Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response

Abstract

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (Pi), in media deficient in both Pi and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during Pi starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.

FIG. 1

Complementation of a phoB mutant with PhoB restores polyP accumulation. Wild-type E. coli MG1655 (A), a corresponding phoB::Tn5 mutant, and the mutant complemented with a phoB plasmid (phoB*) (B) were grown in MOPS-buffered minimal medium containing 0.1 mM P_i (20 μCi of ³²P_i per ml) and 2 μg of each of the 20 amino acids per ml. Cultures (1 ml) were sampled for measurement of growth and polyP content (see Materials and Methods). Growth (OD) and polyP accumulation (PolyP) of the wild type (A) and of the phoB::Tn5 and plasmid-complemented phoB::Tn5 strains (B) are shown.

FIG. 2

PPK activity of the wild type and a phoB mutant. A phoB mutant (MG1655 phoB::Tn5) (B) and the corresponding wild-type strain (A) were grown in low-P_i (0.1 mM) MOPS-buffered minimal medium with 2 μg of each of the 20 amino acids per ml. Cultures (1 ml) were sampled periodically for measurement of growth and PPK activity.

FIG. 3

PolyP accumulation depends on amino acid (aa) concentration in the growth medium. E. coli MG1655 was grown in minimal medium containing 0.1 mM P_i (20 μCi of ³²P_i per ml) and the following concentrations of each of the 20 amino acids: 2 (A), 20 (B), or 0 (C) μg/ml. (D) Cells grown in minimal medium with excess P_i (2 mM). Growth and polyP content of the cells were measured as described in the legend to Fig. 1.

FIG. 4

Effect of amino acid supplementation on AP induction. E. coli MG1655 was grown in a P_i-limited (0.1 mM) minimal medium without (A and B) or with (C and D) the addition of 2 μg of each of the 20 amino acids per ml. Panels A and C highlight the initial (up to 4-h) growth period and corresponding AP activity of cells represented in panels B and D, respectively. Note the scales on the y-axes.

FIG. 5

Effects of ΔrelA on growth, polyP accumulation, and (p)ppGpp levels. The wild type and a corresponding relA mutant (MG1655 ΔrelA251) were grown in P_i-limited minimal medium containing 2 μg of each of the 20 amino acids per ml and analyzed for polyP accumulation (A and B), and ppGpp and pppGpp levels (C and D).

FIG. 6

Effects of phoB on polyP accumulation and (p)ppGpp levels. MG1655 wild-type and MG1655 phoB::Tn5 mutant strains were grown in minimal medium containing 0.4 mM P_i and 20 μg of amino acids per ml. At an OD₅₄₀ near 0.2, SHX was added to induce amino acid starvation. At the times indicated after the addition of SHX, samples were collected and analyzed for polyP (A and C) and (p)ppGpp (B and D).

FIG. 7

Effect of overexpression of relA on polyP accumulation and (p)ppGpp levels. E. coli MG1655 containing either pALS14 (IPTG-inducible control plasmid with inactive relA′) (A and C) or pALS13 (IPTG-inducible active relA′⁺) (B and D) was grown in a P_i-limited minimal medium containing 2 μg of each of the 20 amino acids per ml, as described in the legend to Fig. 5. At the times indicated, IPTG (1 mM) was added to the growing culture, which was examined for further growth (OD₅₄₀), polyP accumulation (A and B), and ppGpp and pppGpp levels (C and D).

FIG. 8

Hypothetical scheme for polyP accumulation and function. PolyP accumulation depends on PhoB and (p)ppGpp synthesized either by the relA (stringent-response) pathway or by the phosphate starvation-inducible spoT pathway. The inhibition by (p)ppGpp of exopolyphosphatase PPX as one means of polyP accumulation and the dependence of rpoS expression on polyP are mentioned in the text. Dashed arrows indicate either a direct or an indirect effect on the synthesis or accumulation of the gene products.