCTATU~T~TCTGGT~CTFFATATVfAAGTCCTGTATCAGGATCATTAACATTTFCAAATGAATC~TCTGCAAATAATCAA 1548 I K S I F S G S L Y L S P V S G S L T F S N E S S A N N Q AAATGGAATGTAGAATATATGGCTGAAAATAGAT~AAAATCTCFAATGTAGCAGAACCAAATAAGTATTTAAGTTACGATAA 1634 K W N V E Y M A E N R C F K I S N V A E P N K Y L S Y D N CTTTGGATTTATTTCT-~rAGATrCATTATCfAATAGATGCTACTGGTfTCCTATTAAAATCGCTGTAAATACTTATATTATGTrAA 1720 F G F I S L D S L S N R C Y W F P I K I A V N T Y I M L GTTTAAATAAAGTGAATGAATFAGATTATGCCTGGGACATTfATGATACTAATGAAAATA]TfTAAGTCAGCCACTACT~CTACrA 1806 S L N K V N E L D Y A W D I Y D T N E N I L S Q P L L L L... ->hem21.5/b CCTA,~r i-r rgatatatacaattcaaatcaaatgttcaaacitgaaaaaatataaaggagaaaagggtatgaattcatctataaaaa 1892 P N F D I Y N S N Q M F K L E K I SD M N S S I K AAATITATAATCATATACAAGAAAAAGTTATAAACTATAGTGATACTA ITGATITAGCTGATGGTAATTATGTAGTTAGCAGAGGG 1978 K I Y N H I Q E K V I N Y S D T I D L A D G N Y V V S R G GATGGATGGATATTATCTAGA 1999 D G W I L S R Fig. 2. Nucleotide sequence of the 5' end of the toxin gene locus. The putative Shine- Dalgano sequences are indicated by SD. The sequence determined has been submitted to the EMBL/Genbank Database (accession number U24431). 1144

5 BIOCHEMISTRYnnd MOLECULAR BIOLOGY INTERNATIONAL (A) 33kD hemagglut inin component MEHYSTIONSINI~IVTISCKANTDLFFYQVPGNGNVSLFQQTIINYLERW MSQ- -TNANDLRNNEVF ISPSNNTNKVLDK I SQSEVKLNVKLSGANQKqV RI I YDSNKAAYKIKSt4N I YNTNLVLTWNAPTHNI SALQDSNADNQY~LL RLIYDTNKQAYKIKVMD- -NTSLI LT~NAPLSSVSVKTDTNGI]NEIYWYLL :~. ~,:~::~::. ~X: X~:~:: ~ ~. /, X:. ~. 8~:~::~:~,~.. ~ X. X:. ~X ~:~ X{~,~ KDIGNNSFI IASYI<NPNLVL - YADTVARNLKLSTLNNSSYI KF I IEDYVI QNYISRNVI I PASNqPNLVLQYN- - I DDTLMVSTQTSSSNQFFKFSNCl Y.... ~,,. ~, ~ ~, ~;~. X(~ SDFKNFTCR I SP ILAGGKWQQVSMrNLAVNLYI WNNDLNQKIFr I IYNEE EALKNRNCKLQTQLNSDRFLSK- NLNSQIVLWQWFDSSRQKWI IEYNEr.. ~ ~.. ~ ~ ~ ~ ~ ~-X~ KAAYQFFNK ILSNGVLTWIFSDGNTVRVSSSAQNDAQYWL I NPVSI)NYDR KSAYTL-KCQENKRYLTWIONSNNYVETYQSTDSLIQYWNI NYLDNDASK Yr ITNLRYKTKVLDLYGGQTADGTTI QVFNSNGGDNQI WTMSNP YI LYNLQDTNRVLDVYNSQI ANGTHV I VDSYIONTNQQW I INLI. X~.. ~f~. ~. ~ ~.. ~.., ~ ~X~ ~.. (B) 17kD hemagglutinin component MSAERTFLPNGNYNIKSIFSGSLYLSPVSGSLTFSNESSANN0k'WNVEYbt MSSERTFLPNGNYKIKSLFSDSLYLTYSSGALSFSNTSSLDNQK~J_~yI AI~CFKISNVAEPNKYLSYDNFGFISLDSLSNRCYWFPIKIAVNTYIML SSSNGFRFSNVAEPNKYLAYNDYGFIYLSSSSNNSLWNPIKIAINSYIIC. ~.. ~-~:~. ~... ~ ~ ~ ~ ~. ~-. ~ SLNK~LDYAWDIYDTNENILSOPLLLLPNFDIYNSNQMFKLEKI TLSIVNVTDYAWTIYDNNNNITDQPILNLPNFDINNSNQILKLI~ ~. ~X~ I ~ ~c~ ~. ~,"; I.~$'. ~ ~X~X~ ~.. g~ ~. Fig. 3. Alignments of the translated hem/b genes with hemagglutinins. Asterisks indicate identical amino acid residues common to the two proteins. Dots indicated similar amino acid residues common to the two proteins. 23S ~ Polycistronic RNA 16S -4~ Fig. 4. Northern blot analysis of C.botuliuum RNAs. The positions of the 23s (2.9 kb) and 16s (1.5 kb) rtlna are indicated by continuous arrows. The polycistronic transcripts are indicated by dotted arrow. 1145

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL transcript predicted by considering the total sum of the three hem/b genes, and so the genes for hemmgglutinin components of botulinum progenitor toxin might constitute an operon which is co-regulated in the level of transcription. ACKNOWLEDGEMENT This work was supported by a grant from the Genetic Engineering Research Fund of the Ministry of Education, ROK. (1994). REFERENCE 1. Sugiyama, H. (1980). Microbiol. Rev. 44, Somers, E., DasGupta, B.R. (1991) J. Prot. Chem.10, Suggi, S., Ohishi, I., and Sakaguchi, G. (1977) Infect. Immun.16,9t Ohishi, I. and Sakaguchi, G. (1980) Infect. Immun. 28, Ohishi, I., Suggi, S., and Sakaguchi, G. (1977) Infect. Immun.t6, Thomson, D.E, Brehm, J. K., Oultran, J. D., Swinfield, T. J., Shone, C. C., Atkinson, T., Melling, J. and Minton, N.P. (1990) Eur. J. Biochem. 189, Binz, T., Kurazono, H., Wille, M., Frevert, J., Wernars, K.and Niemann, H. (1990) J. Biol. Chem. 265, Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Brehm, J. K., Atkinson, T., and Minton, N. P. (1992) Appl. Environ. Microbiol. 58, Kimura, K., Fujii, N., Tsuzuki, K., Murakami, T., Indoh, T., Yokosawa, N., Takeshi, K., Syuto, B. and Oguma, K. (1990) Biochem. Biophys. Res. Commun. 171, Binz, T., Kurozono, H., Popoff, M. R., Eldund, M. W., Sachaguchi, G., Kozaki, S., Kreiglstein, K., Henschen, A., Gill, D. M., and Niemann, H. (1990) Nucleic Acids Res. 18, Poulet, S., Hauser, D., Quanz, M., Niemann, H., and Popoff, M. (1992). Biochem. Biophys. Res. Commun. 183, Campbell, K., Collins, M. D., and East, A. K (1993) Biochem. Biophys. Act. 1261, Fairweather, N.F., and Lyness, V.A. (1986) Nucleic Acid Res. 14, Tsuzuki, K., Kimura, K., Fuji, N., Yokosawa, V,. Indoh, T., Murakani, T., and Oguma, k., (1990) Infect. Immun. 58, Fuginaga, Y.,Inoue, K., Shmazald, S., Tomochika, K., Tsuzuki, K., Fugii, N., Wadanabe, T., Ohyama, T., Takeshi, K., Inoue, K. and Oguma.K (1994) Biochem. Biophisic. Res. Commun.205, DasGupta, B.R. and Sugiyama, H. (1976) Infect. Immun. 14, Jung, H.H., Rhee, S.D., and Yang. KH. (1992) FEMS Microbiol. Lett. 91, Chomczynsld, P. and Sacci, N. (1987) Analytic. Biochem. 162,