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 (PCymRC/CymRGV) 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. ImportanceThis work establishes optimised cumate-inducible vectors for use in Burkholderia cenocepacia, addressing the need for alternative inducers to available carbohydrate systems. We show cumate-inducible vectors allow precise control of gene expression even within eukaryotic cells, providing a new and orthogonal way to temporally control protein induction. Utilising cumate- based induction, we demonstrate the importance of O-linked protein glycosylation for optimal intracellular replication in B. cenocepacia, highlighting its potential to be used to explore host- pathogen interactions. Combined, this work shows cumate-inducible vectors extend the range of studies which can be undertaken to dissect B. cenocepacia physiology and virulence.