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Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae.
Journal article

Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae.

  • Frey J Institute for Veterinary Bacteriology, University of Berne, Switzerland.
  • 1992-03-01
Published in:
  • Research in microbiology. - 1992
Actinobacillus pleuropneumoniae
Chloramphenicol O-Acetyltransferase
Chloramphenicol Resistance
Escherichia coli
Genetic Vectors
In Vitro Techniques
Mannheimia haemolytica
Plasmids
Promoter Regions, Genetic
Replicon
Transformation, Bacterial
English We have constructed a pair of broad host range expression vectors, pJFF224-NX and pJFF224-XN, based on plasmid RSF1010, which enable cloning and efficient expression of genes in Actinobacillus pleuropneumoniae and Pasteurella haemolytica and in Escherichia coli. The vectors consist of the minimal autonomous replicon of the broad host range plasmid RSF1010 and a type II chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. In addition, they contain a gene expression cassette based on the E. coli bacteriophage T4 gene 32 promoter region and a transcription stop signal, which are separated by a segment of multiple cloning sites in both orientations. Electroporation and subsequent selection for chloramphenicol resistance was used for the introduction of the vectors in A. pleuropneumoniae and P. haemolytica. A promoterless xy/E gene from the Pseudomonas putida TOL plasmid was cloned onto pJFF224-NX. This plasmid enabled efficient expression of active catechol2,3oxygenase in A. pleuropneumoniae and P. haemolytica. It was stably maintained in A. pleuropneumoniae without antibiotic selection, showing less than 0.1% loss after 100 generations, while native RSF1010 and other RSF1010-based vectors were unstable in this host.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://roar.hep-bejune.ch/global/documents/63878
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