## 2 Maintaining Intracellular pH under Acidic Conditions  
Under acidic conditions, bacteria use a variety of metabolic and homeostatic mechanisms to help maintain the cytoplasmic pH (pH\({}_{\text{i}}\)) within a range that is consistent with growth and survival. Both _Listeria monocytogenes_ and _Escherichia coli_ rely on several decarboxylation systems to protect the cell from a precipitous drop in pH. These systems depend on the activity of cytoplasmic pyridoxal-5'-phosphate (PLP)-containing amino acid decarboxylases which consume one proton and release one CO\({}_{2}\) for every molecule of substrate amino acid, thus helping maintain the cytoplasmic pH [12]. With some exceptions, the reaction products are usually extruded from the cell through specific inner membrane antiporters in exchange for an extracellular amino acid. These decarboxylation systems include the glutamate decarboxylase (GAD) and the arginine decarboxylase (ADI) systems, which are present in both bacteria [13; 14; 15; 16; 17]. _E. coli_ also has two additional amino acid decarboxylation systems: the lysine decarboxylase CadA [18] and the ornithine decarboxylase SpeF [19]. _L. monocytogenes_ can catalyze two consecutive decarboxylation reactions to produce acetoin from pyruvate, consuming two protons in the process [20]. In _E. coli_, the GAD system is composed of two isoenzymes, namely GadA and GadB, and the glutamate/\(\gamma\)-aminobutyric acid (GABA) antiporter GadC [14] (Figure 1A). The _gad_ genes are induced in the stationary phase when cells are grown in rich media or during both growth phases when cells are grown in minimal media at pH 5.5 [21]. Decarboxylases have optimal enzymatic activities at acidic pH and their activities decrease sharply as pH increases. For GadA/B optimal pH is 3.7-3.8, indicating that these enzymes will be fully active under extreme acidic conditions [10]. Glutamine can also be imported into the cytoplasm by GadC and converted to glutamate by the amidohydrolase YbaS, with the release of an ammonia group. Free ammonia can also neutralize protons, thus Gln transport and YbaS also contribute to acid resistance in _E. coli_ through the GAD system [22]. In _L. monocytogenes_, the GAD system comprises three homologous glutamate decarboxylases, GadD1, GadD2, and GadD3 and two antiporters GadT1 and GadT2, encoded at three distinct genetic _loci_[15; 23] (Figure 1B). The _gadD1/T1_ operon is part of the stress survival islet 1 (SSI-1), a hypervariable region of the _L. monocytogenes_ genome that is absent in most serotype 4 _L. monocytogenes_ clinical strains [24]. The GadD1/GadT1 system seems to be active during mildly acid stress whereas GadD2/GadT2 plays a role in survival under severe acid pH [25; 26]. The _gadD3_ gene is upregulated during acid stress and is \(\sigma^{\text{B}}\)-dependent [23; 27; 28]. Moreover, the GadD3 system produces cytoplasmic GABA using the intracellular pool of glutamate, even in the absence of a functional GABA/Glu antiporter [23]. Figure 1: Cont. Figure 1.— Schematic representation of the sensory, protective, and regulatory mechanisms triggered by environmental low pH conditions in _E. coli_ (**A**) and _L. monocytogenes_ (**B**). Red arrows represent upregulated (+) or downregulated (–) gene expression modulated by either periplasmic or cytoplasmic acidic pH. Decarboxylation and deamination reactions (represented by blue arrows) consume a proton (H\({}^{+}\)) and produce CO\({}_{2}\), or produce ammonia (NH\({}_{3}\)), respectively. Ammonia can accept protons and yield ammonium (NH\({}_{4}\)+), thus contributing to pH homeostasis. (**A**) EvgS is thought to act as the periplasmic acidic pH sensor in _E. coli_ and is responsible for the initiation of a complex signal transduction pathway that activates GadE and ultimately results in the upregulation of acid tolerance mechanisms. (**B**) It is hypothesized that the stressosome acts as the cytoplasmic pH sensor in _L.