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

Gardner, Stewart G.; Johns, Kristine Dawn; Tanner, Rebecca; McCleary, William R.

doi:10.1128/jb.00029-14

Cited by 90 publications

(121 citation statements)

References 64 publications

Supporting

Mentioning

111

Contrasting

Unclassified

Order By: Relevance

“…It has been suggested that PhoU modulates the transport activity of PstSCAB; however, the current evidence for this is inconclusive. Two-hybrid data were consistent with a direct interaction between PhoU and PstB (19). Deletion of phoU resulted in increased P i and polyphosphate (polyPi) accumulation by six species, and this accumulation appeared to be PstSCAB dependent in at least three of these species (7)(8)(9)(10)(11)(12)).…”

supporting

confidence: 54%

“…Relative to the positive Zip-Zip control and negative PhoU-Zip and PhoR-Zip controls, we failed to detect a PhoU-PstB, PhoU-PhoB, or PstB-PhoR interaction but did observe a PhoR-PhoR interaction (Table 2) as expected for a histidine kinase (41). The S. meliloti PhoU protein was observed to interact with itself and with PhoR (Table 2) as was previously reported for the E. coli PhoU protein (19). The strength of all interactions, including that seen with the positive control, appeared lower in the P i -starved cells than in the P i -replete cells; however, the PhoU-PhoR interaction displayed a greater decrease in strength in the P i -starved cells that was statistically significantly different from the results seen with the other interactions ( Table 2).…”

Section: Resultsmentioning

confidence: 49%

“…It was hypothesized that PhoU detects the conformational state of PstSCAB and transmits this to PhoR (5). Two-hybrid data are consistent with a direct interaction between PhoU and PhoR (19,30). However, this model of regulation has not yet been experimentally tested.…”

mentioning

confidence: 73%

“…As in E. coli (19,30), the S. meliloti PhoU and PhoR proteins interacted based on the results determined with a two-hybrid screen (Table 2). This interaction appeared to be influenced by environmental P i concentrations (Table 2), perhaps suggesting that the PhoR kinase activity was induced by a disassociation of the PhoU-PhoR interaction.…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

et al. 2017

View full text Add to dashboard Cite

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphatefree media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Sitedirected mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import.IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.KEYWORDS Pho regulon, PhoU, PstSCAB, Sinorhizobium meliloti, environmental adaptation, modulation of TCS, phosphate homeostasis, phosphate sensing, phosphate transport, transport modulation

show abstract

supporting

confidence: 54%

Section: Resultsmentioning

confidence: 49%

mentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The mechanism by which the Pst transport system and PhoU repress PHO is still unclear. It has recently been shown that PhoR interacts with PstB and PhoU, suggesting that the external signal of P i availability is transduced through direct contact between these proteins (9).…”

mentioning

confidence: 99%

Ugp and PitA Participate in the Selection of PHO-Constitutive Mutants

et al. 2015

View full text Add to dashboard Cite

Mutations that cause the constitutive expression of the PHO regulon of Escherichia coli occur either in the pst operon or in the phoR gene, which encode, respectively, a high-affinity P i transport system and a histidine kinase sensor protein. These mutations are normally selected on glycerol-2-phosphate (G2P) as the carbon source in the presence of excess P i . The emergence of early PHO-constitutive mutants, which appear after growth for up to 48 h on selective medium, depends on the presence of phoA, which codes for a periplasmic alkaline phosphatase, while late mutants, which appear after 48 h, depend both on phoA and on the ugp operon, which encodes a glycerophosphodiester transport system. The emergence of the late mutants hints at an adaptive mutation process. PHO-constitutive phoR mutants appear only in a host that is mutated in pitA, which encodes an alternative P i transport system that does not belong to the PHO regulon. The conserved Thr 217 residue in the PhoR protein is essential for PHO repression. IMPORTANCEOne of the principal ways in which bacteria adapt to new nutrient sources is by acquiring mutations in key regulatory genes. The inability of E. coli to grow on G2P as a carbon source is used to select mutations that derepress the PHO regulon, a system of genes involved in the uptake of phosphorus-containing molecules. Mutations in the pst operon or in phoR result in the constitutive expression of the entire PHO regulon, including alkaline phosphatase, which hydrolyzes G2P. Here we demonstrate that the ugp operon, another member of the PHO regulon, is important for the selection of PHO-constitutive mutants under prolonged nutritional stress and that phoR mutations can be selected only in bacteria lacking pitA, which encodes a secondary P i transport system. The PHO regulon of Escherichia coli consists of more than 50 genes and operons that respond to orthophosphate (P i ) limitation (1, 2). The best-documented ones are the phoA and phoE genes, the pstSCAB-phoU (or pst) and ugpBAECQ operons, and the regulatory phoBR operon. phoA encodes a periplasmic alkaline phosphatase (AP), phoE encodes an anion-specific porin, and the first four genes of the pstSCAB-phoU operon code for an ABCtype high-affinity P i transport system which, together with phoU, also plays a role in the regulation of the PHO genes. Similar to the pst operon, the ugpBAEC genes encode an ABC transport system for sn-glycerol-3-phosphate and glycerophosphodiesters, while the fifth gene of the operon, ugpQ, encodes a phosphodiesterase that hydrolyzes glycerophosphoryl diesters (3, 4). The phoBR operon codes for the two-component system that controls the PHO regulon. Null mutations in phoB show a PHO-negative phenotype, while phoR mutations cause the constitutive expression of the PHO regulon (5).Instead of the Ϫ35 sequences, members of the PHO regulon carry in their promoters a sequence known as the PHO box (6). Under conditions of P i limitation, the histidine kinase PhoR autophosphorylates and transfers the P i moiety to t...

show abstract

Revisiting regulation of potassium homeostasis in Escherichia coli: the connection to phosphate limitation

et al. 2017

View full text Add to dashboard Cite

Two‐component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K+ homeostasis. In Escherichia coli, the histidine kinase KdpD senses K+ availability, whereas the response regulator KdpE activates synthesis of the high‐affinity K+ uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate‐responsive two‐component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two‐hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross‐regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K+ and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K+ and phosphate homeostasis and provides a mechanism for fine‐tuning of the balance between positively and negatively charged ions in the bacterial cell.

show abstract

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

Cited by 90 publications

References 64 publications

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

Ugp and PitA Participate in the Selection of PHO-Constitutive Mutants

Revisiting regulation of potassium homeostasis in Escherichia coli: the connection to phosphate limitation

Contact Info

Product

Resources

About