Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus

Studies on the potential virulence genes of the ovine footrot pathogen Dich elobacter nodosus have been hindered by the lack of a genetic system for th is organism. In an attempt to accomplish the transformation of D, nodosus c ells, we constructed a plasmid that contained part of a native D. nodosus p lasmid and carried a tetracycline resistance gene that was located between the D. nodosus rmA promoter and terminator. This plasmid was used to transf orm several D. nodosus strains to tetracycline resistance. Analysis of two independent transformants from each parental strain showed that in nearly a ll of these derivatives, the plasmid was not replicating independently, but that the tetracycline resistance gene had inserted by homologous recombina tion into one of the three rrn operons located on the chromosome. In most o f the transformants, double reciprocal crossover events had occurred. These results are highly significant for genetic studies in D. nodosus and for f ootrot pathogenesis studies, since by using reverse genetics it will now be possible to examine the role of putative D. nodosus-encoded virulence gene s in the disease process. (C) 1998 Federation of European Microbiological S ocieties. Published by Elsevier Science B.V. All rights reserved.