Analysis of Neurotoxin Cluster Genes in Clostridium botulinum Strains Producing Botulinum Neurotoxin Serotype A Subtypes

Clostridium botulinum comprises a heterogeneous group of strains, all of which have the distinctive characteristic of producing botulinum neurotoxin (BoNT). BoNTs are the most potent neurotoxins known to man and are considered a potential weapon for bioterrorism (3). BoNTs are characterized as category A select agents and are considered potential bioterrorism threats (3). BoNTs can be immunologically distinguished using homologous antitoxins into seven serotypes, designated A to G. Among these serotypes there is considerable genetic variation, as demonstrated by the recognition of at least 47 subtypes identified thus far (15, 28). Previous studies have determined that the BoNT genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the BoNT gene that are organized as neurotoxin clusters (4, 7, 10, 11, 13, 14, 17, 18, 19, 23). Available data indicate that there exist two primary types of neurotoxin clusters: (i) a hemagglutinin (HA) cluster and (ii) an Orfx cluster with genes (orf genes) of unknown function. The HA cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The Orfx cluster consists of genes encoding Orfx3, Orfx2, Orfx1, P47, P21, and NTNH. The known serotypes associated with HA clusters are BoNT/A1, BoNT/B, BoNT/C, BoNT/D, and BoNT/G, while serotypes BoNT/E, BoNT/F, and BoNT/A2 are associated with Orfx clusters; the E serotype cluster is unique as it lacks p21 (4, 10, 13, 14, 19, 25, 27). However, results from previous studies indicate that different BoNT/A subtypes possess either an HA cluster or an Orfx cluster associated with the expressed bont gene, depending on the subtype and strain (7, 8, 11, 12). It has been shown that the HA cluster proteins can form toxin complexes of various sizes including LL toxin (∼900 kDa), L toxin (∼500 kDa), and M toxin (∼ 300 kDa), comprised of various combinations of HA proteins, NTNH, and BoNT (∼150 kDa) (17, 18, 23). The biological and structural roles of the complex proteins are not completely characterized, although it is believed that they serve the role of protecting BoNT from digestive enzymes and adverse conditions present in the gastrointestinal tract (23). The function of orfx genes and their role in the neurotoxin cluster are still unknown, but it has been postulated that they perform a function similar to their HA cluster counterparts because the location and orientation of the Orfx and HA cluster genes relative to the BoNT gene in the neurotoxin cluster are analogous (7). A regulatory gene called botR (21, 22) has also been identified in the HA cluster. botR encodes a sigma 70 factor which positively regulates the expression of BoNT and the HA proteins (9). The Orfx cluster has an analogous gene, p21, which appears to be a sigma 70 factor, as determined by amino acid homology BLAST analysis. BoNT/A is of particular importance and interest since it causes the most severe form of human botulism, is the most significant threat in bioterrorism, and has been increasingly used as a pharmaceutical modality (1, 6). During the past 5 years, a focus of our laboratories has been the study of C. botulinum strains that produce novel BoNT/A subtypes including an A3 strain producing BoNT/A3, an A4 strain that is a bivalent strain producing both BoNT/A4 and BoNT/B (2), and a strain (termed HA− Orfx+ A1) that produces BoNT/A1 with a neurotoxin cluster arrangement normally associated with the BoNT/A2. This paper presents a comprehensive comparison of the individual neurotoxin cluster genes and bont genes among different C. botulinum subtype A strains on both nucleotide and amino acid levels. In order to study the neurotoxin cluster genes in these subtypes, we have identified and sequenced each of these strains' neurotoxin cluster genes and have compared the results on nucleotide and amino acid levels to the known sequences from ATCC 3502 (a BoNT/A1 producer) (7, 26), Kyoto F (a BoNT/A2 producer) (7), and NCTC 2916 (a BoNT/A1 producer with a unexpressed BoNT/B gene) (10, 16, 24) to determine their genetic relationships and possible genetic lineages. Our laboratory showed that the genes encoding BoNT/A3 and BoNT/A4 are located on large plasmids; this is the first demonstration that genes encoding BoNT/A are present on extrachromosomal elements (20). Prior to our discovery in these strains and the subsequent confirmation of our findings (27), the gene encoding BoNT/A1 was expected to be located on the chromosome by analysis of the genome of C. botulinum ATCC 3502 (26). Previous studies of BoNT clusters have emphasized the arrangement of the genes and, particularly, the amino acid sequences of the proteins; here, we also address the nucleotide sequence relationships and possible mechanisms of acquisition and rearrangement of the BoNT and neurotoxin cluster genes of the A3 and A4 subtypes. In addition, we describe a novel neurotoxin cluster, HA− Orfx+ A1, in the present study and discuss possible mechanisms of toxin complex acquisition and function.