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Applied and Environmental Microbiology, November 2005, p. 7196-7202, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7196-7202.2005

Construction of In-Frame aroA Deletion Mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by Using a New Temperature-Sensitive Plasmid

Fred M. Tatum* and Robert E. Briggs

Respiratory Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010

Received 23 May 2005/ Accepted 19 July 2005

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, 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 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive 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.

* Corresponding author. Mailing address: 2300 Dayton Road, Ames, IA 50010. Phone: (515) 663-7639. Fax: (515) 663-7458. E-mail: ftatum{at}nadc.ars.usda.gov.

Applied and Environmental Microbiology, November 2005, p. 7196-7202, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7196-7202.2005






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