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Applied and Environmental Microbiology, February 2009, p. 573-582, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01046-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Christopher D. Rice, Jacob E. Pollard, Zachery T. Lewis, and William R. McCleary*

Microbiology and Molecular Biology Department, Brigham Young University, Provo, Utah 84602-5253

Received 6 May 2008/ Accepted 24 November 2008

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 PstSCAB2 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 Ptac promoter and the lacOID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized PphoB::Ptac and PpstS::Ptac 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 PphoB::Ptac {Delta}phoU strain as a platform to begin characterizing new phoU mutations in plasmids.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Biology, Brigham Young University, 775 WIDB, Provo, UT 84602-5253. Phone: (801) 422-6215. Fax: (801) 422-0519. E-mail: bill_mccleary{at}byu.edu

{triangledown} Published ahead of print on 1 December 2008.

Applied and Environmental Microbiology, February 2009, p. 573-582, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01046-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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