Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential

Abstract

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.

FIG. 1.

Clc Cl⁻ transporters and acid resistance. (A) Cells (EK592 and EK590) were grown to stationary phase in LB MOPS (pH 8.0) and LB MES (pH 5.5) and diluted 1:1,000 into EG pH 2.5 without exogenous amino acids to test AR1. (B) Cells were grown in LBG to stationary phase and diluted 1:1,000 into EG pH 2.5 medium with and without 1.6 mM sodium glutamate to test AR2.

FIG. 2.

The F0/F1 proton-translocating ATPase is required by AR1. (A) Cells (EK227 and EF996) were grown in LB MOPS (pH 8.0) or LB MES (pH 5.5) to stationary phase and diluted 1:1,000 into EG pH 2.5 medium without exogenous amino acids (pH 5.5 induces AR1 function). (B) The same cells were grown in LBG to repress AR1 and diluted 1:1,000 into EG pH 2.5 medium, with or without 1.6 mM sodium glutamate. (C) Cells were grown in BHIG to repress AR1 and diluted 1:1,000 into EG pH 2.5 medium with or without 1 mM l-arginine. *, below the level of detection.

FIG. 3.

GadC is the antiporter for glutamate-dependent acid resistance. (A) Western blot assay. Cells were grown to stationary phase in minimal EG medium, and extracts were prepared with a French press. Crude extracts were separated into soluble and insoluble membrane pellets by ultracentrifugation. Samples of each fraction were electrophoresed through a 10% polyacrylamide gel electrophoresis gel and probed with anti-GadC antibody. (B) GadC is required for intact cells to convert glutamic acid to GABA. Cells were grown in LBG to stationary phase, washed in minimal EG pH 7.0 medium, and resuspended in EG at different pH values. Radiolabeled glutamic acid was added to suspensions of intact cells placed at pH 2.5 and to suspensions of cells permeabilized with 0.1% Triton X at pH 4.4, the reported internal pH of acid-challenged cells. Substrate (striped bars) and product (solid bars) present in filtered supernatants were separated by paper chromatography and identified by staining with 0.3% ninhydrin. WT, EK227; gadC, EF962; gadAB, EF522.

FIG. 4.

Intracellular pH optima of glutamate and arginine decarboxylases correlate to the internal pH of acid-stressed cells. EK227 cells were grown to stationary phase in LBG. Conversions of glutamate and arginine to GABA and agmatine, respectively, were carried out essentially as described in the legend for Fig. 3. Cells, either intact or Triton X solubilized, were resuspended at different pH values, and radiolabeled substrate was added.

FIG. 5.

A specific internal pH is not the only requirement for acid stress survival. Cells (EK227) were grown in LBG to stationary phase. A 1-ml aliquot of the culture was harvested and resuspended in 200 μl of EG pH 2.3 medium with 40 mM sodium glutamate or pH 2.7 medium without exogenous glutamate. (A) Internal pH measurements were made at 0, 30, and 60 min after acid challenge. (B) ΔpH (inset) and survival were measured in the presence (hatched bars) and absence (solid bars) of glutamate. ΔpH was calculated by subtracting the internal pH from the external pH.

FIG. 6.

Model for amino acid-dependent acid resistance. Acid stress at pH 2.5 results in illicit entry of H⁺, which decreases pH and increases positive charge. As pH_i drops to around 5, arginine decarboxylase will start to consume protons and convert +1-charged arginine to +2-charged agmatine, further increasing the positive charge. An antiporter will not completely drain agmatine from the cell, as it is continually being made during decarboxylation. In this model, the evolution of CO₂ does not contribute toward internal pH or charge since (i) the proton donor to make carbonic acid is water, not a proton; (ii) carbonic anhydrase will not function at pH 4.5; and (iii) at this internal pH bicarbonate will not form (pK_a = 6.1). The role of the Clc H⁺:Cl⁻ antiporter is unknown but may help expel H⁺, limit excess internal positive charge, and aid in returning the cell to an inside negative charge as external pH returns to neutrality.