Analysis of plasmid DNA from Pasteurella haemolytica and construction of cloning vectors

Pasteurella haemolytica, a Gram-negative bacterium, is the principal causative agent of pneumonic pasteurellosis in cattle and sheep. The presence of plasmids in various isolates of this species and their relationship to antibiotic resistance and to the health status of the source animal were investigated. Plasmids conferring resistance to ampicillin were analyzed and the plasmid-encoded p-lactamase enzymes characterized. A suitable plasmid was identified for the construction of shuttle cloning vectors for P. haemolytica. Thirty five typable and untypable isolates of P. haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype Al, three of serotype A2 and one untypable) contained plasmid DNA. Isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic, or from animals in contact with pneumonic, cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing Al strains exhibited ampicillin resistance, which was shown by transfer studies (transformation and conjugation) to be plasmid-mediated. Plasmid DNA prepared from ampicillin-resistant E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol or by modifying the growth conditions. Terrific broth or broth supplemented with 1% yeast nitrogen base was found to be the best growth medium for plasmid amplification. All Apft plasmids had a similar size (~4. 3 kb), and one of them (pPH843) was highly amplifiable and more stable in E. coll compared to the other plasmids (pPH2, pPH33 and pPH821). An improved method was developed for the large-scale purification of covalently closed circular plasmid DNA molecules over a wide size range. The protocol used an alkaline-lysis procedure followed by acid-phenol extraction and included several modifications to previously reported methods. Two principal modifications were the replacement of NaCl by MgCl in the extraction buffer and vortexing instead of shaking the crude DNA suspensions to more efficiently remove chromosomal and other non-CCC plasmid DNA and to improve yield of purified CCC DNA forms. The Apft plasmids from P, haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 p-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae, but a part of the DNA sequence <~0. 7 kb) from the Haemophilus plasmid was not present in the Pasteurella plasmid. This suggested that one plasmid had been derived from the other during the course of evolution. From substrate profiles and isoelectric focusing data, the p-lactamases encoded by the P. haemolytica plasmids were found to be indistinguishable from the ROB-1 0- lactamase. A restriction map of plasmid pPH843 was constructed and the putative Apft, oriV, and mob regions of the plasmid were located by deletion and fusion experiments. The plasmid was converted into a series of possible cloning vectors (pAKAlb, pAKA15-l, pAKA16, pAKA16-l and pAKA17) by insertion at different points in the plasmid of the lacZ alpha-peptide-coding gene and a multiple cloning site (for insertional inactivation of beta-galactosidase activity) from the E. coli vector pIC20H and an IncP mobilization function from plasmid RK2. Transconjugants obtained by transfer of the constructs from E, coli to P. haemolytlca were found to be unstable on subculture, although they were stable upon transfer to another E, coli strain. This indicated that P. haemolytlca might have a restriction/modification system which affected plasmid DNA previously propagated in E. coli.