ABSTRACT 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.

Dichelobacter 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 D. 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 at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.