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J Bacteriol, March 1998, p. 1277-1286, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the Escherichia coli Phosphate Regulon

Andreas Haldimann, Larry L. Daniels,dagger and Barry L. Wanner*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received 30 September 1997/Accepted 2 January 1998

Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated ParaB promoter or the rhamnose-regulated PrhaB promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of ParaB or PrhaB fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a PrhaB fusion is more tightly regulated than a ParaB fusion in that a PrhaB-phoR+ fusion but not a ParaB-phoR+ fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both ParaB-phoB+ and PrhaB-phoB+ fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise Delta phoU mutations. However, we unexpectedly found that such Delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797-6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by using ParaB-phoB+, PrhaB-phoB+, or PrhaB-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ Delta phoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, Lilly Hall of Life Sciences, West Lafayette, IN 47907. Phone: (765) 494-8034. Fax: (765) 494-0876. E-mail: BLW{at}bilbo.bio.purdue.edu.

dagger Present address: Biotechnology Centre, Faculty of Pure and Applied Sciences, University of the West Indies, Mona, Kingston 7, Jamaica.




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