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

ABSTRACT 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.