Inorganic polyphosphate and the induction of rpoS expression

Abstract

Inorganic polyphosphate [poly(P)] levels in Escherichia coli were reduced to barely detectable concentrations by expression of the plasmid-borne gene for a potent yeast exopolyphosphatase [poly(P)ase]. As a consequence, resistance to H2O2 was greatly diminished, particularly in katG (catalase HPI) mutants, implying a major role for the other catalase, the stationary-phase KatE (HPII), which is rpoS dependent. Resistance was restored to wild-type levels by complementation with plasmids expressing ppk, the gene for PPK [the polyphosphate kinase that generates poly(P)]. Induction of expression of both katE and rpoS (the stationary-phase sigma factor) was prevented in cells in which the poly(P)ase was overproduced. Inasmuch as this inhibition by poly(P)ase did not affect the levels of the stringent-response guanosine nucleotides (pppGpp and ppGpp) and in view of the capacity of additional rpoS expression to suppress the poly(P)ase inhibition of katE expression, a role is proposed for poly(P) in inducing the expression of rpoS.

Figure 1

(A) Starvation-induced poly(P) accumulation. KT1008 cells, harboring either the plasmid with the PPX1 gene (pTrcPPX1) (see text) or the control vector (pTrcHisB) were starved in nitrogen-free MOPS minimal medium (27). (B) Poly(P) levels reduced by poly(P)ase overproduction are restored by extra copies of the ppk gene. KT1008 cells harbored pairs of plasmids as follows: PPX1 gene (pLGPPX1) and ppk gene (pBC29) (▪); control vector for PPX1 (pLGHisB) and control vector for ppk (pUC18) (○); and control vector for PPX1 (pLGHisB) and ppk gene (pBC29) (□). Poly(P) values for cells harboring both the PPX1 gene (pLG PPX1) and the control vector for ppk (pUC18) (•) were below detectable levels (0.1 nmol/mg of protein).

Figure 2

Effects of poly(P)ase overproduction on H₂O₂ sensitivity in catalase-deficient strains. The wild-type strain (CSH7) in A, or the katG mutant (NY001) in B, or the katE mutant (UM178) in C, harbored either the plasmid with the PPX1 gene (pTrcPPX1) (•) or the control vector (pTrcHisB) (○). Cells were exposed to 42 mM H₂O₂ at 25°C; viable cell numbers were determined by plating onto LB agar.

Figure 3

Expression of the katE–lacZ operon fusion monitored by β-galactosidase activity. KT1008EL cells harboring either plasmids with the PPX1 gene (pTrcPPX1) or the control vector (pTrcHisB) were starved in a M9 minimal medium (25). β-Galactosidase activity was measured in Miller units (28).

Figure 4

Visualization of catalase activities (HPI and HPII). Electrophoresis of extracts of wild-type (WT) and CA10 (ppk⁻) cells grown to exponential or stationary phase was performed on an 8.5% acrylamide nondenaturing gel; extracts of stationary-phase CA10 cells complemented with plasmids bearing ppk (pBC29) or rpoS (pMMkatF3) were also tested.

Figure 5

Levels of RpoS (σ³⁸) in KT1008EL cells harboring plasmids with either the PPX1 gene or the control vector during starvation. The plasmids used were pTrcPPX1 or pTrcHisB. Proteins were subjected to Western blotting with anti-σ³⁸ antiserum and visualized with an alkaline phosphatase-conjugated second antibody.

Figure 6

Expression by the rpoS–lacZ operon fusion monitored by β-galactosidase activity (Miller units). KT1008SL cells harbored either plasmids with the PPX1 gene (pTrcPPX1) or the control vector (pTrcHisB). (A) Induction of rpoS transcription was monitored after starvation in an M9 minimal medium (25). (B) Expression of rpoS-lacZ fusion was tested during growth in LB.