ELSEVIER FEMS Microbiology Letters 140 (1996) 151-158

Genetic characterisation of the botulinurn toxin complex of Clostridium botulinum strain NCTC 29 16

Ian Henderson * , Sarah M. Whelan, Tom 0. Davis, Nigel P. Minton

Department of Molecular Microbiology, Research Division, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts. SP4 OJG, UK

Received 26 March 1996; revised 24 April 1996; accepted 25 April 1996

Abstract

An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5’ to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames. Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex. The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutuns, UviA from C. pelfringens and Orftxel located upstream of the C. d@ciZe toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins. Primer extension and Northern blotting analysis demonstrate that the genes are expressed as two divergent operons [HA34, HA17, HA701 and [NTNH/A, type A toxin gene], with the Ortx gene expressed singly. Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (S-AlllTagGTITACAAAA-3’ and 5’-ATGlTATATgTaA-3’1, separated by 12 bp, that span the putative -35 and - 10 promoter regions. Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci. It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C. botulinurn strains and in that of the tetanus toxin gene.

Keywords: Regulation: Clostridium botulinurn; Botulinum neurotoxin A (BoNT/A); Progenitor toxin complex

1. Introduction kDa) chain linked by a single disulfide bridge. The

Pathogenic Clostridium botulinum (physiological groups I-IV>, and strains of C. butyricum and C. baruti produce seven antigenically distinct bo- tulinum neurotoxins (BoNTs), serotypes A-G, repre- sentative genes for which have been sequenced [I].

Synthesised as single polypeptide chains, BoNTs are subsequently proteolytically cleaved to yield a di- chain composed of a light (50 kDa) and a heavy (100

heavy chain is involved in the targeting of the phar- maceutically active light chain to the appropriate

cellular target [2]. The stability of the botulinum neurotoxins is cru-

cial to their toxicity, and to this end they are pro- tected from the external environment by other ‘non- toxic’ proteins, forming a progenitor toxin complex. Its size and composition varies depending on neuro- toxin serotype, host strain and environmental factors

* Corresponding author. Tel.: +44 (1980) 612 361; Fax: +44

(1980) 610 898; E-mail: 100020,2133@compuserve.ccom

[3,4] and ranges from the M-form (300 kDa; 12S), to the L-form (500 kDa; 16S), and finally to the LL form (900 kDa; 19s). The larger progenitor toxins

0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PII SO378-1097(96)00172-3

152 I. Henderson rt ul./ FEMS Microhiolq~ Letters 140 (19961 ljl-15X

can resist greater extremes of pH and temperature

than the purified neurotoxin, and appear to be ideally

suited to the conditions encountered on passage through the stomach of the host, having a much

greater oral toxicity than the neurotoxin alone [51. The LL form has only been observed from prote-

olytic strains (group I) of C. botulinum expressing

BoNT/A, while the L form is associated with

BoNT/B (group I) and group III C. botulinum strains

expressing BoNT/C and BoNT/D. BoNT/E and

BoNT/F only occur in the M form. Proteins with haemagglutinating activity have been detected in the

larger L and LL form progenitor complexes of types

A, B, C and D, and genes with the potential to express haemagglutinins have been detected in strains

that harbour the BoNT/G encoding plasmid [6]. In the present study, we report the identification

and characterisation of the proteins that form the

type A progenitor toxin complex of the C. botulinum

strain NCTC 2916, as well as the identification of

sequences that may play a role in regulating expres-

sion of their encoding genes.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth condi-

tions

Chromosomal DNA was from C. botulinum

NCTC 2916. All recombinant manipulations were

performed in Escherichia coli TGl [ A(Zac-proAB)

thi supE hsdA5 F’[traD36 proAB i lacIY lacZA-

~151. Cloning vectors used were pMTL22 [7], the Ml3 phages mp8 and mp9 [S], and pCRlOO0 [9]. E.

coli was routinely grown in L-broth [lo] supple- mented with ampicillin (50 p_g/ml) when required. C. botulinum NCTC 2916 was grown in USA11 medium as previously described [ 111.

2.2. Recombinant DNA methods

Chromosomal DNA purification and plasmid se- quencing methodology were as previously docu- mented Ill]. DNA manipulation was performed us- ing standard techniques [lo]. DNA modifying and restriction enzymes were used according to manufac- turers’ instructions (Northumbria Biochemicals Ltd.,

UK; Bioline UK Ltd.). Radiolabels ([o-35S]dATP,

[a- 3’ P]dATP and [ y- 3’ P]rATP) were purchased from DuPont NEN, UK. Oligonucleotides were synthe-

sised using an Applied Biosystems 380A DNA syn-

thesiser.

2.3. PCR reaction conditions

PCR mixtures contained 10 ng DNA template, 0.1

mM of dATP, dCTP, dGTP and dTTP, 10 mM

Tris-HCl, 50 mM KCl, 3 mM MgC12, 30 nmol of primer and 2.5 U of Taq DNA polymerase. Amplifi-

cation was through an initial denaturation step of

95°C followed by 30 cycles of (1) 96°C for 1.5 min, (2) 37°C for 3 min, (3) 72°C for 3 min, in an MJ

Research PTC- 100 thermocycler.

2.4. Southern blotting and in situ hybridisation

The detection of DNA sequences transferred to

charged nylon membranes was performed using

methods described elsewhere [ 11 I.

2.5. Northern blotting

RNA isolation and agarose gel electrophoresis

were undertaken as previously described [12]. The RNA was blotted from electrophoretograms on to

Hybond H+ nylon membrane (Amersham Intema-

tional, UK) according to the manufacturer’s instruc- tions. Blots were prehybridised (in 10 ml of 0.2 M

NaH,PO,, 0.3 M Na,HPO,, 0.5 M EDTA, 7% (w/v) SDS) for 30 min prior to the addition of the

labelled probe. DNA probes from appropriate plas- mids (see Fig. 1) were labelled with [o~-~‘P]~ATP

using a Megaprime labelling kit (Amersham Intema- tional). Blots were incubated with probes for at least 4 h at 50-60°C, and excess probe removed by three washes (2 X SSC, 0.1% (w/v) SDS at 50-60°C). Blots were air dried briefly and autoradiographed at - 70°C against Kodak film.

2.6. Primer extension

Primer extension was performed essentially as previously described [ 131. Oligonucleotides were end-labelled using T4 polynucleotide kinase and [y- 32 P]dATP, extracted with phenol:chloroform (1: I),

I. Henderson et al./ FEMS Microbiology Letters 140 (1996) 151-158 153

Restriction H E XTH HE T H HP T P

map

I l II I I I I I I I I 1

pNTNHA

probe 1

probe 2

Plasmids ~257 p544.__-._ l._ 9! ’ pCBA2

pCBA4 I pCBA3 1

Open reading

frames HA70 HA17 HA34 OrfX NTNH/A BoNT/A

Transcriptional -

analysis

0 2Kb I I

Fig. 1. Structure of the BoNT/A gene locus of C. botulinum NCTC 2916. Open arrows indicate the six open reading frames at the locus.

Solid arrows represent a transcriptional map of the locus. The plasmids used to sequence the region 5’ to BoNT/A are ~257, ~544 and

~933. These were generated by the respective insertion into pMTL22 of: (i) a 3 kb HindIII fragment: (ii) a 2.3 kb EcoRI/TaqI fragment;

and (iii) a 2 kb EcoRI/HindIII fragment. Details of the other plasmids are presented in [ 1 I]. The restriction enzyme sites arc H: HindIIl, E:

EcoRI, T: TaqI, X: XbaI, P: PstIr

and precipitated at - 20°C with 2.5 vol ethanol, 0.1 vol sodium acetate (pH 5.3) and 1 pl glycerol

(Boehringer Mannheim Ltd., UK). The pellet was resuspended in 10 ~1 of 3 X PEB buffer (30 mM

PIPES pH 6.4, 1.5 mM EDTA pH 8.0, 1.2 M NaCl),

to which was added 15 p,l sterile distilled water and 5 p,l of RNA solution. This was incubated at 80°C for 5 min and then for 3 h at 55°C. After centrifuga-

tion and brief air drying, the pellet was resuspended in 20 p,l of extension buffer (1 mM dATP, dCTP, dGTP and dTTP, 25 p,g/ml actinomycin D (Sigma Chemical Company, UK), 1 ~1 of placental RNase

inhibitor (RNasin-Clontech, UK) and 2 ~1 of 10 X reverse transcriptase buffer (500 mM Tris-HCl pH 8.3, 500 mM KCl, 40 mM DTT, 100 mM MgSO,)). Extensions were performed at 42°C for 1 h after the addition of 20 U of AMV reverse transcriptase (Life

Sciences, Inc., UK). Extension products were precip- itated using ethanol/sodium acetate, and analysed

by polyacrylamide gel electrophoresis [lo].

2.7. Nucleotide sequence

Data have been deposited in the EMBL/Genbank database under Accession number L42537.

3. Results and discussion

3. I. Cloning of the toxin complex genes

One of the genomic clones (pCBA4) obtained during the previous [ll] characterisation of the BoNT/A gene (botA) of C. botulinurn NCTC 2916,

154 I. Henderson et al. / FEMS Microbiolog?: Letters 140 (I9961 151-158

carried 1734 bp of DNA 5’ to the toxin gene (Fig. 1).

The sequence included a 43 bp non-coding region

immediately 5’ to borA, preceded by a putative open reading frame (ORF) with no recognisable start

codon. Studies elsewhere had indicated the presence

of genes encoding proteins (Hn+) with haemaggluti-

nating activity 5’ to both botA and botB [ 14,151. In

an attempt to characterise the upstream region from

the BoNT/A gene, botA-specific primers were used

in conjunction with primers based on a determined [15] AHnf peptide sequence to PCR amplify the

region upstream of botA from pCBA4 DNA. The resultant 4 kb DNA fragment was cloned into

pCRlOO0 to give the plasmid pNTNHA (Fig. I>.

Two subfragments from the insert of pNTNHA were

then used as probes in Southern blot experiments

(probes 1 and 2, Fig. 1) to generate a restriction map

of the region of the clostridial genome adjacent to botA. This enabled three specific restriction frag-

ments (see Fig. 1) to be targeted and cloned without risk of cloning a functional botA gene. Nucleotide

sequence analysis of the inserts of the resultant three plasmids (~257, ~544 and ~933) revealed five ORFs, encoding the proteins HA70, HA1 7, HA34, OrfX

and NTNH/A (Fig. 1).

of the gene, relative to botA, Northern blotting ex- periments were undertaken. Although a discrete

NTNH/A-specific mRNA product was not detected,

a ‘smear’ was evident on autoradiographs, originat-

ing at a point equivalent to RNA of some 7.5 kb in

size. In contrast, botA-specific probes detected two

discrete mRNA species, of 4 kb and 7.5 kb in size.

This suggests that the two genes form an operon

from which both bicistronic and monocistronic tran- scription occurs (Fig. 1). This conclusion was sup-

ported by the demonstration of two transcriptional start points in primer extension studies, although the

cDNA product obtained in the case of NTNH/A

was very weak (Fig. 2). The labile nature of neuro-

toxin in the absence of NTNH would suggest that

co-expression of the two genes would be advanta- geous. Additional expression of botA may simply be

to overcome mRNA instability of such a large bi- cistronic transcript, or serve another, as yet un-

known, function.

3.2. Organisation of the non-toxic, non-haemaggluti-

nating gene and botA

Immediately 5’ to botA is a gene encoding a protein of 1193 amino acids (138 2 18 Da). This

protein, NTNH/A, shares homology with the non-

toxic, non-haemagglutinating proteins found in type C, E and F progenitor toxin complexes [ 161. It is larger than NTNH/E and NTNH/F, by 33 amino

acids, and shows greatest homology with NTNH/C (Table 1). To assess the transcriptional organisation

The transcriptional start site of botA gene was

mapped to a position 20 nucleotides upstream of the ATG codon (see Fig. 4). Binz et al. [17] mapped the start point in strain 62A as being 118 nucleotides 5’

to botA. Though this discrepancy between the two studies may be attributable to strain differences, the

upstream regions of both genes are identical for at

least 340 bp. The primer used by Binz et al. 1171, however, spanned the botA ATG codon. Small tran- scripts products beginning 20 nucleotides upstream

may, therefore, not have been detected in their exper- iments.

3.3. Organisation of the haemagglutinin genes

On the opposite DNA strand to the NTNH/A

gene, at a distance of 923 bp, reside three contiguous

Table 1 Comparison of NTNH proteins and OrfX homologues by percentage similarity (lower left-hand triangle), and percentage identity (upper

right-hand triangle) of amino acid sequences

Type NTNH 0IT-X

A Cl E F Ofl P21Bp P2lBnp orf22

A _ 65 58 61 OtfX _ 91 88 52

Cl 76 _ 58 61 P21Bp 98 89 52 E 15 75 71 P2lBnp 89 90 _ 47 F 78 16 84 _ ort22 67 68 63 _

I. Henderson et al. /FEMS Microbiology Letters 140 (1996) 151-158 155

ORFs, encoding HA34/A (291 amino acids; 33 826 Da), HA17/A (147 amino acids; 17 035 Da) and HA70/A (625 amino acids; 71 144 Da), separated from each other by 69 and 13 nucleotides, respec- tively (Fig. 1). All three proteins are homologous to proteins encoded by ORFs adjacent to botC1

(antp34/C 1, antpl7/C 1 and antp70/C 1 respec- tively) [ 181. In addition, the translated N-terminal sequence of HA17 is identical to the previously determined [15] sequence of a purified type A Hn+ peptide. The finding that antp34/Cl possesses haemagglutinating activity [ 141 strongly suggests that HA34 has a similar activity.

Positions 16-25 of the deduced amino acid se- quence of HA70 correspond exactly to the deter- mined N-terminal sequence of the 21.5 kDa protein purified from type A progenitor toxin by Somers and DasGupta [15], while residues 204-213 match ex- actly the N-terminal sequence of the 57 kDa compo- nent of the type A complex. This suggests that HA70 is initially produced as a single protein and is subse- quently processed into these smaller polypeptides. Intriguingly, the smaller of the two proteins shows some homology with the receptor binding/cytotoxic domain of the C. perfringens enterotoxin (20% iden- tity/33% similarity in a 202 amino acid overlap)

A G C T P

T

ii A T

tG A

:

:

(i)

The close proximity of the HA genes is indicative of transcriptional linkage. Northern blotting detected a single 3.2 kb transcript, using probes derived from either gene. Furthermore, in primer extensions, a cDNA product was only obtained using a HA34- specific primer (Fig. 2), and not with primers based on HA17 or HA70. These results suggest that the genes form a tricistronic operon.

3.4. Putative regulatory factors

The genes of the botulinum toxin complex of strain NCTC 2916 are arranged as two divergent polycistronic operons. At the BoNT/Cl locus, tran- scriptional start sites for both antp34/Cl and antp139/Cl lie within an 80 bp intergenic region between the two putative operons. In strain NCTC 2916, the equivalent intergenic region is interrupted by a small ORF encoding a protein of 178 amino acids (21654 Da), OrfX. A homologous protein (Orf22) is specified by a gene located downstream of the antp34/Cl operon of type C strains. Equivalent ORFs have also been detected in proteolytic and non-proteolytic C. botulinurn strains expressing BoNT/B (P21Bp and P21Bnp respectively), as well as in strains that harbour the BoNT/G encoding plasmid (Stacey, Collins and East, personal commu-

; A T A

+T

G A A G

(ii)

Fig. 2. Primer extension analysis of the promoter regions controlling the expression of the two major operons at the BoNT/A locus, (i)

[NTNH/A, BoNT’/A] using the primer 5’-CTCTAACTACTACAACA-3’ and (ii) [HA34, HA17 and HA701 using the primer 5’-ACGT-

TACCGGCAACTIG-3’. Arrows indicate the primary cDNA product of primer extension present in lane P.

156 I. Henderson et al. / FEMS Microbiology Letters 140 ( 1’996) I.5 I - IS8

BOX 1 BOX 2

(( _ 3 6 II II _ 1 0 *

C0IlSfZIlSU.S

(a.1

(b)

(c)

Gram +ve vegetative promoter

ATTTTagQTTTACMAA RatRRYNtRRNt ATGTTATATgZkA

TTTACA________2~~t___________t~tgtt

TTTACA--------18nt--------ttatat

TTTACA--------16nt------tgttat

TTGACA____________________TATaT

1. GATATGTCAAAGTATTTGTATTTATGGTCATTTAAATAATT----Snt--AAGAGG---Bnt-ATG-BoNT/A

J

Fig. 3. Transcriptional analysis of the genes at the BoNT/A locus and putative regulatory sites in the promoters of the two major operons at

the BoNT/A, BoNT/Cl and TeNT gene loci. Arrows indicate transcriptional initiation sites (this study for the NTNH/A, HA34, BoNT/A

and Oltx genes, and [ 18,231 for the remainder). Boxes 1 and 2 represent conserved sequences in the promoter regions indicated. the

consensus of which is presented below the boxed DNA sequences. The sequences (a-c) indicate putative - 35 and - 10 promoter sites of

boxes 1 and 2. The transcriptional start sites for the BoNT/A and OrfX genes are also presented.

nication) (Table 1; Fig. 3A). All of these proteins, and OrfX, possess a weak helix-turn-helix motif at

their C-terminus and a p1 of approximately 10.4.

These features are indicative of a DNA-binding func-

tion. Furthermore, a database search revealed signifi-

cant homology between OrfX and the MsmR regula-

Orfx

Orftxel

UviA

Orfx

OrftXel

UviA

Orfx

0rftxe1

UvlA

Fig, 4. A comparison of the OrfX protein sequence with related proteins. (A) P2lBp and P2IBnp from proteolytic and non-proteolytic

strains respectively of C. botulinurn expressing BoNT/B; Orf22 from C. botulinurn expressing BoNT/C 1. (B) Orftxel from C. difJiciZe

VP1 10463; UviA from C. perfringens. Boxed sequences represent identical/similar amino acids. The putative helix-turn-helix motif (HTH)

is indicated below the C-terminal portion of these proteins.

I. Henderson et al. / FEMS Microbiology Letters 140 (19%) 151-158 157

tory protein [ 191 of Streptococcus mutans (22% iden-

tity in a 177 amino acid overlap), and UviA from C.

pe$ringens. UviA is homologous to the FixJ family of proteins that possess features in common with the

- 35 recognition domains of u factors, including a” of E. coli and oD of Bacillus subtilis (Fig. 3B)

[20,21]. In common with UviA, Ofl is also homol-

ogous to Orftxel of the C. diflcile strain VP1 10463

(Fig. 3B). Intriguingly, the gene for this protein is

located immediately upstream of the gene encoding

toxin B [22].

The physical characteristics of OrfX, and its strong

association with botulinum genes, suggest a role in

the regulation of genes concerned with production of

the toxin complex. To date, however, equivalent genes have not been located in clostridial strains

expressing BoNT/E or BoNT/F. However, the C-

terminal portion of an ORF immediately upstream of

the TeNT gene promoter encodes a peptide that is

homologous to the C-terminus of OrfX. No 0$X-

specific mRNA transcript was detected by Northern blotting, but a cDNA product was evident following

primer extension reactions employing a primer based

on the OF sequence. This suggests that under the

culture conditions used to purify total RNA, the ofl

gene is only expressed at a very low level.

An alignment of the promoter regions of the HA34 and NTNH/A genes revealed a further highly

significant feature. A sequence motif was evident spanning the - 35 and - 10 promoter region of the

HA34 gene (composed of box 1 and box 2, Fig. 4) which was also present in an identical position up-

stream of the NTNH/A gene. The same motifs were also located in the equivalent positions of the

BoNT/Cl locus. In this case the intervening orjX is

absent. The two motifs, therefore, exist as contigu- ous inverted repeats. This led to the probably incor-

rect previous assumption [17] that, in the BoNT/Cl locus, they play a role in the formation of a ‘stem- loop’ structure. It seem more likely that these se-

quences represent operator sites at which an un- known transcriptional factor binds. It is tempting to speculate that such a factor could be the product of ofl. Significantly, this same putative operator se- quence is found in an equivalent position adjacent to the TeNT gene (Fig. 4). It seems likely that these sequences play a common role in the regulation of expression of the genes encoding BoNTs and TeNT.

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