The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny

Abstract

Botulinum neurotoxins (BoNTXs) produced by Clostridium botulinum are among the most poisonous substances known. Of the seven types of BoNTXs, genes for type C1 and D toxins (BoNTX/C1 and D) are carried by bacteriophages. The gene for exoenzyme C3 also resides on these phages. Here, we present the complete genome sequence of c-st, a representative of BoNTX/C1-converting phages. The genome is a linear double-stranded DNA of 185,682 bp with 404-bp terminal direct repeats, the largest known temperate phage genome. We identified 198 potential protein-coding regions, including the genes for production of BoNTX/C1 and exoenzyme C3. Very exceptionally, as a viable bacteriophage, a number of insertion sequences were found on the c-st genome. By analyzing the molecular structure of the c-st genome in lysogens, we also found that it exists as a circular plasmid prophage. These features account for the unstable lysogeny of BoNTX phages, which has historically been called "pseudolysogeny." The PCR scanning analysis of other BoNTX/C1 and D phages based on the c-st sequence further revealed that BoNTX phages comprise a divergent phage family, probably generated by exchanging genomic segments among BoNTX phages and their relatives.

Fig. 1.

Genome organization of the c-st phage and the summary of PCR scanning analysis of other BoNTX phages. Horizontal arrows represent ORFs identified on the c-st genome, and ORF numbers or gene products are indicated above each arrow. ORFs were categorized into eight groups according to their predicted functions. TDRs are indicated by striped boxes, IS elements by green boxes, and the putative replication origin (ori) and terminus (ter) by vertical arrows. Four virion component proteins identified by micosequencing analysis of the c-st virion proteins are indicated by asterisks. Results of PCR scanning analysis of phages c-468 and d-1873 are shown below the ORF map. Horizontal thick lines represent segments where PCR products were obtained, and broken lines represent segments where no products were obtained. The segments that yielded PCR products larger than those of c-st are indicated by (+) and smaller by (-). Because circular plasmid prophages were examined in this PCR scanning analysis, segments encompassing the TDRs were portioned to both ends (the connection indicated by horizontal arrowheads).

Fig. 2.

Base composition and GC skew analyses of the c-st genome. The average G + C percent, the GC-skew (G - C/G + C) in the forward strand, and the cumulative GC-skew are represented by blue, red, and green lines, respectively. All were calculated by using a window size of 1,000 bp and a step size of 100 bp. ter, terminus; ori, origin.

Fig. 3.

Structures of the c-st genome in lysogens and phage particles. (A) Locations of hybridization probes and PCR primers on the linear and circular forms of the c-st genome are shown. Among the AatII and SalI sites on the c-st genome, only AatII and SalI sites located closest to the right and left ends, respectively, are shown. Open boxes represent TDRs. (B) Cellular DNAs from strains C-ST, (C)-AO2:c-st, and (C)-AO2 and the phage-particle DNA were examined by PCR using primer pairs 1f/1r (lanes 1-4) and 2f/2r (lanes 5-8). (C)-AO2:c-st is a c-st-lysogen derived from (C)-AO2. Lane M, size makers; lanes 1 and 5, C-ST; lanes 2 and 6, (C)-AO2:c-st; lanes 3 and 7, (C)-AO2; lanes 4 and 8, phage particle. (C) The same set of DNA preparations as in B was digested by AatII and SalI, separated on 1% agarose gels (a), and subjected to the Southern hybridization analyses using probes P1 (b), P2 (c), and P3 (d). Lanes M and 1-4 are as in B.

Fig. 4.

Southern hybridization analysis of the c-st DNAs from lysogens and phage particles. (A) Locations of PvuII sites and hybridization probes and the linear and circular forms of the c-st genome are shown. (B) Cellular DNAs from strains C-ST, (C)-AO2:c-st, and C-AO2 and the c-st phage-particle DNA were digested with ApaI (lanes 1-4) or PvuII (lanes 4-8), separated by PFGE (a), and subjected to Southern hybridization analysis using probes P2 (b), P4 (c), P5 (d), and P6 (e). Lane M, size makers; lanes 1 and 5, C-ST; lanes 2 and 6, (C)-AO2:c-st; lanes 3 and 7, C-AO2; lanes 4 and 8, phage particle.