The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

Abstract

Disruption of pstS encoding the P i -binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P i -controlled expression is restored when the activity of the P i transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.Growth of Escherichia coli under P i limitation results in the induction of the pho regulon, which includes phoA encoding alkaline phosphatase (18) and the pst operon encoding a P i transporter. This P i transporter consists of a periplasmic P ibinding protein (PstS), two integral membrane proteins (PstA and PstC), and an ATP-binding protein (PstB) (4, 16). Central to the regulation of the pho regulon is a two-component regulatory system encoded by the phoBR operon (21). In addition, the Pst system plays a role in P i regulation, since mutations in any of the genes of the pst operon generally lead to constitutive expression of the pho regulon (21-23). This constitutive expression is not due to decreased intracellular phosphate levels, which were reported to be maintained at a high level under high-P i conditions by a secondary P i transporter, PitA (11,24). Furthermore, the regulatory and transport roles of the Pst system could be uncoupled by specific amino acid substitutions in PstC or PstA (5, 6). Since periplasmic PstS binds P i with high affinity, it could potentially act as the primary sensor of external P i (20). The interaction of P i -loaded PstS with the membrane components of the Pst system might lead to a conformational change, which is sensed by the product of the fifth gene of the pst operon, PhoU. PhoU is not involved in P i transport (15), but probably forms the regulatory link between the P i transporter and the PhoBR system (20).To study the role of PstS in signal perception, we wished to isolate missense mutations in pstA or pstC that allow the Pst system to transport P i in the absence of PstS. In a previous study, such mutants were not obtained, but a third P i transporter, PitB, was discovered (9). In this study, we demonstrate that PitA or PitB activity can restore P i regulation of the pho regulon in the absence of PstS.Pseudorevertants in pitA restore P i regulation in a pstS mutant. To study the role of PstS in P i regulation of the pho regulon, we attempted to isolate mutants in pstA or pstC, allowing the Pst system to transport P i in the absence of PstS. Therefore, strain CE1491, which lacks all three known P i transporters (Table 1), was mutagenized with ethylmethane sulfonic acid, and mutants that could grow on minimal medium plates (9) with 660 M P i as the sole source of phosphate were selected. Emanating from the idea that restored P i transport via the Pst system might also restore P i control of the pho regulon, the mutants obtained were tested for expression of alkaline phosphatase on L broth plates containing the chromogenic substrate 5-bromo-

Molecular Mechanisms of Phosphate Homeostasis in <i>Escherichia coli</i>

“…It may mediate the formation of a signaling complex between the PstSCAB transporter and PhoR [5,64] or it may produce a soluble messenger that is recognized by the cytoplasmic domains of PhoR (consistent with observations reported by Hoffer and Tommassen [67] and by Rao and Torriani [68]). The following section presents new evidence in favor of the Pi-signaling complex model.…”

The PhoU Protein from Escherichia coli Interacts with PhoR, PstB, and Metals To Form a Phosphate-Signaling Complex at the Membrane

“…However, two general classes of models have been suggested: PhoU may mediate the formation of a signaling complex between the PstSCAB transporter and PhoR (2,27), or it may produce a soluble messenger that is recognized by the cytoplasmic domains of PhoR (consistent with observations reported by Hoffer and Tommassen [28] and by Rao et al [29]). No experimental evidence has yet been presented supporting physical interactions between signaling proteins.…”

Induction of the Pho Regulon Suppresses the Growth Defect of an Escherichia coli sgrS Mutant, Connecting Phosphate Metabolism to the Glucose-Phosphate Stress Response

“…PstSCAB is part of the phosphate (Pho) regulon activated by the two-component system PhoBR and induced during growth in phosphate starvation conditions (47,48,53,60,62,67). PstSCAB is also active in a pitA mutant, supporting growth on P i (18,21,48). Although E. coli possesses another low-affinity P i transporter, PitB, it does not support growth in a pst pitA mutant on an inorganic source of phosphate (18,20,48).…”