Plasmids for heterologous expression in Pasteurella haemolytica

Area of Science:

• Molecular Biology
• Microbiology
• Genetic Engineering

Background:

• Development of versatile cloning and expression vectors is crucial for genetic manipulation in diverse bacterial species.
• Existing vectors may lack replication capabilities or selectable markers suitable for both Gram-negative pathogens like Pasteurella haemolytica and model organisms such as Escherichia coli.

Purpose of the Study:

• To construct novel shuttle vectors capable of replicating in both Pasteurella haemolytica and Escherichia coli.
• To engineer vectors with essential features for molecular cloning and gene expression studies in these two bacterial species.

Main Methods:

• Construction of shuttle vectors based on the native P. haemolytica plasmid pYFC1, conferring sulfonamide (SuR) and streptomycin (SmR) resistance.
• Incorporation of a multiple cloning site (MCS) and antibiotic resistance markers (ampicillin, kanamycin, chloramphenicol) functional in both host organisms.
• Development of an expression vector (pNF2176) by linking the MCS to the sulfonamide gene promoter (PsulII) for gene expression studies.
• Creation of a promoter-probe vector (pNF2283) through deletion of the PsulII promoter from an expression construct.

Main Results:

• Successfully constructed shuttle vectors pNF2176 and pNF2214, which replicate in both P. haemolytica and E. coli.
• Demonstrated the functionality of antibiotic resistance markers (ampicillin, kanamycin, chloramphenicol) in both bacterial species.
• Developed an expression vector (pNF2176) enabling cloning and expression of genes under the control of the PsulII promoter in P. haemolytica.
• Generated a promoter-probe vector (pNF2283) for identifying and characterizing novel promoters in P. haemolytica.

Conclusions:

• The developed shuttle vectors provide valuable tools for genetic manipulation and molecular studies in both P. haemolytica and E. coli.
• These vectors facilitate comparative genomics and functional studies across different bacterial species.
• The availability of these vectors enhances the capacity for genetic engineering in veterinary pathogens and related bacteria.