Optimized cumate toolkit for tunable protein expression during in vitro and in vivo studies of Burkholderia cenocepacia

Inducible gene expression is pivotal to dissect bacterial physiology and virulence mechanisms. Across the Burkholderia genus, a limited range of inducible systems currently exist that 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 in eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we have generated an optimized cumate circuit (P CymRC /CymR GV ) which allows the tight and tunable control of protein expression in B. cenocepacia, as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we find that cumate elicits discrete alterations within the B. cenocepacia proteome that are orthogonal to those seen in widely used rhamnose-based induction systems, supporting cumate induction as a complementary approach for protein expression in Burkholderia spp. Leveraging the cell permeability of cumate and the generation of CTX-based chromosomal integration vectors, we show that inducible control of protein expression is achievable during intracellular replication of B. cenocepacia . By controlling intracellular expression of O-linked protein glycosylation, we demonstrate that protein glycosylation contributes to optimal intracellular replication of B. cenocepacia . This work demonstrates that cumate inducible systems allow precise and tunable gene expression in Burkholderia even within a host-pathogen context. IMPORTANCE This work establishes optimized cumate-inducible vectors for use in Burkholderia cenocepacia , addressing the need for alternative inducers to available carbohydrate systems. We show that cumate-inducible vectors allow precise control of gene expression even within eukaryotic cells, providing a new and orthogonal way to temporally control protein induction. Utilizing cumate-based induction, we demonstrate the importance of O-linked protein glycosylation for optimal intracellular replication in B. cenocepacia , highlighting the potential of cumate systems 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.