THE GLUTAMATE-BASED ACID RESISTANCE SYSTEM IN ESCHERICHIA COLI: CHARACTERIZATION OF THE TWO MAJOR STRUCTURAL COMPONENTS

The glutamate-based (gad) system is by far the most efficient acid resistance system in commensal and pathogenic enteric bacteria, like E. coli, L. monocytogenes and S. flexneri [1]. It ensures survival for more than a 2-h exposure to a strongly acidic environment (pH < 2.5), such as that of the stomach. The structural components of the system are the enzyme glutamate decarboxylase (Gad) and its cognate glutamate/-aminobutyrate (GABA) antiporter (GadC). The Gad reaction contributes to pH homeostasis by consuming intracellular H+ while GABA export, via GadC, contributes to the local alkalinization of the extracellular environment. The E. coli chromosome contains two distantly located genes, gadA and gadB, encoding biochemically undistinguishable isoforms of Gad. The gadC gene, located downstream of gadB, is co-transcribed with it [2]. E. coli GadA/B have an acidic pH optimum for activity, which dramatically drops above pH 5.0. Moreover, upon changing the pH from 5 to 6, the enzyme undergoes a pH-dependent conformational change involving the reversible uptake of protons upon acidification [3, 4]. We are currently carrying out a broad-scope biochemical characterization of the gad system. In particular our interest is focused on establishing how the the structural components of this system work in the cell. As a first step, we successfully crystallized and solved the GadB structure both at pH 7.6 (neutral-pH form) and at pH 4.6 (low-pH form) [5]. GadB is a hexameric protein (a trimer of dimers with P32 symmetry). Comparison of the low-pH and the neutral-pH forms shows that the overall structure of the hexamer remains the same, but very significant changes occur at the N- and C-termini and in a -hairpin region spanning residues 300-313. The N-terminal region of each of the six subunits, disordered at neutral pH, assumes a -helical conformation at low pH, forming two short three-helical bundles parallel to the three-fold axis of the hexamer. In the neutral-pH form, the C-terminus of each GadB subunit ends into the active site and blocks it. In the low-pH form, the C-terminus is disordered and the active site is freely accessible to the substrate. Upon exposure to strong acids, the E. coli cell membrane becomes leaky to H+[1]. We demonstrated that under these circumstances GadB, typically localized in the cytoplasm at neutral pH, is detected and assayed (>50 %) in the membrane fraction at acidic pH [5]. The structural basis for this behaviour resides in the N-terminal region of GadB. In the present work we provide structural and biochemical evidence for the existence of a binding site for anions, such as chloride, known to affect significantly GadB activity [3]. The physiological relevance of this binding will be discussed. As a second step of our program, we undertook the large-scale production and characterization of GadC. We purified GadC by affinity chromatography. The protein was purified from the membrane fraction with a yield of more than 1 mg per L of bacterial culture. Biophysical characterization of GadC will be presented.