Molecular genetic construction of vaccine strains of pasteurellaceae

Abstract

Tools for genetically engineering Pasteurellaceae are provided. Replication-conditional plasmids which are useful for the Pasteurellaceae have been isolated and characterized. The plasmids can be utilized for delivery of DNA segments into the Pasteurellaceae in situations where control of extra-chromosomal replication desired, such as in achieving allelic exchange or site-directed mutagenesis.A restriction endonuclease, HsoI, was isolated from a bovine lung isolate of Haemophilus somnus. The enzyme was found to be a true isoschizomer of HinPI, a commercially available enzyme originally isolated from Haemophilus influenzae PI. Commercially available HhaI methyl transferase was found to protect against cleavage by both enzymes. Methylation of foreign plasmid DNA was found to enhance transformation of Haemophilus somnus in excess of four orders of magnitude.

We claim:  
     
       1. A method of introducing a DNA segment to a Pasteurellaceae genome comprising: 
       administering to a Pasteurellaceae cell selected from the group consisting of P. Multocida, P. Haemolytica, and H. somnus, a recombinant construct comprising the DNA segment and plasmid PBB192 which is temperature-conditional for replication in the Pasteurellaceae cell to form transformants;  
       subjecting the transformants to a non-permissive temperature;  
       screening the transformants for the presence of the DNA segment; and  
       screening the transformants for the absence of the plasmid.  
     
     
       2. The method of  claim 1  wherein the DNA segment is a transposable element. 
     
     
       3. The method of  claim 1  wherein the DNA segment is a mutant form of the genomic DNA of the Pasteurellaceae. 
     
     
       4. The method of  claim 3  wherein the mutant form is a deletion. 
     
     
       5. The method of  claim 3  wherein the mutant form is an insertion. 
     
     
       6. Isolated and purified bacterial cells of Pasteurella multocida aroA strain ATCC  55892 . 
     
     
       7. Isolated and purified bacterial cells of Haemophilus somnus or Pasteurella multocida made by the process of  claim 1 . 
     
     
       8. Isolated and purified bacterial cells of Haemophilus somnus or Pasteurella multocida made by the process of  claim 2 . 
     
     
       9. Isolated and purified bacterial cells of Haemophilus somnus or Pasteurella multocida made by the process of  claim 3 . 
     
     
       10. Isolated and purified bacterial cells of Haemophilus somnus or Pasteurella multocida made by the process of  claim 4 . 
     
     
       11. Isolated and purified bacterial cells of Haemophilus somnus or Pasteurella multocida made by the process of  claim 5 . 
     
     
       12. The isolated and purified bacterial cells of  claim 6  which is formulated with adjuvants. 
     
     
       13. The isolated and purified bacterial cells of  claim 7  which is formulated with adjuvants. 
     
     
       14. The isolated and purified bacterial cells of  claim 8  which is formulated with adjuvants. 
     
     
       15. The isolated and purified bacterial cells of  claim 9  which is formulated with adjuvants. 
     
     
       16. The isolated and purified bacterial cells of  claim 10  which is formulated with adjuvants. 
     
     
       17. The isolated and purified bacterial cells of  claim 11  which is formulated with adjuvants.