Biochimica et Biophysica Acta, 1216 (1993) 487-491 487 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90593 Short Sequence-Paper

Nucleotide sequence of the gene coding for Clostridium botulinum ( Clostridium argentinense) type G neurotoxin:

genealogical comparison with other clostridial neurotoxins

Kathryn Campbell, Matthew D. Collins and Alison K. East *

Department of Microbiology, Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Reading RG6 2EF (UK)

(Received l0 August 1993)

Key words: Neurotoxin; Gene sequence; (C. argentinense); (C. botulinum)

The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 5'-end of the gene was obtained using a primer based on a conserved region of the nontoxic-nonhaemagglutinin gene lying upstream of the toxin gene. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues (rmm 149, 147). Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinurn. Tetanus toxin (TeTx) and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity).

Clostridium botulinum comprises a group of Gram- positive sporeforming rod-shaped bacteria, all of which produce a characteristic neurotoxin responsible for the neuroparalytic illness botulism. The species displays considerable metabolic diversity, and four distinct phe- notypic groups (designated I to IV) are recognized [1] which have now been confirmed by rRNA sequence data [2]. Seven immunologically distinct botulinal tox- ins (types A to G) are produced by C. botulinum strains. Toxin types A through F have been implicated in human or animal botulism or both [1]. Toxin type G has been isolated from soil samples [3] and autopsy specimens [4] but has not been clearly implicated as the cause of paralytic illness or death [1]. The organism elaborating type G toxin differs phenotypically from other C. botulinum types, and is now designated a distinct species, C. argentinense [5].

Considerable progress has been made in elucidating botulinal neurotoxin (BoNT) sequences with complete gene sequences now available for C. botulinum type A,B (proteolytic and non-proteolytic), C, D, E and F

* Corresponding author. Fax: +44 734 267917. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X74162.

(non-proteolytic), C. barati type F and C. butyricum type E [6-11]. It was demonstrated in C. botulinum type G that the gene is encoded on a plasmid [12]. In this article we report the complete gene sequence of B o N T / G , the only remaining uncharacterized bo- tulinum toxin type, and the results of a comparative analysis with other clostridial neurotoxins.

C. botulinum type G (strain 113/30, NCFB 3012) was grown anaerobically in TPYCG broth and total genomic DNA prepared as described by Lawson et al. [13]. Primers designed to conserved regions of pub- lished BoNT gene sequences were used to amplify toxin gene fragments from genomic DNA using PCR methodology. The conditions used were as described previously [10] except the PCR profile used was 25 cycles of 92°C for 1 min, 45°C for 1 min and 75°C for 2 min, with a final extension step of 75°C for 10 min. The 5'-end of the BoNT gene was obtained using a primer ( 5 ' - T T T A G T T T C T T A G A T C A A T G G T G G - 3 ' ) de- signed to a conserved region (amino acids 1996 to 2019) of the upstream nontoxic-nonhemagglutinin gene. The 3'-end of the gene was obtained by cloning a 1.9 kb EcoRI fragment and a 1 kb HindIII fragment amplified from total genomic DNA by inverse PCR [14]. PCR products were cloned directly into pCR TM

Vector (TA Cloning Kit, Invitrogen San Diego, USA) which is supplied cut and has T overhangs. Competent

488

TABLE I

Percentage amino acid identities of clostridial neurotoxin L- (upper right-hand triangle) and H- (lower left-hand triangle) chains

TeTx B o N T / A B o N T / A B o N T / B B o N T / B B o N T / C B o N T / D B o N T / E B o N T / E B o N T / F B o N T / F B o N T / G (inf) a (p) (NP) (but) (NP) (bar)

TeTx - 31.8 31.6 51.9 52.1 34.8 34.5 44.4 44.9 45.1 45.6 49.0 B o N T / A 37.9 - 94.9 32.1 32.0 34.3 34.3 34.1 34.5 35.0 35.2 35.1 B o N T / A (inf) 35.6 87.1 - 32.3 32.2 34.3 33.6 34.5 35.0 35.2 35.9 35.3 B o N T / B (P) 37.3 45.6 43.0 - 97.7 33.6 34.7 37.9 38.2 39.2 43.3 61.1 B o N T / B (NP) 37.7 45.0 42.7 90.2 - 33.7 34.6 37.9 38.1 39.5 43.0 60.8 B o N T / C 35.4 33.7 33.4 37.2 35.5 - 46.5 34.8 34.5 35.3 35.3 35.4 B o N T / D 34.5 34.3 34.6 37.6 37.4 57.3 - 36.2 34.9 36.3 34.7 36.6 B o N T / E 34.7 45.2 43.9 40.9 41.4 34.4 36.0 96.0 58.3 55.1 38.5 B o N T / E (but) 34.4 44.8 43.2 40.8 40.8 34.9 35.1 98.1 - 57.6 54.6 39.7 B o N T / F (NP) 35.0 44.6 44.1 41.7 42.8 34.4 36.5 64.9 64.4 - 64.3 40.5 B o N T / F (bar) 37.8 48.5 46.0 42.5 43.5 34.8 35.9 68.8 68.5 73.7 41.9 B o N T / G 37.4 44.1 42.4 56.9 56.0 34.9 37.0 40.7 41.5 38.6 40.2 -

a Abbreviations: NP, non-proteolytic; P, proteolytic; but, C. butyricum; bar, C. barati; inf, infant botulism.

Escherichia coli cells (One Shot cells, TA Cloning Kit, Invitrogen) were transformed and inserted fragments sequenced using dideoxynucleotide chain termination. Primers complementary to plasmid and toxin gene were used to sequence both strands of each insert com- pletely. As the inserts were generated using PCR, the sequence of two clones from different PCR reactions was determined to detect possible errors made by Taq polymerase. Where two clones differed as they did in seven positions, a third clone from a separate PCR reaction was sequenced to determine a consensus se- quence. Distance matrix trees were constructed by the Fitch method using the Wisconsin Molecular Biology software package [15] on a VAX computer.

Fig. 1 shows the PCR and sequencing strategy em- ployed to determine the full nucleotide sequence of the BoNT/G gene of C. botulinum. The sequence, shown in Fig. 2, revealed an open reading frame encoding a protein of 1297 amino acids. To ascertain the extent of

sequence homology a comparative analysis of the de- rived BoNT/G sequence and those of other published clostridial neurotoxins was performed (data not shown) extending previous studies [10,11]. Table I summarises the amino acid identity values of the light (L)-and heavy (H)-chains of BoNT/G and the different BoNTs and tetanus toxin. BoNT/G shows highest sequence identity with BoNT/B (approx. 58% for the complete protein, approx. 61% and 56% for L- and H-chains, respectively). Relatively high relatedness was also evi- dent between BoNT/G and BoNT/A, BoNT/E, BoNT/F and TeTx (approximate range 40-42% iden- tity for the complete protein). A distance matrix analy- sis depicting the genealogical interrelationships of the L- and H-chains of BoNT/G and other neurotoxins is shown in Fig. 3.

Recent studies have shown the BoNT/E of C. bo- tulinum and C. butyricum are very highly related (ap- prox. 97% identity at the amino acid level) and it has

EcoRI Hindll] EcoRI Hindlll

I I I kb

m

PLASMID PRIMERS

pCbotGla+ 658-982

pCbotG 1 a 1026 -918 pCbotG2a

pCbotGlb 699-694 pCbotG2b

pCbotGlc BoNT1-BoNT2 pCbotG2c

• pCbotG 1 d 923-946 • pCbotG2d

. pCbotGle 1023-1024 • pCbotG2e

Fig. 1. Strategy for cloning fragments of the B o N T / G gene. Arrows indicate the position of the primers used to generate the fragments. Clones generated by ' inverse PCR' are indicated by *; in the case of p C b o t G l d / pCbotG2d the template D N A was digested with EcoRl, and

* p C b o t G l e / * pCbotG2e with HindIlI. Only the sites of restriction enzymes involved in the construction of the clones are shown.

489

ATGCCAGTTAATATAAA~ACTTTAATTATAATGACCCTATT~TAATGATGACATTATTATGATGG~CCATTC 75 M P V N I K N F N Y N D P I N N D D I I M M E P F ( 2 5 )

982 ~TGACCCAGGGCCAGG~CATATTAT~AGCTTTTAGGATTATAGATCGTATTTGGATAGTACCAG~AGGTTT 150 N D P G P G T Y Y K A F R I I D R I W I V P E R F ( 5 0 )

ACTTATGGATTTCAACCTGACC~TTT~TGCCAGTACAGGAGTTTTTAGTA~GATGTCTACGAATATTACGAT 225 T Y G F Q P D Q F N A S T G V F S K D V Y E Y Y D ( 7 5 )

CC~CTTATTTA~AACCGATGCTGAA~AAGAT~ATTTTTA~AAC~TGATTAAATTATTTAATAGAATT~T 300 P T Y L K T D A E K D K F L K T M I K L F N R I N ( 1 0 0 )

TCAAAACCATCAGGACAGAGATTACTGGATATGATAGTAGATGCTATACCTTATCTTGGAAATGCATCTACACCG 375 S K P S G Q R L L D M I V D A I P Y L G N A S T P I 2 5 )

CCCGACAAATTTGCAGCAAATGTTGCA~TGTATCTATTAAT~A~TTATCC~CCTGGAGCTG~GATC~ 450 P D K F A A N V A N V S I N K K I I Q P G A E D Q ( 1 5 0 )

ATAAAAGGTTT~TGAC~ATTT~TAATATTTGGACCAGGACCAGTTCTAAGTGAT~TTTTACTGATAGTATG 525 I K G L M T N L I I F G P G P V L S D N F T D S M ( 1 7 5 )

ATTATG~TGGCCATTCCCCAATATCAG~GGATTTGGTGCAAGAAT~TGAT~GATTTTGTCCTAGTTGTTTA 600 I M N G H S P I S E G F G A R M M I R F C P S C L ( 2 0 0 )

~TGTATTTAAT~TGTTCAGG~AAT~AGATACATCTATATTTAGTAGACGCGCGTATTTTGCAGATCCAGCT 675 N V F N N V Q E N K D T S I F S R R A Y F A D P A ( 2 2 5 )

699 CT~CGTT~TGCATGAACTTATACATGTGTTACATGGATTATATGG~TTAAGATAAGT~TTTACC~TTACT 750 L T L M H E L I H V L H G L Y G I K I S N L P I T ( 2 5 0 )

CCAAATACAAAAG~TTTTTCATGCAACATAGCGATCCTGTACAAGCAGAAG~CTATATACATTCGGAGGACAT 825 P N T K E F F M Q H S D P V Q A E E L Y T F G G H (275)

GATCCTAGTGTTAT~GTCCTTCTACGGATATGAATATTTAT~TAAAGCGTTACAAAATTTTCAAGATATAGCT 900 D P S V I S P S T D M N I Y N K A L Q N F Q D I A ( 3 0 0 )

918 AATAGGCTT~TATTGTTTCAAGTGCCCAAGGGAGTGGAATTGATATTT¢CTTATAT~ACA~TATATAAA~T 975 N R L N I V S S A Q G S G I D I S L Y K Q I Y K N ( 3 2 5 )

A~TATGATTTTGTTGAAGATCCT~TGGAAAATATAGTGTAGATAAGGATAAGTTTGATAAATTATATAAGGCC 1050 K y D F V E D P N G K Y S V D K D K F D K L Y K A ( 3 5 0 )

TT~TGTTTGGCTTTACTGA~CTAATCTAGCTGGTG~TATGGAAT~AAACTAGGTATTCTTATTTTAGTGAA 1125 L M F G F T E T N L A G E Y G I K T R Y S Y F S E ( 3 7 5 )

TATTTGCCACCGAT~AAACTG~ATTGTTAGACAATACAATTTATACTC~AATGAAGGCTTTAACATAGCT 1200 Y L P P I K T E K L L D N T I Y T Q N E G F N I A (400)

AGTAAAAATCTC~CGGAATTTAATGGTCAGAATAAGGCGGTAAATAAAGAGGCTTATG~GAAATCAGCCTA 1275 S K N L K T E F N G Q N K A V N K E A Y E E I S L ( 4 2 5 )

G~CATCTCGTTATATATAGAATAGCAATGTGCAAGCCTGTAATGTACAAAAATACCGGTAAATCTG~CAGTGT 1350 E H L V I Y R I A M C K P V M Y K N T G K S E Q C ( 4 5 0 )

ATTATTGTTAAT~TGAGGATTTATTTTTCATAGCTAATAAAGATAGTTTTTCAAAAGATTTAGCTA~GCAGAA 1425 I I V N N E D L F F I A N K D S F S K D L A K A E ( 4 7 5 )

ACTATAGCATAT~TACACA~AT~TACTATAGAAAATAATTTTTCTATAGATCAGTTGATTTTAGATAATGAT 1500 T I A Y N T Q N N T I E N N F S I D Q L I L D N D ( 5 0 0 )

TT~GCAGTGGCATAGACTTACCAAATGAAAACACAG~CCATTTAC~ATTTTGACGACATAGATATCCCTGTG 1575 L S S G I D L P N E N T E P F T N F D D I D I p V { 5 2 5 )

TATATTA~C~TCTGCTTTAA~AAAATTTTTGTGGATGGAGATAGCCTTTTTGAATATTTACATGCTCAAACA 1650 Y I K Q S A L K K I F V D G D S L F E Y L H A Q T ( 5 5 0 )

EcoRl TTTCCTTCTAATATAGA~ATCTACAACTAACGAATTCATTA~TGATGCTTTAAGA~TAATAATA~GTCTAT 1725 F P S N I E N L Q L T N S L N D A L R N N N K V Y ( 5 7 5 )

ACTTTTTTTTCTACAAACCTTGTTGAA~AGCTAATACAGTTGTAGGTGCTTCACTTTTTGT~ACTGGGTA~A 1800 T F F S T N L V E K A N T V V G A S L F V N W V K ( 6 0 0 )

GGAGTAATAGATGATTTTACATCTGAATCCACACAAA~AGTACTATAGATA~GTTTCAGATGTATCCATAATT 1875 G V I D D F T S E S T Q K S T I D K V S D V S I I ( 6 2 5 }

BoNT 1 ~ ~ 923 ATTCCCTATATAGGACCTGCTTTGAATGTAGGA~TQAAACAGCT~AGA~ATTTTAAA~TGCTTTTG~ATA 1950 I P Y I G P A L N V G N E T A K E N F K N A F E I ( 6 5 0 )

GGTGGAGCCGCTATCTT~TGGAGTTTATTCCAGAACTTATTGTACCTATAGTTGGATTTTTTACATTAGAATCA 2025 G G A A I L M E F I P E L I V P I V G F F T L E S ( 6 7 5 ]

TATGTAGGAAAT~AGGGCATATTATTATGACGATATCCAATGCTTT~AGA~AGGGATCA~AATGGACAGAT 2100 Y V G N K G H I I M T I S N A L K K R D Q K W T D ( 7 0 0 )

ATGTATGGTTTGATAGTATCGCAGTGGCTCTC~CGGTTAATACTCAATTTTATACAATAAAAGAAAGAATGTAC 2175 M Y G L I V S Q W L S T V N T Q F Y T I K E R M Y ( 7 2 5 )

AATGCTTTAAAT~TCAATCAC~GCAATAGA~AAATAATAGAAGATCAATATAATAGATATAGTG~GAAGAT 2250 N A L N N Q S Q A I E K I I E D Q Y N R Y S E E D ( 7 5 0 )

AA~TGAATATT~CATTGATTTTAATGATATAGATTTTAAACTTAATCAAAGTATA~TTTAGCAATAAAC~T 2325 K M N I N I D F N D I D F K L N Q S I N L A I N N ( 7 7 5 )

694 ATAGATGATTTTATAAACCAATGTTCTATATCATATCT~TATG~TAG~TGATTCCATTAGCTGTAA~AAGTTA 2400 I D D F I N Q C S I S Y L M N R M I P L A V K K L ( 8 0 0 )

Fig. 2. Complete nucleotide sequence of the BoNT/G gene. The translated amino acid sequence is given under the second nucleotide of each codon. The position of the primers used in PCR to generate clones is indicated by underlining.

490

A/~GACTTTGATGATAATCTTAAGAGAGATTTATTGGAGTATATAGATACAAATGAACTATATTTACTTGATGAA 2475 K D F D D N L K R D L L E Y I D T N E L Y L L D E ( 8 2 5 )

GTA~TATTCTA~ATC~AAGT~ATAGACACCTA~GACAGTATACCATTTGATCTTTCACTATATACC~G 2550 V N I L K S K V N R H L K D S I P F D L S L Y T K ( 8 5 0 )

GACAC~TTTT~TACAAGTTTTT~T~TTATATTAGTAATATTAGTAGT~TGCTATTTT~GTTT~GTTAT 2625 D T I L I Q V F N N Y I S N I S S N A I L S L S Y ( 8 7 5 )

AGAGGTGGGCGTTT~TAGATTCATCTGGATATGGTGCAACTATG~TGTAGGTTCAGATGTTATCTTTAATGAT 2700 R G G R L I D S S G Y G A T M N V G S D V I F N D ( 9 0 0 )

ATAGGA~TGGTC~TTT/~TT~T~TTCTGA/~TAGT~TATTACGGCACATCAAAGTAAATTCGTTGTA 2775 I G N G Q F K L N N S E N S N I T A H Q S K F V V ( 9 2 5 )

TATGATAGTATGTTTGAT~TTTTAGCATT~CTTTTGGGTAAGGACTCCTAAATAT~TAATAATGATATACAA 2850 Y D S M F D N F S I N F W V R T P K Y N N N D I Q ( 9 5 0 )

946 ACTTATCTTC~u~TGAGTATAC~T~TTAGTTGTATAAA~a~ATGACTCAGGATGG~AGTATCTATTAAGGGA 2925 T Y L Q N E Y T I I S C I K N D S G W K V S I K G ( 9 7 5 )

~TAG~T~TATGGACATT~TAGATGTT~TGCAAAATCTA~TC~TATTTTTCG~TATAGTATA~AGAT 3000 N R I I W T L I D V N A K S K S I F F E Y S I K D ( 1 0 0 0 )

BoNT 2 ~TATATCAGATTATATA~TAAATGGTTTTCCAT~CTATTACT~TGATAGATTAGGTAACGCA~TATTTAT 3075 N I S D Y I N K W F S I T I T N D R L G N A N I Y ( 1 0 2 5 )

ATAAATGG~GTTTGA~u&AGTG~u~TTTTAAACTTAGATAG~TTAATTCTAGTAATGATATAGACTTC 3150 I N G S L K K S E K I L N L D R I N S S N D I D F ( 1 0 5 0 )

A~TT~TTAATTGTACAGATACTACT~ATTTGTTTGGATT~GGATTTTAATATTTTTGGTAGAG~TTA~T 3225 K L I N C T D T T K F V W I K D F N I F G R E L N ( 1 0 7 5 )

GCTACAG~GTATCTTCACTATATTGGATTC~TCATCTACAAATACTTTAA~GATTTTTGGGGGAATCCTTTA 3300 A T E V S S L Y W I Q S S T N T L K D F W G N P L ( I I 0 0 )

Hind]II AGATACGATACACAATACTATCTGTTT~TCAAGGTATGCAA~TATCTATATAAAGTATTTTAGTA~GCTTCT 3375 R Y D T Q Y Y L F N Q G M Q N I Y I K Y F S K A 5 ( I I 2 5 )

1023 ATGGGGGAAACTGCACCACGTAC~ACTTT~TAATGCAGCAATAAATTATC~AATTTATATCTTGGTTTACGA 3450 M G E T A P R T N F N N A A I N Y Q N L Y L G L R ( I I 5 0 )

1024 TTTATTATAAAAAAAGCATCAAATTCTCGGAATATAAATAATGATAATATAGTCAGAGAAGGAGATTATATATAT 3525 F I I K K A S N S R N I N N D N I V R E G D Y I Y ( I I 7 5 )

CTT~TATTGAT~TATTTCTGATGAATCTTACAGAGTATATGTTTTGGTGAATTCT~AGAAATTC~ACTCAA 3600 L N I D N I S D E S Y R V Y V L V N S K E I Q T Q ( 1 2 0 0 )

TTATTTTTAGCACCCAT~ATGATGATCCTACGTTCTATGATGTACTACAAATAAAA~ATATTATG~AAAACA 3675 L F L A P I N D D P T F Y D V L Q I K K Y Y E K T ( 1 2 2 5 )

ACATAT~TTGTCAGATACTTTGCGAA~AGATACTA~ACATTTGGGCTGTTTGGAATTGGTAAATTTGTT~A 3750 T Y N C Q I L C E K D T K T F G L F G I G K F V K ( ] 2 5 0 )

GATTATGGATATGTTTGGGATACCTATGATAATTATTTTTGCATAAGTCAGTGGTATCTCAG~GAATATCTGAA 3825 D Y G Y V W D T Y D N Y F C I S Q W Y L R R I S E ( 1 2 7 5 )

~TATA~TAAATTAAGGTTGGGATGT~TTGGCAATTCATTCCCGTGGATG~GGATGGACAGAAT~ 3894 N I N K L R L G C N W Q F I P V D E G W T E * (1297)

Fig. 2 (continued).

been suggested that lateral gene transfer could have occurred between these species [9]. The present analy- sis shows that B o N T / G and BoNT/B are genealogi- cally related. However, the significantly lower sequence

identity between these toxins (approx. 58%) suggests their common ancestry is not particularly recent (as- suming a similar rate of nucleotide change in the various BoNTs). The degree of relatedness between

L-CHAIN H-CHAIN

.F A(infant) ~--A

F(non-proteolytic) F(barati)

[EE(butyricurn) TeTx

G B(non-proteolytic)

B(proteolytic) D

C

m A A(infant)

F(non-proteolytic) F(barati)

[E(butyricum) TeTx

G B(non-proteolytic)

C B(proteolytic)

C

Fig. 3. Distance matrix trees depicting the relationships of light and heavy neurotoxin chains.

B o N T / G and BoNT/B is somewhat lower than that observed between B o N T / F s of C. barati and non-pro- teolytic C. botulinum (approx. 70% identity), but com- parable to that shown between B o N T / E and B o N T / F (approx. 63% identity) [11]. It is known that B o N T / F of C. barati cross-reacts with antibodies raised against type E NTs [16,17]. To our knowledge cross-reactivity has not been observed between B o N T / G and BoNT/B despite a similar overall sequence relatedness. Presum- ably in the case of B o N T / G and BoNT/B there are sufficient epitopic differences in the regions determin- ing antigenic specificity to facilitate production of anti- bodies capable of clearly differentiating between these serotypes. In the H c region which is thought to deter- mine the major antigenic sites, BoNT/B and BoNT/G showed approx. 49% identity compared with approx. 58% for the complete protein.

The present analysis completes the gene sequence determination of a representative of each of the known botulinal neurotoxin antigenic types and will contribute to elucidating the regions determining antigenic reac- tivity of the botulinal neurotoxins.

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