Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5

Abstract

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.

Figure 1. Relative locations of the A2 and F4 toxin gene clusters within the chromosome.

The two locations in the chromosome of strain Kyoto-F where the A2 and F4 toxin gene clusters are located within the chromosome of strain Af84 are shown. The A2 toxin gene cluster of strain Af84 is located within two copies of the arsC gene (in green), as is the case for the A2 toxin gene cluster of strain Kyoto-F. The F4 toxin gene cluster is located within a split pulE gene (in blue). At this site there is no toxin gene cluster within Kyoto-F (or in any other known C. botulinum genome). The genomic locations of the toxin gene cluster are magnified with red lines. The horizontal arrows indicate coding sequences (CDSs) and the black arrows indicate the toxin gene clusters. GenBank locus IDs are labeled below the arrows. The first CDS was given the full GenBank locus ID, followed by an abbreviated ID that uses only 2–3 digits.

Figure 2. Plasmid synteny among pCLJ, pCLQ and pCLK.

The three fully sequenced plasmids are compared by the Artemis Comparison Tool (ACT) . Regions of homology among the pCLJ, pCLQ and pCLK are indicated in red. Regions containing the bont/A4, bont/F5 and bont/A3 orfX toxin gene clusters are in blue. The region containing the bont/B5 ha toxin gene cluster is in yellow. The comparison shows that the bont/F5 toxin gene cluster is found in the same location as the bont/A3 and bont/A4 (inverted) toxin gene clusters in homologous plasmids.

Figure 3. Locations and gene content of the three botulinum toxin gene clusters of C. botulinum strain Af84.

Schematic presentation of the genomic location of the three botulinum toxin gene clusters in strain Af84 (I) and their gene contents (II). The A2 and F4 toxin gene clusters are located on the chromosome. The F5 toxin gene cluster is located on a large 246,124 bp plasmid. All three toxin gene clusters contain the same genes (orfX3, orfX2, orfX1, botR, p47, ntnh and bont) in the same gene arrangement with a 1.2 kb intergenic region, containing a putative degenerated is element, between orfX1 and botR.

Figure 4. Identification of recombination events in the bont/F5 toxin gene cluster.

Comparison of the nucleic sequences of the bont/F5 toxin cluster genes of strain Af84 to their homolog genes in other orfX toxin gene clusters. The comparison shows that the 5′ terminus of the F5 toxin gene cluster, containing the genes orfX3, orfX2, orfX1 and the orfX1-botR intergenic region, is almost identical to the 5′ terminus of the A3 toxin gene cluster in strain Loch Maree. In contrast, the 3′ terminus of the F5 toxin gene cluster, containing the genes botR, p47 and most of ntnh, is 100% identical to the 3′ terminus of the A2 toxin gene cluster in strain Af84. The ntnh/F5 and ntnh/A2 of strain Af84, as well as the ntnh/F5 of strain CDC5479, are 100% identical until nucleotide position 2630; however, from nucleotide 2631 through the reminder of the gene, the ntnh/F5 of strain Af84 becomes 100% identical to the ntnh/F5 of strain CDC54096. The comparisons demonstrate that several recombination events contributed to the creation of the F5 toxin gene cluster in strain Af84. ^aThe numbers below the arrows are percentage identities. Identities higher than 99% are in bold. ^bnucleotide location within the ntnh. ^cNA, not available in GenBank.