The type F6 neurotoxin gene cluster locus of group II clostridium botulinum has evolved by successive disruption of two different ancestral precursors

Abstract

Genome sequences of five different Group II (nonproteolytic) Clostridium botulinum type F6 strains were compared at a 50-kb locus containing the neurotoxin gene cluster. A clonal origin for these strains is indicated by the fact that sequences were identical except for strain Eklund 202F, with 10 single-nucleotide polymorphisms and a 15-bp deletion. The essential topB gene encoding topoisomerase III was found to have been split by the apparent insertion of 34.4 kb of foreign DNA (in a similar manner to that in Group II C. botulinum type E where the rarA gene has been disrupted by a neurotoxin gene cluster). The foreign DNA, which includes the intact 13.6-kb type F6 neurotoxin gene cluster, bears not only a newly introduced topB gene but also two nonfunctional botulinum neurotoxin gene remnants, a type B and a type E. This observation combined with the discovery of bacteriophage integrase genes and IS4 elements suggest that several rounds of recombination/horizontal gene transfer have occurred at this locus. The simplest explanation for the current genotype is that the ancestral bacterium, a Group II C. botulinum type B strain, received DNA firstly from a strain containing a type E neurotoxin gene cluster, then from a strain containing a type F6 neurotoxin gene cluster. Each event disrupted the previously functional neurotoxin gene. This degree of successive recombination at one hot spot is without precedent in C. botulinum, and it is also the first description of a Group II C. botulinum genome containing more than one neurotoxin gene sequence.

Keywords: botulinum toxin gene; genome comparison; horizontal gene transfer; synteny.

Fig. 1.—

Group II Clostridium botulinum chromosomal DNA ACT comparisons of the 50-kb neurotoxin cluster locus of type F6 strain IFR 06/001 (B) with type B strain Eklund17B (A) and type E strain Alaska (C). Red bars denote regions sharing a high degree of sequence identity; the two thinner red bars linking section A with B are due to homology between the two versions of the topB gene, one present as a newly acquired gene, one as the original disrupted version. A similar duplication of homology is seen for the topB gene (CLH_1538) of Alaska (C, box i). Color code for features: 1) red, neurotoxin gene/cluster sequence; 2) blue, bacteriophage DNA; 3) purple, plasmid DNA; 4) yellow, IS4 elements; and 5) green and black, sequences unrelated to mobile DNA elements or toxin cluster genes, with black features apparently acquired horizontally. Boxes lacking arrowheads represent features with disrupted ORFs. Greatest synteny is seen between strain 06/001 (B) and Eklund17B (A): CDS/features 1–8 and 36–42 are homologs of Eklund17B (NRP [Norwich Research Park]) CDSs CB17B1604-1610 and CB17B1610-1617 (all sharing >90% identity). CDS 12 (in black): newly introduced topB gene. CDSs 19–24: neurotoxin cluster genes orf-x3, orf-x2, orf-x1, p47, nontoxic–nonhemagglutinin gene, and type F6 neurotoxin gene, respectively. Features 32 and 35 (red boxes): fragments of type B neurotoxin gene and type E neurotoxin gene, respectively. Chromosomal regions of type E strain Alaska homologous to the 50-kb toxin cluster locus of strain 06/001 are scattered due to poorer synteny; three main regions are depicted (section C). Box i: genes CLH_1532-1548, (88–96% identity). Box ii: CLH_1115-1105 (79–90% identity, note lack of homology with orf-x2, CLH_1114). Box iii: CLH_1908-1901 (95–96% identity). The type E3 neurotoxin gene of Alaska, CLH_1110 shares homology with strain 06/001 at two locations; the 3′-end of the type F6 neurotoxin gene (CDS 24) plus the nonfunctional type E neurotoxin gene fragment (feature 35).