The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

ThearaC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range inEscherichia coli , which has led to its widespread use in other bacteria. Although anecdotal evidence suggests thataraC-ParaBAD is leaky inPseudomonas aeruginosa , neither a thorough analysis of this inducible promoter system inP. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of thearaC-ParaBAD system inP. aeruginosa . Using transcriptional fusions to alacZ reporter gene, we determined that the noninduced expression fromaraC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can inE. coli . Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression inP. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of thearaC-ParaBAD system, we found that thelacI q -Ptac andrhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range thanaraC-ParaBAD . We demonstrated that noninduced expression from thearaC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis inP. aeruginosa , problems that were avoided withrhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement overaraC-ParaBAD inP. aeruginosa .IMPORTANCE We report the shortcomings of the commonly usedEscherichia coli araC-ParaBAD inducible promoter system inPseudomonas aeruginosa , successfully reengineered it to improve its functionality, and show that theE. coli rhaSR-PrhaBAD system is tightly controlled and allows inducible gene expression over a wide range inP. aeruginosa .