GENERATION AND MOLECULAR CHARACTERIZATION OF NEW TEMPERATURE-SENSITIVE PLASMIDS FOR THE PASTEURELLACEAE

Authors: Briggs, Robert; Tatum, Fred

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2005
Publication Date: 11/21/2005
Citation: Briggs, R.E., Tatum, F.M. 2005. Generation and Molecular Characterization of New Temperature-Sensitive Plasmids for the Pasteurellaceae. Applied and Environmental Microbiology. 71(11):7187-7195.

Interpretive Summary: The leading cause of morbidity and mortality sustained by the cattle feedlot industry in the United States is caused by bovine respiratory disease. In the U.S. the bacterium Mannheimia (Pasteurella) haemolytica is principally responsible for the economic losses sustained by the beef cattle industry. Pasteurella multocida and Haemophilus somnus often are the bacterial agents causing substantial losses to the dairy industry. Here we describe the development and characterization of new plasmids which will allow us to engineer new live vaccine candidates against the three previously mentioned bacteria.

Technical Abstract: Temperature-sensitive (Ts) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin-resistant parent plasmid, pD70, isolated from Mannheimia haemolytica Serotype 1. Selected were three plasmids which failed to replicate at or above 420C in M. haemolytica but were fully functional below 330C. The Ts plasmids were shown by sequence analysis to possess clustered mutations within a 112 base pair region within their origins of replication. Site-directed nucleotide mutations were introduced in the wild-type pD70 origin of replication to confirm that the single-base pair substitutions identified resulted in temperature-dependent plasmid replication. The Ts plasmids were shown to exhibit temperature sensitivity in Pasteurella multocida and in Haemophilus somnus. However prior to introduction into H. somnus, the Ts plasmids required in vitro methylation to protect against the organism's endogenous restriction system. In part, the restriction barrier of H. somnus is due to the newly described restriction enzyme, HsoI, which recognizes the sequences, 5' -GCGC-3'. In vitro methylation of plasmid DNA with commercially available HhaI methyltransferase increased transformation efficiencies of H. somnus by four orders of magnitude. Lastly too better characterize this new Ts system the replicon was mapped to determine the sequence required for plasmid maintenance. Deletion analysis revealed that approximately 760 base pairs were necessary for plasmid replication.