Comparison of virulence plasmids among Clostridium perfringens type E isolates

Abstract

Clostridium perfringens type E isolates produce iota-toxin, which is encoded by iap and ibp genes. Using Southern blot analyses, the current study identified iap/ibp plasmids of approximately 97 or approximately 135 kb among eight type E isolates. For most of these isolates, their iap/ibp plasmid also encoded urease and lambda-toxin. However, the beta2-toxin gene, if present, was on a different plasmid from the iap/ibp plasmid. For all isolates, the iap/ibp plasmid carried a tcp locus, strongly suggesting that these plasmids are conjugative. Overlapping PCR analyses demonstrated some similarity between the iap/ibp plasmids and enterotoxin-encoding plasmids of type A isolates. Additional PCR analyses demonstrated that the iap/ibp locus is located near dcm sequences, an apparent plasmid hot spot for toxin gene insertion, and that two IS1151-related sequences are present in the iap/ibp locus. To begin testing whether those IS1151-like sequences can mobilize iap/ibp genes, a PCR assay was performed that amplifies a product only from circular DNA forms that could represent transposition intermediates. This PCR assay detected circular forms containing iap/ibp genes and silent enterotoxin gene sequences, with or without an IS1151-like sequence. Collectively, these results suggest that a mobile genetic element carrying iap/ibp has inserted onto a tcp-carrying enterotoxin plasmid in a type A isolate, creating a progenitor iap/ibp plasmid. That plasmid then spread via conjugation to other isolates, converting them to type E. Further iap/ibp plasmid diversity occurred when either the iap/ibp genes later remobilized and inserted onto other conjugative plasmids or some iap/ibp plasmids acquired additional DNA sequences.

FIG. 1.

Southern blot of a pulsed-field gel electrophoresed with DNA from specified type E isolates and then hybridized with an iap probe (A). After stripping, the blot was rehybridized with a cpe probe (B). After a final stripping, the blot was rehybridized with a cpb2 probe (C). An overlay of the panel B and panel C blots demonstrates that cpb2 is located on a different plasmid than the iota-toxin locus (D). Numbers at left of each blot indicate migration of size markers in kb.

FIG. 2.

Southern blot of a pulsed-field gel electrophoresed with DNA from type E isolate 853 and then digested with specified restriction enzymes for 16 h. The Southern blot was first hybridized with an iap probe and then sequentially stripped and rehybridized with lam, ure, and tcpH probes.

FIG. 3.

Overlapping PCR amplification of the conserved region of pCPF5603/pCPF4969 using a previously described primer battery (21) and one described in this study (see Table S2 in the supplemental material). DNA template included F5603 DNA (top), NCIB10748 DNA (middle), and 853 DNA (bottom). Marker sizes are shown at left.

FIG. 4.

Overlapping PCR amplification of the pCPF4969 (A) or pCPF5603 (B) variable region using DNA from type E isolates NCIB10748 and 853. No substantial amplification was observed using DNA from the other six surveyed type E isolates. Marker sizes are shown at left.

FIG. 5.

Further characterization of the iap/ibp locus arrangement and demonstration that this locus can exist as circular intermediates that could be transposition intermediates. Based upon sequencing of a long-range PCR product connecting dcm to the iap/ibp locus in NCIB10748 (not shown), an overlapping PCR assay was designed and applied to the eight surveyed type E isolates. Results for NCIB10748 are shown in panel A; similar results were also observed with the other seven isolates. Panel B shows results from a PCR assay that used primer sets that amplify products from the iap/ibp locus of NCIB10748 when this locus is present in a small circular form but not in a linear form. The primer sets used are R1 (primers L1-R and P8-F), R2 (L1-R and L1-F), R3 (P1-R and L1-F), and R4 (P1-R and P8-F); locations of primers are shown in panel A. Panel C shows the expected products from small circular forms containing IS1151-2-ibp-iap-silent cpe sequences or ibp-iap-silent cpe sequences. The identity of these products was confirmed by DNA sequencing.