Chloramphenicol resistance in Clostridium difficile is encoded on Tn4453 transposons that are closely related to Tn4451 from Clostridium perfringens

Abstract

The chloramphenicol resistance gene catD from Clostridium difficile was shown to be encoded on the transposons Tn4453a and Tn4453b, which were structurally and functionally related to Tn4451 from Clostridium perfringens. Tn4453a and Tn4453b excised precisely from recombinant plasmids, generating a circular form, as is the case for Tn4451. Evidence that this process is mediated by Tn4453-encoded tnpX genes was obtained from experiments which showed that in trans these genes complemented a Tn4451tnpX delta 1 mutation for excision. Nucleotide sequencing showed that the joint of the circular form generated by the excision of Tn4453a and Tn4453b was similar to that from Tn4451. These results suggest that the Tn4453-encoded TnpX proteins bind to similar DNA target sequences and function in a manner comparable to that of TnpX from Tn4453. Furthermore, it has been shown that Tn4453a and Tn4453b can be transferred to suitable recipient cells by RP4 and therefore are mobilizable transposons. It is concluded that, like Tn4451, they must encode a functional tnpZ gene and a target oriT or RSA site. The finding that related transposable elements are present in C. difficile and C. perfringens has implications for the evolution and dissemination of antibiotic resistance genes and the mobile elements on which they are found within the clostridia.

FIG. 1

Comparative linear maps of Tn4451, Tn4453a, and Tn4453b. The oligonucleotide primers used for PCR analysis are indicated by the short arrows. Restriction sites for CfoI (C), EcoRV (E), HindII (HII), HindIII (HIII), NsiI (N), and Sau3A (S) are indicated. The relative size, location, and direction of transcription of each of the Tn4451-encoded genes are indicated by the longer filled arrows.

FIG. 2

Alignment of the joint of the circular forms of Tn4451, Tn4453a, and Tn4453b. Tn4451 sequences are as reported previously (5, 8). The consensus target sequence which is cleaved by TnpX (8) is also shown. All of the sequences consist of central GA residues (black boxes). Residues which are the same as those found in Tn4451 are shaded (grey boxes). Residues which differ between Tn4453a and Tn4453b are outlined in black.