The chloramphenicol resistance gene catD fromClostridium difficile was shown to be encoded on the transposons Tn4453a and Tn4453b, which were structurally and functionally related to Tn4451 fromClostridium 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-encodedtnpX genes was obtained from experiments which showed that in trans these genes complemented a Tn4451tnpXΔ1 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 Tn4451. 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 targetoriT or RSA site. The finding that related transposable elements are present in C. difficile andC. perfringens has implications for the evolution and dissemination of antibiotic resistance genes and the mobile elements on which they are found within the clostridia.

Dichelobacfer nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent 0. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. It was concluded that the divergence of the benign and virulent strains at this locus was a multi-step process. Several potential ORFs were identified a t the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli. 1

The vrl locus is preferentially associated with virulent isolates of the ovine footrot pathogen, Dichelobacter nodosus. The complete nucleotide sequence of this 27.1-kb region has now been determined. The data reveal that the locus has a G+C content much higher than the rest of the D. nodosuschromosome and contains 22 open reading frames (ORFs) encoding products including a putative adenine-specific methylase, two potential DEAH ATP-dependent helicases, and two products with sequence similarity to a bacteriophage resistance system. These ORFs are all in the same orientation, and most are either overlapping or separated by only a few nucleotides, suggesting that they comprise an operon and are translationally coupled. Expression vector studies have led to the identification of proteins that correspond to many of these ORFs. These data, in combination with evidence of insertion of vrl into the 3′ end of an ssrA gene, are consistent with the hypothesis that the vrl locus was derived from the insertion of a bacteriophage or plasmid into the D. nodosusgenome.

Sequence analysis of the Clostridium butyricum chloramphenicol acetyltransferase (CAT) gene, catB, showed that it encoded a CAT monomer of 219 amino acids with a molecular weight of 26,114. Comparison of the deduced amino acid sequence of the CATB monomer to those of sixteen other CATs showed that it was most closely related to the CATQ monomer from Clostridium perfringens.

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