Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates

Abstract

Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.

FIG. 1.

Genetic organization of the cpe locus in C. perfringens type A isolates. (I) Type A isolates carrying a cpe plasmid, including the sequenced ∼9-kbp XbaI cpe-containing fragment of the cpe plasmid from sporadic diarrhea isolate F4969 (A) and the deduced plasmid cpe locus of isolates F4013 (B). (II) The previously described (5) chromosomal cpe locus of food poisoning strain NCTC8239 is shown for comparison. Broad bars show ORFs; arrows under ORFs indicate orientation. Numbers shown for F4969 indicate nucleotide bases. Long thin bars depict the PCR products amplified with each primer pair (see the text).

FIG. 2.

Comparison of IS1470-like sequence in isolate F4969 and the downstream IS1470 sequence in the type A chromosomal-cpe food poisoning strain NTCT8239. #, putative start codon; +, putative stop codon. Underlined nucleotides are homologous between the IS1470 sequences downstream of the NCTC8239 cpe gene and the IS1470-like sequence of isolate F4969. ∗, missing nucleotide.

FIG. 3.

PCR analyses of sequences upstream of the plasmid cpe gene of type A isolates. (A) Results of a dcm-specific PCR assay (expected ∼0.4-kbp product marked by arrow) with primers internal to F4969 dcm sequences. (B) Results of a PCR assay performed with primers designed to amplify a product from the DNA region between dcm sequences and sequences ∼400 bp downstream of them (based upon sequencing of the cpe-containing XbaI fragment of F4969). In this PCR assay, F4969 DNA yields an ∼0.9-kbp PCR product (small arrow), which is consistent with sequencing results for the F4969 plasmid cpe locus; note that one cpe-negative type A isolate, 297442, gave an ∼2.0-kbp product (large arrow). (C) PCR analysis of the association between dcm gene and IS1469 genes. An ∼2.1-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene. (D) PCR analysis of the association between dcm sequences and the cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. An ∼3.5-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. Numbers on the left indicate migration of molecular size markers.

FIG. 4.

Southern blot analyses of cpe-positive C. perfringens type A isolates. Results shown are for type A isolates carrying a cpe plasmid hybridized with a cpe probe, a dcm probe, or an IS1470-like probe. The uppermost bands in panel C lie in the gel wells. The F5603 lane shows hybridization of the probes to incompletely digested DNA. Numbers on the left indicate migration of molecular size markers.

FIG. 5.

Southern blotting analysis of dcm sequences in C. perfringens type A food poisoning isolates. Note that the probe, generated by PCR with primers to F4969 dcm sequences and F4969 sequences ∼400 bp downstream of the dcm sequences, does not hybridize with DNA from any type A food poisoning isolates tested. However, this probe does react with DNA isolated from F4969.

FIG. 6.

PCR and Southern blotting analyses of putative cytosine methyltransferase gene sequences (dcm sequences) in other C. perfringens types. (A) PCR analyses with primers to internal dcm sequences; (C) Southern blotting results with a F4969 dcm-specific probe generated with the same primer pair and pKCpeFX28 template DNA. (A) This PCR assay generated an ∼0.4 kbp product (arrow) in C. perfringens types A to E which is similar in size to the PCR product this primer pair generates with most type A strains (Fig. 3); those PCR products also hybridized to a dcm-specific probe (C). (B) PCR analysis using primers based on dcm sequences and sequences ∼400 bp downstream from dcm sequences; (D) Southern blotting analysis with a dcm-specific probe. An ∼0.9-kbp product was detected in type A isolate F4969 (single arrow); otherwise, ∼2.0-kbp products were detected in type B, D, and E isolates (double arrow). Those products also hybridized with an F4969 dcm-specific probe. Numbers on the left indicate migration of molecular size markers.

FIG. 7.

PCR analyses of the region between the cpe gene and downstream IS1470-like sequences. (A) PCR product amplified from specified type A isolates using primers for cpe gene sequences and sequences present in IS1470-like sequences of F4969, which generate an ∼2.2-kbp product (arrow at right) with DNA from F4969. Numbers on the left indicate migration of molecular size markers. (B) PCR products amplified from specified type A isolates using primers specific for internal IS1470-like sequences. This reaction generated the expected 0.9-kbp product from F4969 DNA (arrow). Numbers on the left indicate migration of molecular size markers.

FIG. 8.

PCR detection of IS1151 sequences in C. perfringens type A isolates. (A) PCR results obtained with primers specific for IS1151 sequences which amplified (arrow at right) the expected ∼0.6-kbp product with total DNA from the previously sequenced IS1151 of type E isolate 853 (1). Numbers on the left indicate migration of molecular weight markers. (B) PCR results obtained with a primer specific for cpe sequences and a reverse primer specific for IS1151 sequences. Sequencing of the ∼1.2-kbp product obtained with F4013 DNA confirmed the inclusion of sequences spanning from cpe to IS1151. (C) Southern blot results obtained by hybridizing XbaI-digested DNA from type A isolates carrying a cpe plasmid with an internal IS1151-like probe.