The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements

Abstract

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

Keywords: Clostridium parabotulinum; arsC; lycA; neurotoxin; orfX; plasmids; pulE.

FIGURE 1

Core genome SNP phylogeny of C. parabotulinum strains with orfX toxin clusters. The bont gene types, gene cluster locations, and geographic locations of the strains are color coded according to the legend. Additional information is listed in Supplementary Table 1 with the genomes in the order they appear in this figure.

FIGURE 2

Homologous recombination (HR) events that have exchanged or altered bont genes/gene clusters, resulting in the generation of diverse toxin subtypes. (A) C-terminal sequence of selected orfX ntnh genes showing homologous and disparate sequences that illustrate the recombination event placing bont/A2/A3 genes within bont/F gene clusters, or vice versa. Sequences showing complete identity are in black font, bont/A-specific sequences are in blue font, bont/F sequences are in green font, and single mutational differences are shown in red font. (B) Comparisons of bont/F2:bont/F3 and bont/F1:bont/F8 gene sequences showing areas of identity, possibly gained through HR events, and regions where sequences differ (shaded in gray).

FIGURE 3

Chromosomal and plasmid C. parabotulinum orfX bont gene cluster locations. The sites are numbered according to their base pair location within the BoNT/A3 SU1169 genome and plasmid pCBG from BoNT/A2B5 CDC 1436.

FIGURE 4

Dendrogram showing the relationship between bont/A and bont/F lycA gene sequences located within the chromosome and plasmids. Chromosomally located lycA genes are in black font and plasmid-borne genes are in blue font. Two major lycA variants with nucleotide differences of ∼6% can be discerned. The tree is midpoint rooted.

FIGURE 5

Dendrogram comparing sequences from arsC1, arsC2, and arsC3 genes associated with selected BoNT/A and BoNT/F toxin clusters. The ars genes are numbered 1, 2, or 3 according to their locations in relation to the bont gene cluster. Sequences from strains that contain all three arsC genes are shown in green font, sequences in blue font are from strains with arsC1 and arsC2 sequences, and sequences in black font are from strains with arsC2 only. The arsC3 gene differs by ∼20% in nucleotide residues from arsC1 and arsC2. The tree is midpoint rooted.

FIGURE 6

Arrangements of conserved genes surrounding chromosomally located bont gene clusters that are adjacent to the ars operon. Conserved genes are numbered 1–31 with #9–16 representing the bont gene cluster (colored red with the lycA gene in green) and #17–24 representing the ars operon (colored blue). Supplementary Table 2 lists the genes/encoded proteins corresponding to the numbers shown in Figure 6. Conserved genes that flank this location are colored orange; conserved hypothetical genes are gray; and non-conserved hypothetical genes are black. Panel (A) represents an arrangement where the ars operon is located downstream from the bont gene cluster while panels (B–G) represent arrangements where the ars operon precedes the bont gene cluster. Strains representing these arrangements include: (A) most Argentinean and Australian BoNT/A2 strains; (B) BoNT/F1 strains; (C) OrfX BoNT/A1 strains; (D) BoNT/A1(B) strains; (E) African BoNT/A2 strains; (F) Italian BoNT/A2 trains; and (G) Italian BoNT/F8 It 357.

FIGURE 7

Illustration of the integration location of chromosomally located bont/F3 and bont/F4 cluster genes, which is distinct from the ars operon location. At this site the bont gene clusters in panels (B,C) (#9–16, shown in red with the lycA gene in green) are inserted within a split pulE gene (#4 and 4’, purple). Supplementary Table 3 lists the genes/encoded proteins corresponding to the numbers shown in Figure 7. Additional conserved known genes are colored orange and conserved hypothetical genes are in black. Transposase genes are in light green. (A) Insertion region in BoNT F5 BrDura showing a complete pulE gene and an absence of the bont gene cluster. (B) The same region in BoNT F4 SU1425, showing an approximately 35 kb region between the truncated segments of the pulE gene. (C) The 92.5 kb DNA sequence in BoNT F3 SU0160 with the identical sequence found in BoNT F4 plus an inserted segment containing an intact bacteriophage.

FIGURE 8

Arrangements of conserved genes surrounding orfX bont gene clusters that are located within plasmids. Conserved genes representing the bont gene cluster are numbered #1–9, colored red with the lycA gene in green. Conserved genes that flank this location are colored orange, including the HepA/SNF, Tnase, and DNA helicase genes (#9–11); conserved hypothetical genes are gray; and non-conserved hypothetical genes are black. Supplementary Table 4 lists the genes/encoded proteins corresponding to the numbers shown in Figure 8. Panels (A,B) illustrate arrangements where the bont gene cluster is in the same orientation as the surrounding genes, while panels (C–F) show arrangements where the bont genes are in opposite orientation. Examples of these arrangements are found with (A) the bont/A2 gene cluster in strain BoNT/A2B3 It 87; (B) the bont/A2 gene cluster in BoNT/A2B5 CDC 1436; (C) the bont/F2 gene cluster in BoNT/B5F2 An436; (D) the bont/A2 gene cluster in BoNT/A2B7 It 92; (E) the bont/F5 gene cluster in BoNT/A2F5 BrDura, BoNT/F5 SU0634F, and BoNT/A2F4F5 AF84; and (F) the bont/A3 gene cluster in BoNT/A3 Loch Maree.

FIGURE 9

Illustrations of conserved DNA sequences that are shared between chromosomal and plasmid located bont clusters. With BoNT A2 strains, conserved sequences comprise only the actual bont cluster and lycA genes, while the conserved sequence with BoNT A3 strains includes additional surrounding genes. The bont cluster genes are colored red, the lycA gene is green, and other conserved known genes are orange. Genes that are unique to chromosomal or plasmid-borne bont/A2 gene clusters are shown in purple. Conserved hypothetical genes that are common to both plasmid-borne bont/A2 and plasmid and chromosomally located bont/A3 are in gray and conserved hypothetical genes that are exclusive to bont/A3 are in black. Specific gene identifications are listed in Supplementary Tables 2, 4 and are related to Figure 6A (bont/A2 chromosome), 8B (bont/A2 plasmid), and 8F (bont/A3 chromosome and plasmid).