Construction of a tightly-controlled expression system for use in Pseudomonas

Transcriptional analysis of czcCBA gene cluster in Pseudomonas aeruginosa PAO1 revealed that the promoter of this operon is tightly controlled by Zn. The promoter activity is undetectable in the uninduced condition but reaches high levels when induced. We used the czcCBA promoter to construct a tightly-controlled expression vector series, pLY vectors (pLY-A, pLY-B and pLY-C). These differed by just one base pair at the multiple cloning sites, allowing convenient in-frame cloning of any genes for expression. Using the luxCDABE reporter, this expression system was shown to be controlled by Zn in a dose-dependent manner in Pseudomonas; cloning the sacB gene encoding levansucrase onto the pLY-C vector enabled Zn-controlled growth of P. aeruginosa PAO1. These pLY vectors provide a useful tool for functional characterization of genes and controlled production of difficult or toxic proteins in Pseudomonas sp.