Improved Dynamic Range of a Rhamnose-Inducible Promoter for Gene Expression in spp.

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In and related species, the l-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in Burkholderia cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene stem-loop and engineered transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increases in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in , creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis, , and and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia. The utility of the modified system was demonstrated as we recovered viable mutants to control , , and , whereas the unmodified promoter was unable to control . The modified expression system allowed control of an essential gene depletion phenotype at lower levels of l-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for and related species. Species of are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. Thus, we sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly expressed essential genes in B. cenocepacia. The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into and related bacteria.