Generation of an optimised Cumate toolkit for tuneable protein expression during in vitro and in vivo studies of Burkholderia cenocepacia

Inducible gene expression is pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (P _CymRC /CymR _GV ) which allows the tight and tunable control of protein expression within B. cenocepacia, as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show that inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allow precise and tuneable gene expression in Burkholderia even within a host-pathogen context.