Improvement in the efficiency of natural transformation of Haemophilus parasuis by shuttle-plasmid methylation.

Some Haemophilus parasuis strains display resistance to transformation with Escherichia.coli-derived plasmids. This property limits the application of genetic approaches previously developed for H. parasuis. The present study showed that natural transformation with the shuttle plasmid pS2UK led to allelic exchange in H. parasuis strains SH0165 and CF7066. Furthermore, natural transformation with pS2UK yielded allelic exchange mutants in 10 of 17 H. parasuis strains, similar to results using the suicide plasmid pK2UK. Subsequently, 17 H. parasuis strains were transformed with pS2UK by electroporation and 13 obtained the transformants harboring the complete plasmid molecules. As a result, natural transformation of homologous blank strains with the H. parasui-derived plasmids significantly improved the transformation efficiency targeted at obtaining allelic exchange mutants. In addition, shuttle plasmids pS1UG and pSHUK that carried the different homologous arm sequences also displayed the increased transformation efficiency after they were replicated in homologous H. parasuis cells. The approach described here not only improved the efficiency of natural transformation of H. parasuis, but also enlarged the range of transformable H. parasuis strains, thereby enabling application of H. parasuis-specific genetic manipulation techniques in a wider range of isolates.