CONSTRUCTION OF ISOGENIC MUTANTS OF PASTEURELLA-HAEMOLYTICA BY ALLELIC REPLACEMENT

We describe methods for the mutagenesis of cloned Pasteurella haemolyt ica (Gram(-)) genes and for the construction of P. haemolytica mutants by allelic exchange. We used these methods to construct isogenic muta nts of P. haemolytica which no longer synthesize three membrane lipopr oteins (Lpp). A single genetic locus, consisting of three tandemly arr anged genes encoding 28-30-kDa membrane Lpp, was replaced with a mutat ed locus which carries the beta-lactamase-encoding Ap(R) gene from a 4 .2-kb P. haemolytica plasmid. The inactivated locus was introduced int o P. haemolytica by electroporation of a plasmid which carries the mut ated locus, but is incapable of replicating in P. haemolytica. Souther n and Western blot analyses indicate that the wild-type locus was repl aced by the mutated locus through a double-crossover recombination eve nt and that the membrane Lpp were no longer produced by the mutant str ain. These methods should be useful in constructing mutant loci which can be used to analyze the roles for various P. haemolytica proteins i n the pathogenesis of bovine pneumonic pasteurellosis.