Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa

The functional and physiological characterization of bacterial genes required for growth and/or cell survival is limited by the inability to generate deletion mutants lacking the specific gene of interest. This limitation can be circumvented by generating conditional mutants in which the loss of the endogenous copy of the gene is compensated by the introduction of the wild-type allele under the control of an inducible promoter, which allows for tightly regulated expression of the gene of interest. Besides the confirmation and/or functional investigation of essential genes, conditional mutants can also be useful to investigate the effect of finely controlled expression of nonessential genes. In this chapter, we describe a method that can be used to generate stable and unmarked conditional mutants in Pseudomonas aeruginosa.