Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB

Abstract

Escherichia coli and other enterobacteria exploit the H þ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5 0 -phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H þ /Cl À exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Compensations for Diminished Terminal Oxidase Activity in Escherichia coli

“…Of considerable importance is the dramatic up-regulation (65-fold) of the GABA/glutamate antiporter GadC, which facilitates both utilization of glutamate as a carbon source and the generation of a pH gradient via uptake of glutamate and export of GABA. The reaction catalyzed by the GadB glutamate decarboxylase enzyme, encoded by the most dramatically up-regulated gene in cydB cells, is marked with a dashed line because this process is poorly catalyzed above pH 4.5 (41,42). These observations are consistent with the hypothesis that E. coli may uncouple catabolism from ATP synthesis by shutting down NADH synthesis, consuming NADH and ubiquinol electroneutrally, and translocating protons via GABA/glutamate antiport.…”

“…5). Under conditions of low internal pH, the highly up-regulated GadB glutamate decarboxylase will become active (41,42) and catalyze the synthesis of GABA, which can be used by GadC for the removal of protons from the cell. However, under the experimental conditions for the current microarray data, the internal pH of both cydB and wild type cells is slightly alkaline (supplemental Fig.…”

“…However, GadB is relatively inactive at neutral pH (41,42). To determine whether exponentially growing cydB cells have a favorable environment for GadB activity, the internal pH of wild type and cydB strains was measured.…”

Characterization of Glutamate Decarboxylase from a High γ-Aminobutyric Acid (GABA)-Producer,Lactobacillus paracasei

“…In LAB and E. coli, GABA production is among the most important mechanisms for achieving acid resistance. [26][27][28] Since Lb. paracasei increases acidity via growth, it has been suggested that it produces GABA in order to increase the pH of the growth medium.…”

Structural Insights into Inhibition of Bacillus anthracis Sporulation by a Novel Class of Non-heme Globin Sensor Domains

“…Thus, pXO1-118 crystallized in sodium chloride, whereas pXO2-61 required the presence of 1.0 M sodium iodide. Iodide is a strongly chaotropic agent that disfavors complex formation and increases the solubility of hydrophobic moieties (38), consistent with the expulsion of fatty acid from the protein. The higher pH required for crystal growth of pXO2-61 (pH 7.5 versus 5.4) may also disfavor binding (see below).…”