Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector

Abstract: The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glässer''s disease in pigs, using a novel H. parasuis–Escherichia coli shuttle vector. A 4.2kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5×102 CFU/μg of DNA. Transformation efficiency was notably increased (1.3×105 CFU/μg of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. [Copyright &y& Elsevier]