USE OF NEW METHODS FOR CONSTRUCTION OF TIGHTLY REGULATED ARABINOSE AND RHAMNOSE PROMOTER FUSIONS IN STUDIES OF THE ESCHERICHIA-COLI PHOSPHATE REGULON

Escherichia coli genes regulated by environmental inorganic phosphate (P-i) levels form the phosphate (Pho) regulon, This regulation require s seven proteins, whose synthesis is under autogenous control, includi ng response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the P-i-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 sy nthesis from their normal controls by placing each under the tight con trol of the arabinose-regulated P-araB promoter or the rhamnose-regula ted P-rhaB, promoter, To do this, we made allele replacement plasmids that may be generally useful for construction of P-araB or P-rhaB fusi ons and for recombination of them onto the E. coli chromosome at the a raCBAD or rhaRSBAD locus, respectively, Using strains carrying such si ngle-copy fusions, we showed that a P-rhaB fusion is more tightly regu lated than a P-araB fusion in that a P-rhaB-phoR(+) fusion but not a P -araB-phoR(+) fusion shows a null phenotype in the absence of its spec ific inducer, Yet in the absence of induction, both P-araB-phoB(+) and P-rhaB-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 a ction, We also used these fusions to study PhoU, Previously, we had co nstructed strains with precise Delta phoU mutations, However, we unexp ectedly found that such Delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol, 175:6797-6809, 1993). Th ey 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 P-araB-phoB(+), P-rhaB-phoB(+), or P-rhaB-pho R(+) fusions, we were able to overcome the extremely deleterious growt h defect of a Pst(+) Delta phoU mutant, The growth defect is apparentl y a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.