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The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot

Molecular and Cellular Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia¹

Author for correspondence: Brian Cheetham. Tel: +61 2 6773 3394. Fax: +61 2 6773 3267. e-mail: bcheetha{at}metz.une.edu.au

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. The authors have previously characterized two genetic elements, the intA (vap) and intB elements, which integrate into the genome of D. nodosus. In the virulent strain A198 there are two copies of the intA element. One copy is integrated into the 3’ end of the tRNA-ser_GCU gene, close to the aspartokinase (askA) gene, and the second copy is integrated into the 3’ end of the tRNA-ser_GGA gene, next to the polynucleotide phosphorylase (pnpA) gene. In this study, a new genetic element was identified in the benign strain C305, the intC element, integrated into the 3’ end of the tRNA-ser_GCU gene, next to askA. The intC element was found in most D. nodosus strains, both benign and virulent, which were examined, and was integrated into tRNA-ser_GCU in most strains. Between the askA and tRNA-ser_GCU genes, a gene (designated glpA), was identified whose predicted protein product has very high amino acid identity with RsmA from the plant pathogen Erwinia carotovora. RsmA acts as a global repressor of pathogenicity in E. carotovora, by repressing the production of extracellular enzymes. In virulent strains of D. nodosus the intA element was found to be integrated next to pnpA, and either the intA or intC element was integrated next to glpA. By contrast, all but one of the benign strains had intB at one or both of these two positions, and the one exception had neither intA, intB nor intC at one position. The loss of the intC element from the virulent strain 1311 resulted in loss of thermostable protease activity, a virulence factor in D. nodosus. A model for virulence is proposed whereby integration of the intA and intC genetic elements modulates virulence by altering the expression of glpA, pnpA, tRNA-ser_GCU and tRNA-ser_GGA.

View larger version (27K): [in this window] [in a new window]   Fig. 1. vap regions 1, 2 and 3 of D. nodosus strain A198 compared with the intC element in D. nodosus strain C305. The numbers show the distance in kb from the left-most NruI site in strains A198 or C305. Restriction sites shown are BamHI (B), EcoRI (E), HindIII (H), KpnI (K), NruI (N), SacI (S) and XhoI (X). The major potential genes are indicated by open arrows. The tRNA-ser genes are shown by black triangles. Repeated sequences (Cheetham et al., 1995 ) are indicated as follows: 19 bp att sites (small open boxes), 103 bp repeats, or partial copies thereof (small shaded boxes), 102 bp repeats, or partial copies (small black boxes) and the putative origin of replication (large shaded box). The large open boxes show regions which are not found in strain C305.

Abbreviations: HSL, N-(3-oxohexanoyl)-L-homoserine lactone

The GenBank accession number for the intC element in strain C305 beginning at the left-most EcoRI site (Fig. 1) is Y15939.

^a Present address: Roche Diagnostics Australia Pty Ltd, 31 Victoria Avenue, Castle Hill, NSW 2154, Australia.

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