Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed transcription under the control of the rhamnose-inducible promoter system (designated P ) and found that P promoter activity was highly dependent upon . Vfr dependence was also observed for the arabinose-inducible promoter (designated P ). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the P and P promoters . Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in P and P promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible P (P ) and rhamnose-inducible P (P ) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a mutant background, the issue is more pervasive, considering that transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic P promoter is not subject to Vfr regulatory control.