Ugp and PitA participate in the selection of PHO-constitutive mutants

Abstract

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 Pi 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 Pi. 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 Pi transport system that does not belong to the PHO regulon. The conserved Thr(217) residue in the PhoR protein is essential for PHO repression.

Importance: One 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 Pi transport system.

FIG 1

Growth of ΔphoA and Δugp mutants on TG2P plates. Bacteria were streaked on TG2P (minimal medium with G2P as the sole carbon source supplemented with the AP substrate X-P) and grown for 48 h at 37°C. (A) MG1655; (B) ΔphoA::Cm; (C) Δpst::Km; (D) ΔphoA::Cm Δpst::Km; (E) MG1655; (F) Δugp::Cm; (G) Δpst::Km; (H) Δugp::Cm Δpst::Km.

FIG 2

Selection of PHO-constitutive mutants on TG2P. Bacteria were seeded on TG2P plates and incubated for 14 days at 37°C. The appearance of colonies was monitored daily. The data points were calculated by dividing the number of colonies observed on each day by the effective number of CFU seeded on each plate (about 10⁹). The data represent the mean ± SEM from 20 independent experiments.

FIG 3

Selection of PHO mutants in a pitA background. Bacteria were seeded on TG2P plates as described in the legend to Fig. 2. The data represent the mean ± SEM from 8 independent experiments.

FIG 4

AP activity of the various PHO-constitutive mutants. Bacteria grown overnight in medium A without P_i (−) and medium A with P_i (+) were assayed for AP activity. K10, wild-type K10; phoR69 (strain C3), K10 high-AP phoR69 spontaneous mutant; phoR129 (strain JV1) and phoR130 (strain JV2), K10 low-AP phoR nonsense mutants; pst (strain JV6), K10 pst spontaneous mutant; MG1655, wild-type MG1655; pitA::Km, MG1655 pitA::Km (strain EG2); phoR217 (strain RI65), MG1655 high-AP phoR spontaneous mutant; pst (strain RI81), MG1655 pitA::Km pst spontaneous mutant; phoR129 (strain TP01), MG1655 into which the phoR129 mutation was transduced. Each bar represents the mean ± SEM from 3 independent experiments.