Poster Presentation The Prato Conference on the Pathogenesis of Bacterial Infections of Animals 2016

The development of an anhydrotetracycline inducible gene expression system for use in Actinobacillus pleuropneumoniae (#64)

Leon G Leanse 1 , Janine T Bosse 1 , Yanwen Li 1 , Paul R Langford 1
  1. Imperial College London, London, LONDON, United Kingdom

Inducible gene expression systems are valuable tools that permit the regulation of bacterial genes. These systems are particularly useful in the context of verifying and characterising putative essential genes selected from high-throughput mutant library screens such as Transposon Directed Insertion-site Sequencing (TraDIS). With respect to the Pasteurellaceae family, there has only been one study involving Haemophilus influenzae that used a xylose-inducible promoter to regulate gene expression. Here we report the construction of a tightly regulated anhydrotetracycline (aTc) gene expression system for use in the economically significant porcine pathogen, Actinobacillus pleuropneumoniae (APP). We have exploited a Tn10-derived regulatory system for the expression of the proton dependent antiporter protein TetA, found endogenously in the APP serovar 7 strain AP76. Using the broad host range plasmid pLS88 as the backbone, we cloned in the sequences encoding the tetracycline repressor protein, TetR, and the 2 cognate operator binding sites from AP76, and coupled this with a reporter gene encoding Nanoluc luciferase. Unique restriction sites were introduced upstream of the reporter gene in order to permit the cloning of any gene of interest for regulation in tandem with the Nanoluc luciferase. The resulting vector, pTetRnLuc, was conjugally transferred into MIDG2331, a genetically tractable serovar 8 strain of APP. Growth in the presence of different concentrations aTc demonstrated titratable repression of luciferase activity, with >250-fold repression in the absence of aTc. In conclusion, these promising results suggest that pTetRnLuc will be useful not only for verification of putative essential genes, but also for regulated expression of genes for complementation where gene-dosage may be an issue.