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
* Corresponding author. Tel.: +44 (1980) 612 361; Fax: +44
(1980) 610 898; E-mail: 100020,2133@compuserve.ccom
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
[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
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 152 I.Henderson rt ul./
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
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
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.,
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FEMS Microhiolq~ Letters 140 (19961 ljl-15X
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
of The detection of DNA sequences transferred to
charged nylon membranes was performed using
methods described elsewhere [ 11I.
2.5. Northern blotting
RNA isolation and agarose gel electrophoresis
condi- 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-
32P]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
I l II I I I I I I I I 1
map

pNTNHA

probe 1

probe 2

p544.__-._ l._ 9! ’ pCBA2

Plasmids ~257 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 Sciences, Inc., UK). Extension products were precip-
vol sodium acetate (pH 5.3) and 1 pl glycerol itated using ethanol/sodium acetate, and analysed
(Boehringer Mannheim Ltd., UK). The pellet was by polyacrylamide gel electrophoresis [lo].
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), 2.7. Nucleotide sequence
to which was added 15 p,l sterile distilled water and
5 p,l of RNA solution. This was incubated at 80°C Data have been deposited in the EMBL/Genbank
for 5 min and then for 3 h at 55°C. After centrifuga- database under Accession number L42537.
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 3. Results and discussion
Chemical Company, UK), 1 ~1 of placental RNase
inhibitor (RNasin-Clontech, UK) and 2 ~1 of 10 X 3. I. Cloning of the toxin complex genes
reverse transcriptase buffer (500 mM Tris-HCl pH
8.3, 500 mM KCl, 40 mM DTT, 100 mM MgSO,)). One of the genomic clones (pCBA4) obtained
Extensions were performed at 42°C for 1 h after the during the previous [ll] characterisation of the
addition of 20 U of AMV reverse transcriptase (Life BoNT/A gene (botA) of C. botulinurn NCTC 2916,

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 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).
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
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.
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 _

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 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 [ 141strongly 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)
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 G C T P

ii
A ;
T A
tG T
A A
+T
:

:
G
A
A
G

(i) (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.

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 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 ATTTTagQTTTACMAA RatRRYNtRRNt ATGTTATATgZkA

(a.1 TTTACA________2~~t___________t~tgtt

(b) TTTACA--------18nt--------ttatat

(c) TTTACA--------16nt------tgttat

Gram +ve
vegetative TTGACA____________________TATaT
promoter

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

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, These features are indicative of a DNA-binding func-
and OrfX, possess a weak helix-turn-helix motif at tion. Furthermore, a database search revealed signifi-
their C-terminus and a p1 of approximately 10.4. 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. botulinurnexpressing BoNT/B; Orf22 from C. botulinurnexpressing 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.

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