The Gram-negative anaerobic pathogen 'Dichelobacter nodosus' is the principal causative agent of footrot in sheep. The 'intA', 'intB' and 'intC' elements are mobile genetic elements which integrate into two tRNA genes downstream from 'csrA' (formerly 'glpA') and 'pnpA' in the 'D. nodosus' chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of 'pnpA'. We have proposed a model in which virulence in 'D. nodosus' is controlled in part by the integration of genetic elements downstream from 'csrA' and 'pnpA', altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the 'intD' element, which is 32 kb in size and contains an integrase gene, 'intD', several genes related to genes on other integrated elements of 'D. nodosus', a type IV secretion system and a putative mobilisation region, suggesting that the 'intD' element has a role in the transfer of other genetic elements. Most of the 'D. nodosus' strains examined which contained the 'intD' gene were benign, with 'intD' integrated next to 'pnpA', supporting our previous observation that virulent strains of 'D. nodosus' have the 'intA' element next to 'pnpA'.

'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof 'D. nodosus' strains are classified as benign, intermediate or virulent depending upon the severity of disease they cause in sheep. Previous work has led to the identification of a virulence-associated DNA region in 'D. nodosus' virulent strain Al98, called the 'vap' element which may have a role in virulence. However, there is no transformation system for 'D. nodosus' which precludes direct testing of the role of these and other genes in virulence. In this work, a native 'D. nodosus' plasmid pDN1 was isolated, sequenced, characterised and modified to contain appropriate antibiotic resistance markers and a multiple cloning site. Derivatives of pDN1 were subsequently used in transformation experiments in an effort to develop a transformation system for 'D. nodosus'. In the absence of a transformation system, more indirect methods were utilised to determine whether the 'vap' genes of 'D. nodosus' have a role in virulence. Past investigations have concentrated on analysis of the 'vap' genes at the DNA level. In this work, northern blot experiments were undertaken to determine whether the 'vap' genes are expressed at the RNA level in the virulent and/or benign strains in which they are present. Results indicated that in general, the 'vap' genes are expressed in the virulent and benign strains in which they are present, and although differential expression of the 'vap' genes was observed, the differences observed were not related to the virulence of the isolates. In addition to the 'vap' element, part of an 'intB' element was previously identified. In an effort to further characterise the 'intB' element, chromosome walking 4.2 kb downstream of regions which were determined previously was undertaken. Results suggest that these downstream sequences, which include genes 'gepC-gepG' are not part of an integrated genetic element. In addition, Southern blot analysis was utilised in order to determine the prevalence, arrangement and the integrity of the 'intB' element in seventeen strains of 'D. nodosus'.

The anaerobic bacterium 'Dichelobacter nodosus' is the principal causative agent of ovine footrot, a mixed bacterial infection of the hoof. Although the bacterium only survives for a few days in soil, this period is crucial for transmission of the disease as sheep are infected by walking through soil or pasture contaminated with infectious bacteria. The 'D. nodosus' genome is only 1.3Mb in size and has a dearth of genes encoding regulatory proteins. A series of genetic elements which integrate into the genome has been identified and we have proposed that these integrated genetic elements control the expression of adjacent genes encoding global regulators of virulence. The intA, intB, intC and intD elements integrate next to csrA or pnpA while the vrl integrates next to ssrA. CsrA, PnpA and the ssrA gene product, a 10SaRNA, have been shown to act as global virulence regulators in other bacteria. We have also identified a bacteriophage, DinoHI, which is integrated into the genome of some 'D. nodosus' strains. Sequence analyses suggest that there are many possible interactions between these integrated genetic elements. The vrl contains a copy of the DinoHI packaging site, indicating that the vrl may be transferred between strains by the bacteriophage. DinoHI and the intB element have a common repressor gene, suggesting that maintenance of the integrated state of these two genetic elements is co-ordinately controlled. Similarly, a DNA segment resembling the bacteriophage P4 immunity region is present on the intA, intC and intD elements and may be responsible for maintaining these three genetic elements in the integrated state. The features of the intD element suggest that it is self-transmissible and also capable of mobilising the intA element. Exchange of sequences between these genetic elements may also occur. We discuss here evidence for a network of interactions between these genetic elements with implications for the control of virulence in 'D. nodosus'.