High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials.

Coward C,Dharmalingham G,Abdulle O,Avis T,Beisken S,Breidenstein E,Carli N,Figueiredo L,Jones D,Khan N,Malara S,Martins J,Nagalingam N,Turner K,Wain J,Williams D,Powell D,Mason C. (2020)

FEMS microbiology letters

The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

FEMS microbiology letters

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