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...
To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.
In Clostridium perfringens, conjugative plasmids encode important virulence factors, such as toxins and resistance determinants. All of these plasmids carry a conjugation locus that consists of 11 genes: intP and tcpA to tcpJ. Three proteins, TcpA, a potential coupling protein, TcpF, a putative ATPase that is similar to ORF15 from Tn916, and TcpH, which contains VirB6-like domains, are essential for conjugation in the prototype conjugative plasmid pCW3. To analyze the functional domains of TcpH, a putative structural component of the mating-pair formation complex and deletion and site-directed mutants were constructed and analyzed. The results showed that the N-terminal 581 residues and the conserved 242 VQQPW 246 motif were required for conjugative transfer. Bacterial two-hybrid and biochemical studies showed that TcpH interacted with itself and with TcpC. An analysis of the tcpH mutants demonstrated that the region required for these interactions also was localized to the N-terminal 581 residues and that the function of the C-terminal region of TcpH was independent of protein-protein interactions. Finally, immunofluorescence studies showed that TcpH and TcpF were located at both cell poles of donor C. perfringens cells. The results provide evidence that TcpH is located in the cell membrane, where it oligomerizes and interacts with TcpC to form part of the mating-pair formation complex, which is located at the cell poles and is closely associated with TcpF.
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