Mutations in the major outer membrane protein gene from Histophilus somni by an allelic exchange method

Abstract

Functional roles of the major outer membrane protein (MOMP) gene from the bovine pathogen Histophilus somni have remained to be elucidated due to lack of mutagenesis methods easily applicable to this gene. In this study, the direct use of PCR-amplified mutated DNA flanking by an antibiotic selection marker for transformation of H. somni was applied to accomplish the site-directed mutagenesis via homologous recombination in H. somni non-pathogenic strain 129Pt and pathogenic strain 2336. A protocol for unmarking the antibiotic resistance gene from the created MOMP mutant by using a temperature-sensitive plasmid vector was also established. In both strains, no significant phenotypic difference was observed between the wild-type strain and its isogenic mutant expressing the exchanged MOMP in growth in broth medium and antibiotic susceptibility. However, the mutant 129Pt expressing MOMP from strain 2336 was significantly more susceptible to bacterial killing by fresh normal bovine serum than its wild-type parent strain. Serum susceptibility of the mutant 2336 expressing MOMP from strain 129Pt was significantly lower than its wild-type parent strain although the susceptibility difference was considerably less than that found between the 129Pt wild-type and MOMP mutant strains. From further assays using MOMP mutants that express chimeric MOMP consisting of the amino-terminal part that contains loops L1–L3 from one strain and the carboxyl-terminal part that contains loops L4–L8 from another strain, the C-terminal part of MOMP was found to affect serum susceptibility of H. somni.