Precise regulation of gene expression is essential for dissecting molecular basis of bacterial physiology, virulence and post-translational modifications [1]. Within Burkholderia species, few inducible systems combine tight regulation with minimal interference to cellular processes [2]. Whereas basal transcription can mask phenotypic effects or trigger unintended pathway activation, making precise control essential for reliable functional studies [3]. To address this, we developed a next generation cumate inducible expression tool for Burkholderia cenocepacia, engineered for strong yet tightly repressed, tunable control of gene expression. This system offers precise regulation while maintaining proteome integrity, providing a powerful platform for functional, regulatory, and glycosylation-focused studies in Burkholderia and related organisms.
Through targeted mutagenesis and functional screening of the cumate regulator (CymR) and promoter elements, we engineered an optimized construct (PCymRC/CymRGV) that delivers precise on/off control (minimal leakiness) and broad dynamic responsiveness. Using sfGFP and O-linked glycoprotein reporters, we demonstrate rapid induction and minimal background activity across a wide range of expression levels supported by proteomics analysis. To evaluate global cellular effects, we applied quantitative proteomics to compare the cumate system with the commonly used rhamnose-inducible promoter. The cumate circuit produced minimal and orthogonal proteomic changes, confirming its suitability as a non-perturbative system for studying protein function and regulation. We further adapted the optimized system into a CTX-based chromosomal integration vector, enabling robust, stable and inducible expression during intracellular infection of eukaryotic host cells for precise control of glycosylation-associated pathways [4]. Leveraging this control, we uncovered that O-linked protein glycosylation is critical for optimal intracellular replication as evident by low bacterial survival within THP1 infection model, linking regulated expression directly to pathogen biology.
Together, this work delivers a powerful and flexible genetic tool for Burkholderia related glycobiology research. This new cumate-inducible system combines tight regulation, tunability and proteomic stability, offering new precision for studying bacterial gene function, glycosylation and host pathogen interactions in complex biological contexts.