FEMS MicrobiologyLetters96 (1992) 225-230 © 1992Federation of European MicrobiologicalSocieties0378-1097/92/$05.00 Published by Elsevier

225

FEMSLE 05048

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum Alison K. East, P.T. Richardson, D. Allaway, M.D. Collins, T.A. Roberts and D a p h n e E. T h o m p s o n Department of Microbiology,AFRC Institute of Food Resea:ch. Reading Laborat~.'y, Readi;;g, UK

Received5 June 1992 Accepted 24 June 1992

Key words: Clostridium botulinum; Type F neurotoxin; Nucleotide sequence; Polymerase chain reaction

1. SUMMARY

2. INTRODUCTION

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridiura bomlinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.

Within the genus Clostridium, organisms producing protein toxins able to cause paralysis symptomatic of botulism are deemed to be Clostridium botulinum. Outbreaks of botulism in humans and animals have resulted in the classification of the neurotoxin into seven serologically different forms identified as type A ( B o N T / A ) to type G (BoNT/G). B o N T / A , B o N T / B and B o N T / E are the main causative agents of human botulism, with rar,e incidents attributed to B o N T / F and B o N T / G [1]. The disease is most often the result of ingestion of toxin formed in foods that have been inadequately processed a n d / o r stored at inappropriate temperatures. Another source of the disease is after colonization of the gut by clostridia in infants of less than approx. 6 months, consequently known as infant botulism [1,2]. Occasionally botulism is the result of infection of a wound. There have been reports of BoNT being produced by clostridia that

Correspondence to: D.E. Thompson, Department of Health, Eileen House, 80-94NewingtonCauseway, London, SEI 6EF, UK.

226 phenotypicaily are not C botulinum viz. C butyticum and C barati [1,2]. Type F neurotoxin is produced by C. botulinum Group I (proteolytic strains), Group II (non-proteolytic strains) and by C. barati [3]. Comparison of the complete amino acid sequences from BoNT genes (BoNT/A [4,5], BoNT/C [6], B o N T / D [7], BoNT/E [8] and BoNT/E of C. butyricum [9]), and partial sequence (BoNT/B [10]), with tetanus toxin (req'x) [11] has revealed conserved regions thought to be implicated in toxin function [10]. Primers based on data from these conserved regions were used to clone and sequence the gene encoding B o N T / F from non-proteolytic C. botulinum using polymerase chain reaction (PCR). We report here the nucleotide sequence of a gene, which when translated, gives an amino acid sequence similar to those of BoNTs already reported [5-10].

3. MATERIALS AND METHODS 3.1. Cultures and preparation of DNA C botulinum strain 202F (ATCC 23387) was grown anaerobically in TPYCG (trypticase 50 g / l , bactopeptone 5 g / l , yeast extract 3 g/I, cysteine HCI 0.5 g / I and glucose 5 g / i ) broth and DNA prepared as described by Farrow et al. [12]. All manipulations of C. botulinum and cloned fragments were carried out under GMP II conditions. E. coli SURE (Stratagene) was used as the recipient for transformation. Plasmid DNA was prepared from E. coli by the alkaline lysis method of Birnboim and Doly (see ref. 13) and for sequencing, by the method of Treisman as described by Sambrook et al. [13]. 3.2. PCR amplification PCR was performed with a Biometra thermocycler using the following conditions: 95°C for 5 rain, followed by 25 cycles of: 94°C for 45 s, between 37 and 45°C for 45 s and 58°C for 2 min, followed by 62°C for 10 rain and then 4°C. Primers were made on an Applied Biosystems model 391 DNA synthesiser and used at a final concentration of 4 ng//~l. Deoxynucleotides (BCL) were present at a final concentration of 200 mM and 1

unit Taq polymerase (Amersham International) was used according to the manufacturers instructions. Template DNA was at a final concentration of 20 ng//xi except for 'inverse PCR' when the concentration was 50 n g / # ! of restriction enzyme-digested and re-ligated DNA. For 'inverse PCR' [14], DNA was prepared using restriction enzymes (BCL) according to the manufacturer's instructions and ligations performed using a 'Ligation System' kit (Amersham International).

3.3. Cloning and transformation Products of PCR were cloned into the EcoRV site of pBlueseript KS + (Stratagene) following modification and purification of the fragment by Klenow (BCL), 'Geneclean' (Stratateeh), kinase (Gibeo-BRL), phenol extraction and ethanol precipitation [13]. An alternative vector system was used to construct pCBF2 and pCBF8; DNA fragments generated by PCR were cloned directly, without enzymic modification, into 'T-vector' (D. Evans, University of Reading, personal communication) digested with Xcml (New England Biolabs). This digestion generates a single 3' overhanging T on the vector which base pairs with the A, often added to the 3' end of PCR fragments during the reaction [15]. Transformation into E. coil competent cells, prepared as described by Inoue et al. [16], was carried out as described by Sambrook et al. [13]. Plasmids containing inserts of the expected size were sequenced following testing of the recombinant-ceU lysates by the mouse bioassay [17], to ensure that no toxic polypeptides were produced.

3.4. Sequence determination Sequencing of plasmid DNA was carried out using dideoxy-nucleotidc chain termination. Primers complementary to both the plasmid and toxin were used to sequence both strands of each insert completely. As the inserts were generated using PCR, the sequence of two fragments synthesised in different PCR reactions was determined. Where two sequences differed, as they did in three positions, a third clone was sequenced and in each case confirmed one of the sequences.

227 4. RESULTS AND DISCUSSION To ensure that no toxic polypeptides were produced only fragments encoding part of BoNT/F were cloned in E. coli. Figure 1 shows the position of the primers used to amplify regions of DNA which were subsequently cloned. No recombinant plasmid was toxic in the mouse bioassay [17]. The open reading frame codes for a protein of 1274 amino acids with a molecular mass of 146 708. This calculated value compares well with the measured value of 150000 for BoNT/F purified from proteolytic C. botulinum [18]. The protein encoded by BoNT/F shows high homology with TeTx and other BoNTs. Since these toxins are usually cleaved in cico and as the two chains have different functions in toxic activity [10], the proteins were divided into light and heavy chains for comparative purposes. Comparing BoNT/F with light and heavy chains for toxins where the sequence is known (data not shown), the light chain of B o N T / F is more like that of BoNT/E, showing 58.3% and 57.6% identity with the toxins of C. botulinum and C. butyricum respectively, than TeTx (45.1% identity) and BoNT/A (35.1% identity). The heavy chain also showed greater homology with the BoNT/E from C. botulinum (64.9% identity) and C. butyricum (64.4% identity), with BoNT/A and TeTx having identities 44.6% and 35.0% respectively. The high sequence identity of BoNT/F and BoNT/E may explain SaI/3AI

I

the cross-reactivity of antiscra raised against either type toxin with both BoNT/F and BoNT/E. Within the toxin sequence there are regions of highly conserved amino acids thought to be important for structure and function (see ref. 10). In the light chain, the region noted by Niemann, DPhhnLhHELnHnnHxLYG, where h is a hydrophobic residue, n is uncharged and x is any amino acid [10], is also present in BoNT/F (Fig. 2, residues 220-239). The cysteine residues involved in disulphide linkage of the heavy and light chains are found in all the toxins (Fig. 2, residues 429 and 445). The number of residues between the cysteines varies between eight (BoNT/B) and 27 (TeTx); BoNT/F has 15 amino acids. As with BoNT/E, this BoNT/F is encoded by a non-proteolytic C botulinum, it is therefore unlikely to undergo proteolytic cleavage by an endogenous viotease, but a possible cleavage site for exogenous proteases is between residues 439 and 440 (Fig. 2). The C-terminus of the heavy chain, a region noted for its lack of homology in BoNTs [10], is very well conserved between BoNT/F and BoNT/E. It has been suggested that this region is involved in the binding of BoNT to a ganglioside receptor [10]. The high homology between BoNT/E and BoNT/F may indicate that they share a common receptor. Analysis of sequences upstream of BoNT/F revealed a possible Shine-Dalgarno sequence 5'AGGGGG-3' (Fig. 2, nucleotides 132-137), eight nucleotides upstream of the proposed start of

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Fig. I. Strategy for cloningfragmentsof the BoNT/F gene. Arrows indicate the positionof the primers used to generate the fragments.Clonesgeneratedby "inversePCR" are indicatedby *: in the case of pCBFI*/pCBF2* the chromosomalDNA was digested with Sau3AL for pCBF6* with EcoRI, and pCBF8*/pCBF9* with EcoRV. Only the sites of restriction enzymes involvedin the constructionof clonesare shown.

228

translation. Further upstream, nucleotides 33-38 and 47 (Fig. 2) are identical to those mapped as the - 1 0 and transcriptional start by Binz et al. for B o N T / A using primer extension [5]. The - 3 5 region suggested by the same authors for B o N T / A of 5'-TTAACCC-Y is present virtually unchanged (5'-TFAACGC-3') in the same position in BoNT/F (Fig. 2, nucleotides 5-11). However, further analysis of this upstream region of the BoNT/F gene shows that it contains the Y-end of an open reading frame (Fig. 2, nu-

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cleotides 1-130 and data not shown), translation of which into protein sequence gives the C-terminal 57 amino acids (Fig. 3). The translated sequence shows homology with the non-toxic-nonhaemagglutinin component of C. botulinum type C [19], the gene for which is found in the same position, upstream of the toxin gene. Published sequences reveal the presence of such open reading frames in type A [5], type E [8] and C. butyricum [9], although not commented upon by the authors• As these translated open reading

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CAGGATTTAKATATTATTAC TA GTGC TAT GAAGGAAAAAATATATAACAATC TT TT AGCT 1020 (292) Q D L N I i T S A M K E 8 1 Y N N L L A

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Fig. 2. Complete nuci=otide sequence of the B o N T / F gene. The translated amino acid sequence of B o N T / F is given under the second nucleotide of each codon. The position of primers used in PCR to generate the clones is indicated by underlining; * above a nuclcotidc indicates a mismatch between the primer and the D N A sequence. The possible position of cleavage between the light and heavy chains is indicated. The cystcinc residues arc underlined at amino acids 364 and 445, Nucleotides ] to 130 form part of the open reading frame translated to give the sequence shown in Fig. 3. The sequence has been given the Genbank database accession number M92906.

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229 ATAAAAACAGCAATAGAATATAAATATAATAATTATACTT~'AGA2";A%AAAAATAGACTT 242: GAATCTGAATATAATATCAATAATATAGAAGAAGAATT";AATA~AAAAGTTTCTTTAGCA ; 4 '%c' ATGAA~@.~TATAGAAAGATTTATGACAGAAAGTT-TATA

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)tMDI< YFSLS

ISNC]~'~LTYMNVN Z S N 4 = Z~T L T Y N

DGDYH

I C L S M K D E N Y N L$ IKN N V N V "L' L ~ ~ g N

DYN D Y w

frames are homologous with the non-toxic-nonhaemagglutinin component of BoNT/C (Fig. 3) it is possible that there is a toxin operon encoding the components of the progenitor botulinum toxin complex in types A and E and C. butyricum, as in type C [19].

i

TypeV 'Type A

NMI(:

TypeE Type E{but)

WVXCD M v Z

Fig.

C

3. C o m p a r i s o n

terminus protein

:PgK

NES

w M x c s ~

of open encoded

non-haemagglutinin

of the reading

by type

amino frames

acid

nucleotide

of the

of BENT.

described

of the

type E (bu0

I

sequence

upstream

C has been

from published

v

L * . ~ W S ~

PKK YLMXLKN P K K S "g

component

For type A, ~,pe E and was derived

YLWILK

[~Msxv

LNHD'I L N H

sequences

ACKNOWLEDGEMENTS

as non-toxic-

progenitor

the amino

CThe

toxin

[19].

acid sequence [5,8,9]. Type

E ( b u t ) r e f e r s t o t y p e E o f C. b u t y r i c u m .

We thank Mrs L. Taylor of Terry Research Station for testing the E. coli clones in the mouse bioassay.

2~ REFERENCES [1] McCroskey, L.M., Hatheway, C.L. Woodruff, B.A., Greenberg, J.A. and Jurgenson. P. (1991) J. Clin. Microbiol. 29, 2618-2620. [2] McCroskey, L.M., Hatheway, C.L., Fenicia, L., Pasolini, B. and Aureli, P. (1986) J. Clin. Microbiol. 23, 201-202. [3] Hatheway. C.L (1989) In: Botulinum neurotoxin and tetanus toxin, (Simpson, L.L., Ed.), pp. 3-24. Academic Press, New York. [4] Thompson, D.E., Brehm, J.K., Oultram, J.D., Swinfield, T.-J.. Shone. C.C., Atkinson, T., Melling, J. and Minton, P. (1990) Eur, J. Biochem. 189, 3-81. [5] Binz, T., Kurazono. H., Wille, M., Frevert, J., Wernars, K. and Niemann, H. (1990)J. Biol. Chem. 265, 9153-9158. [6] Hauser, D., Eklund, M.W., Kurazono, H., Binz, T., Niemann, N., Gill, D.M., Boquet, P. and Popoff, M.R. (1990) Nucleic Acids Res. 18, 4924. [7] Binz, T., Kurazono, H., Popoff, M.R., Eklund, M.W., Sakaguchi, G., Kozaki, S., Krieglstein, K., Henschen, A., Gill, D.M. and Niemann, H. (1990) Nucleic Acids Res. 18, 5556. [8] Whelan, S.M., EImore, M.J., Bodsworth, N.J., Atkinson, T. and Minton, N.P. (1992) Eur. J. Biochem. 204, 657667.

[9] Poulet, S., Hauser, D., Quanz, M., Niemann, H. and Popoff, M.R. (1992) Biochem. Biophys. Res. Commun. 183. 107-113. [10] Niemann, H. (1991) In: Sourcebook of Bacterial Protein Toxins (AIouf, J.E. and Freer, J.H., Eds.), pp. 303-348. Academic Press, London. [11] Fairweather, N.F. and Lyness, V.A. 0986) Nucleic Acids Res. 14, 7809-7812. [12] Farrow, J.A.E., Jones, D., Phillips, B.A. and Collins, M.D. (1988) J. Gen. Microbiol. 129, 1423-1432. [13] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N'Y. [14] Ochman, H., Gerber, A.S. and Hartl, D.L. (1988) Genetics, 120, 621-623. [15] Clark, J.M. (1988) Nucleic Acids Res, 16, 9677-9686. [16] lnoue, H., Nojima, H. and Okayama, H. (1990) Gene 96, 23-28. [17] Hauschild, A.H.W. (1989) In: Foodborne bacterial pathogens, (Doyle, M.P., Ed.), pp. 111-189. Marcel Dekker, New York. [18] Yang, K,H. and Sugiyama, H. (1975) Appl. Microbiol. 29, 598-603. [19] Tsuzuki, K., Kimura, K., Fujii, N., Yokosawa, N. and Oguma, K. (1992) Biochem. Biophys. Res. Commun. 183, 1273-1279.