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

Abstract

ABSTRACT The araC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range in Escherichia coli , which has led to its widespread use in other bacteria. Although anecdotal evidence suggests that araC-ParaBAD is leaky in Pseudomonas aeruginosa , neither a thorough analysis of this inducible promoter system in P. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of the araC-ParaBAD system in P. aeruginosa . Using transcriptional fusions to a lacZ reporter gene, we determined that the noninduced expression from araC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can in E. 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 in P. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of the araC-ParaBAD system, we found that the lacI q - Ptac and rhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range than araC-ParaBAD . We demonstrated that noninduced expression from the araC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis in P. aeruginosa , problems that were avoided with rhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement over araC-ParaBAD in P. aeruginosa . IMPORTANCE We report the shortcomings of the commonly used Escherichia coli araC-ParaBAD inducible promoter system in Pseudomonas aeruginosa , successfully reengineered it to improve its functionality, and show that the E. coli rhaSR-PrhaBAD system is tightly controlled and allows inducible gene expression over a wide range in P. aeruginosa .