Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

Abstract

Background: Clostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.

Results: The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids.

Conclusions: The completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.

Figure 1

Circular representation of the replicons characterised from the BKT015925 C. botulinum group III genome. The circles represent (from the outside in): 1, plus strand genes; 2, minus strand genes (mobile elements are marked in red); 3, CDSs coding for phage proteins (brick red), plasmid proteins (light red) and toxins (orange); 4, rRNA genes (blue) and tRNA genes (green). The two inner circles display GC content and GC skew. (A) The chromosome of BKT015925, (B-F) the plasmidome of BKT015925, (G) p6CSt from C-Stockholm.

Figure 2

Phylogenomic analysis of strains of C. botulinum group III and in relation to other Clostridia. (A) Heat plot (based on the ASC method) showing the results of pairwise average nucleotide distance calculations between the available completed and draft genomes from C. botulinum group III and representative genomes from other C. botulinum groups and Clostridium species.(B) Phylogenetic tree derived from the similarity matrix shown in (A) using the neighbor-joining method.

Figure 3

Genetic synteny and replication-directed translocations visualized by MUMmer alignments. (A) A comparison of the chromosomes of C. botulinum group III (BKT015925) and C. novyi-NT. The conserved gene synteny and the low replication-directed translocation activity between the chromosomes are obvious. (B) Four selected pairs of genomes with the same genetic distance to each other as BKT015925 and C. novyi-NT show how much the amount of symmetrical inversions can vary.

Figure 4

Comparative analysis of the plasmidome. Comparisons of the three largest plasmids in BKT015925 with previously completed plasmids from C. botulinum group III, performed with Artemis Comparison Tool (ACT). (A) The BoNT-carrying prophage (p1BKT015925) compared to the equivalent, previously released C-Stockholm phage (B) The 99 kb and 80 kb plasmids (p2BKT015925 and p3BKT015925) compared to each other and to plasmids pC2C203U28 (very similar to pCLG1) and pCLG2 from strain C203U28 and D-1873, respectively.

Figure 5

The transposon activity of high-copy mobile elements. The mobile elements from the chromosome of BKT015925 and V891 were mapped on a transposon-free consensus chromosome generated in silico. The positions of the elements are displayed with arrows. The high-copy elements displayed are ISCbo1 (red), ISCbo5 (blue), ISCbo4 (green), ISCbo2 (brown), and a tandem reverse-transcriptase; ISCbo3 (purple). ISCbo2 has in several cases been inserted into the ISCbo3 element.