Functional Identification of Conjugation and Replication Regions of the Tetracycline Resistance Plasmid pCW3 fromClostridium perfringens

Abstract

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.Conjugative plasmids are self-replicating molecules that encode their own transfer to recipient strains, usually by a type IV secretion apparatus. Conjugative plasmids from gram-positive bacteria, such as Enterococcus, Staphylococcus, and Bacillus spp., employ similar mechanisms (23); however, the absence of an outer membrane and the presence of a much thicker peptidoglycan layer necessitates a requirement for different conjugation complexes. Integrative conjugative elements (ICEs), or conjugative transposons, are also capable of encoding their own conjugative transfer, but these elements are not self replicating and require genomic integration for their stable maintenance. The best known ICEs are Tn916 from Enterococcus faecalis and Tn1545 from Streptococcus pneumoniae, but little is known about their mechanisms of conjugation (45). ICEs have been identified from many gram-positive bacteria, including the pathogenic clostridia. These elements include the Tn916-like conjugative element Tn5397 from Clostridium difficile and the defective element CW459tet(M) from Clostridium perfringens (46).The 47-kb tetracycline resistance plasmid pCW3 (49) carries a novel tetracycline resistance operon (56) and is the paradigm conjugative plasmid from C. perfringens. It is closely related to pIP401, which carries the same tetracycline resistance genes, and also carries Tn4451, an integrative mobilizable element that confers chloramphenicol resistance (5). Deletion of Tn4451 from pIP401 (3) results in a plasmid that has a restriction profile identical to that of pCW3. Comparative restriction...

Toxin Plasmids of Clostridium perfringens

“…Similarly, the netB-derived plasmids pJIR3536 and pNetB-NE10 share ϳ35 kb of conserved backbone (Fig. 2) with pCPF5603 and pCW3 (13,19,235,236). As mentioned above, some type E iota-toxin-encoding plasmids share substantial similarity with pCPF5603 (18), while others more closely resemble pCPF4969 (23).…”

“…All conjugative C. perfringens plasmids identified to date have the tcp locus, which has been demonstrated to be essential for conjugative transfer of pCW3 (236,(242)(243)(244)(245). Furthermore, either sequence analysis (13,14,23) or Southern hybridization analysis (14)(15)(16)(17)(18) indicated that many C. perfringens type B to E strains contain multiple large plasmids carrying toxin genes (cpb, etx, iapA/iapB, cpb2, and tpeL) and a tcp locus, which we assume is a reasonable predictor of their conjugative potential.…”

“…4). Both the pCPF5603-and pCPF4969-like toxin plasmids contain two regions (dam-rep) and tcp, which are also present on the pCW3 tetracycline resistance plasmid (13,14,18,23,227,235,236). Since the tcp region has homology with Tn916, which is a conjugative transposon, it is conceivable that a Tn916-like transposon may have integrated into a plasmid, creating a conjugation-capable precursor plasmid (13,236).…”

“…Finally, Southern blots provided evidence that pMRS4969 carried regions that were also present on pCW3, which at the time had not been sequenced. It was postulated that these regions were involved in conjugative transfer, a postulate that was subsequently validated (236).…”

“…Analysis of the conjugation mechanism in C. perfringens has focused on the tetracycline resistance plasmid pCW3 (224,227,228), which is 47,263 bp and encodes 51 potential open reading frames (236). As mentioned above, a conjugation locus of 11 genes, intP to tcpJ, has been designated the transfer of clostridial plasmid (tcp) locus.…”