Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes

Abstract

Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.

Figure 1.

Circular representations of the genome of C. botulinum. The circles represent (from the outside in): (1 and 2) All CDS (transcribed clockwise and anticlockwise); (dark blue) pathogenicity/adaptation; (black) energy metabolism; (red) information transfer; (dark green) surface-associated; (cyan) degradation of large molecules; (magenta) degradation of small molecules; (yellow) central/intermediary metabolism; (pale green) unknown; (pale blue) regulators; (orange) conserved hypothetical; (brown) pseudogenes; (pink) phage and IS elements; (gray) miscellaneous. (3) (Blue) CDSs shared with sequenced clostridia. (4) (Red) C. botulinum-unique CDSs relative to the sequenced clostridia. (5) Virulence factors discussed in the text; (brown) streptolysin; (red) neurotoxins; (blue) six metalloproteases; (black) type IV pilus; (green) flagellar and chemotaxis operons; (orange) flagellar glycosylation island. (6) RNA genes; (blue) rRNAs; (red) tRNAs; (purple) stable RNAs. (7) G+C content (plotted using a 10-kb window). (8) GC deviation [(G − C)/(G + C) plotted using a 10-kb window]; (khaki) values >1; (purple) values <1.

Figure 2.

Schematic representation of the C. botulinum chitinolytic system. (CBD) Chitin-binding-domain; (SS) signal sequence; (FN3) fibronectin type III domain; (GH18) family 18 domain of glycosylhydrolases; (LysM) LysM domain.

Figure 3.

Chitinase assay. Chitin degradation was assessed on chitin-overlaid agar plates as described in Methods. Small zones of clearing are evident around colonies of proteolytic C. botulinum strains Hall A (A) and 213B (B), but not around colonies of nonproteolytic C. botulinum strain CDC 7854 (C).

Figure 4.

Organization of the accessory gene regulator (agr) loci in clostridia and S. aureus. Similar genes are shown in the same color. Significant functions are: (blue) autoinducer maturation protein (AgrB); (red) autoinducer peptide (AgrD); (green) two-component sensor kinase (AgrC); (pink) two-component response regulator (AgrA); (light blue and orange) membrane proteins; (purple) signal transduction proteins with GGDEF and HD domains; (yellow) two-component sensor kinases.

Figure 5.

Comparative genomic analysis of strains of proteolytic C. botulinum and C. sporogenes strains using microarrays. Horizontal colored bars indicate array competitive hybridization. Vertical lines represent CDS present (yellow lines), absent or highly diverged (blue lines), and uncertain (gray lines). The plasmid CDS are on the left, followed immediately by the first gene in the chromosome (CBO001), and the last chromosomal CDS (CBO3650) is on the right. For strains of proteolytic C. botulinum, the toxin types are included in square brackets; two strains of C. sporogenes (NCIMB 8053 and NCIMB 10696) are also included. The position of the two prophages (Φ-Cb1 and Φ-Cb2) and the potential flagellin glycosylation island (FGI) are indicated by arrows.