Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

Abstract

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.

FIG. 1.

P_i transport as assessed by using P_i-sufficient MG1655 (▪), P_i-limited MG1655 (•), P_i-sufficient MT4 (▵), and P_i-limited MT4 (○) cultures. Cells were grown in MOPS medium containing either 2 mM P_i (P_i sufficient) or 0.1 mM P_i (P_i limited). OD, optical density at 600 nm.

FIG. 2.

Growth (A) and P_i uptake (B) of E. coli MG1655, MG1655(pBC29), MT4, and MT4(pBC29). Strains MG1655 (▪), MG1655(pBC29) (•), MT4 (▵), and MT4(pBC29) (○) were grown in MOPS medium containing 2 mM P_i at 28°C. OD₆₀₀, optical density at 600 nm.

FIG. 3.

PolyP granules in MT4(pBC29) detected by DAPI fluorescence. Strain MT4(pBC29) was grown overnight on 2×YT medium, stained with DAPI at a final concentration of 50 μg/ml, and observed with a fluorescence microscope (Olympus BX-40).

FIG. 4.

PolyP granules in Synechocystis sp. strain PCC6803 and phoU mutant SPU101 detected by DAPI fluorescence. Strains PCC6803 and SPU101 were grown in BG-11 medium at 30°C under light (5,000 lx) with 1% CO₂ for 3 days and stained with DAPI. Cells were observed with either visible light (A and B) or UV light (C and D).

FIG. 5.

Growth (A) and P_i uptake (B) of Synechocystis sp. strain PCC6803 and phoU mutant SPU101. Strains PCC6803 (•) and SPU101 (○) were grown in BG-11 medium at 30°C under light (5,000 lx) with 1% CO₂. OD₇₅₀, optical density at 750 nm.