Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2005
Publication Date: 11/15/2005
Citation: Tatum, F.M., Briggs, R.E. 2005. Construction of in-frame aroa deletion mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus using a new temperature-sensitive plasmid. Applied and Environmental Microbiology. 71(11):7196-7202. Interpretive Summary: The leading cause of morbidity and mortality sustained by the cattle feed lot industry in the North America is bovine respiratory disease. In the United States, Mannheimia haemolytica is the organism most commonly isolated from feedlot calves with pneumonia. Other bacteria causing substantial losses to both the dairy and beef cattle industries are Pasteurella multocida and Haemophilus somnus. While there has been considerable effort to use vaccines and therapeutic treatments to mitigate disease caused by these bacteria, economic losses attributed to them exceed one billion dollars annually in the United States. Currently, most vaccines against these organisms are of the killed variety. However, live bacterial vaccines are in many cases more effective at eliciting protection. Herein we describe production of new live vaccine strains of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus that contain no foreign DNA.
Technical Abstract: A temperature-sensitive (Ts) plasmid was generated from the endogenous streptomycin-resistance plasmid of Mannheimia haemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus. Ts replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 300C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a non-permissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.