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Applied and Environmental Microbiology, October 2009, p. 6124-6131, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.00809-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae{triangledown}

Janine T. Bossé,1 Andrew L. Durham,1,{dagger} Andrew N. Rycroft,2 J. Simon Kroll,1 and Paul R. Langford1*

Molecular Infectious Diseases Group, Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom,1 Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mimms, Herts AL9 7TA, United Kingdom2

Received 9 April 2009/ Accepted 30 July 2009

We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE ({sigma}E) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of {sigma}E, among a bank of random transposon mutants, as well as to detect induction of {sigma}E following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMC-Express, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent.

* Corresponding author. Mailing address: Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom. Phone: 44 (0)207 594 3359. Fax: 44 (0)207 594 3984. E-mail: p.langford{at}imperial.ac.uk

{triangledown} Published ahead of print on 7 August 2009.

{dagger} Present address: Airways Disease Section, National Heart and Lung Institute, Imperial College London, London SW3 6LY, United Kingdom.

Applied and Environmental Microbiology, October 2009, p. 6124-6131, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.00809-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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