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 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...

Phosphate uptake by the phosphonate transport system PhnCDE

“…PHO-constitutive mutants can be isolated by plating wild-type bacteria on glycerol-2-phosphate (G2P) as the sole C source. We have previously observed that the frequency of such mutants in bacteria carrying both Δ pitA and Δ ugp knockouts is extremely low [13]. This is because as most PHO-constitutive mutations occur in one of the five genes of the pst operon, a Δ pitA pst strain would lack a functional Pi-uptake system and will thus not be able to grow with Pi as a phosphorus source.…”

“…Of the four tested transport systems, only Ugp was unable to support growth and take up Pi. In a previous study, we have shown that the emergence of PHO-constitutive mutants on plates containing glycerol-2-phosphate (G2P) as the sole carbon source partially depends on the presence of Ugp [13]. The hypotheses raised to explain this phenomenon were that Ugp was required for the uptake of either G2P or Pi.…”

Competition for nutritional resources masks the true frequency of bacterial mutants

“…Pst is encoded by the pstSCAB-phoU genes, or in short pst operon. Mutations in any one of the five pst operon genes result in the constitutive expression of the PHO regulon, i.e., maximal synthesis of all PHO proteins even under conditions of excess Pi [13,14]. Thus, the Pst system, besides being the principal Pi-uptake system in E. coli, also acts as a repressor of the PHO regulon [15][16][17].…”

“…The glycerol moieties released by AP activity thereby enter the cell mostly by facilitated diffusion via GlpF [ 18 , 19 ]. Thus, the constitutive expression of AP caused by the pst mutation enables E. coli to grow on G2P medium [ 14 , 20 ]. It should be added that some mutations in phoR also result in the constitutive expression of the PHO regulon and likewise confer the ability to grow on G2P.…”

“…It should be added that some mutations in phoR also result in the constitutive expression of the PHO regulon and likewise confer the ability to grow on G2P. However, all PHO-constitutive mutations (PCMs) derived from strain MG1655 hitherto analyzed were in the pst operon [ 14 ].…”