Employment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB ₂ ABC Transporter in Escherichia coli

Abstract

Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P i ). Under these conditions, the high-affinity PstSCAB 2 protein (i.e., with two PstB proteins) is the primary P i transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR twocomponent 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 P tac promoter and the lacO ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P phoB ::P tac and P pstS ::P tac strains to characterize phenotypes resulting from various ⌬phoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB 2 transporter, as well as its abundance within the cell. In addition, we used the P phoB ::P tac ⌬phoU strain as a platform to begin characterizing new phoU mutations in plasmids.

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

“…All previous studies that directly measured the rate of P i transport by PstSCAB in the presence and absence of PhoU detected little (ϳ20%) to no difference (8,9,20). However, those studies focused on cells grown under low-P i conditions.…”

“…Second, phoU mutants display poor growth, loss of viability, and genetic instability. This led to attempts at developing E. coli and C. crescentus strains to overcome these obstacles (8,20). However, those systems remained imperfect; the E. coli systems continued to display genetic instability in high-phosphate medium (20), while the C. crescentus system did not overcome the expression differences or the viability phenotype (8).…”

“…Yet there is little direct evidence that PhoU modulates the transport rate of PstSCAB. One study found a mere 20% increase in the rate of PstSCAB-mediated P i transport in an Escherichia coli ΔphoU mutant relative to the wild-type strain (20), and a separate study failed to detect a difference (7). Additionally, no difference in the rates of PstSCAB-mediated P i transport was detected in comparing wild-type strains and phoU mutants of Pseudomonas aeruginosa or Caulobacter crescentus (8,9).…”

RpoS proteolysis is controlled directly by ATP levels in Escherichia coli

“…1). The speed at which this degradation initiated raised the possibility that a small molecule or mechanical signal may be the cellular indicator of carbon source availability.Glucose and phosphate transport into the cell is monitored by the proteins GlcIIa and PhoU, respectively (Deutscher et al 2006;Rice et al 2009), and the slowing of phosphate transport upon phosphate limitation signals production of a small RNA that stimulates rpoS translation (Ruiz and Silhavy 2003). To test whether metabolite transport alone could trigger RpoS degradation, the glucose analog a-methylglucoside was added to starved cells.…”

Crystallization and preliminary X-ray characterization of a catalytic and ATP-binding domain of a putative PhoR histidine kinase from the γ-radioresistant bacteriumDeinococcus radiodurans

“…One of the first proposals was that it could interact with the PhoU protein from the phosphate-transport (Pst) system; however, the experimental data are not sufficient to support this hypothesis. It has been observed that the Pst system plays a role in this regulation, with PhoU being a negative regulator of the Pho system (Rice et al, 2009).…”