Employment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB2 ABC Transporter in Escherichia coli

Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (Pi). Under these conditions, the high-affinity PstSCAB(2) protein (i.e., with two PstB proteins) is the primary Pi transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P-tac promoter and the lacO(ID) regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P-phoB::P-tac and P-pstS::P-tac strains to characterize phenotypes resulting from various Delta phoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB2 transporter, as well as its abundance within the cell. In addition, we used the P-phoB::P-tac Delta phoU strain as a platform to begin characterizing new phoU mutations in plasmids.