Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of 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: 11/1/2005
Citation: Briggs, R.E., Tatum, F.M. 2005. Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae. Applied and Environmental Microbiology. 71(11):7187-7195.

Interpretive Summary: The leading cause of morbidity and mortality sustained by the cattle feed lot industry in the North America is bovine respiratory disease (BRD). The three bacteria chiefly responsible for causing BRD are Mannheimia haemolytica, Pasteurella multocida and Haemophilus somnus. Herein we have developed a widely applicable method for constructing unmarked mutations in this family of bacteria using a newly engineered temperature-sensitive plasmid, derived from an endogenous plasmid of M. haemolytica pD70. The broad utility of this system was demonstrated by generating unmarked, in-frame aroA mutants of M. haemolytica, P. multocida, and H. somnus. This system represents a broadly applicable means for constructing unmarked mutants of Pasteurellaceae species which can be used to further our understanding of the mechanisms of disease pathogenesis and for generating new vaccines.

Technical Abstract: Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism’s restriction enzyme HsoI (recognition sequence 5’-GCGC-3’) characterized herein.