Construction of isogenic mutants of Pasteurella haemolytica by allelic replacement

We describe methods for the mutagenesis of cloned Pasteurella haemolytica (Gram −) genes and for the construction of P. haemolytica mutants by allelic exchange. We used these methods to construct isogenic mutants of P. haemolytica which no longer synthesize three membrane lipoproteins (Lpp). A single genetic locus, consisting of three tandemly arranged genes encoding 28–30-kDa membrane Lpp, was replaced with a mutated locus which carries the β-lactamaseencoding Ap R gene from a 4.2-kb P. haemolytica plasmid. The inactivated locus was introduced into P. haemolytica by electroporation of a plasmid which carries the mutated locus, but is incapable of replicating in P. haemolytica. Southern and Western blot analyses indicate that the wild-type locus was replaced by the mutated locus through a doublecrossover recombination event and that the membrane Lpp were no longer produced by the mutant strain. These methods should be useful in constructing mutant loci which can be used to analyze the roles for various P. haemolytica proteins in the pathogenesis of bovine pneumonic pasteurellosis.