Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential

Enteric organisms that colonize and cause disease in the human intestine must first endure a transient but extreme acid challenge in the stomach. The normal human stomach presents an antimicrobial acid environment averaging pH 2, with an emptying time of approximately 2 h (53). As a result, acid-sensitive pathogens like Vibrio cholerae must be ingested in massive numbers (10 to 100 million) to increase the possibility that some will survive and enter the intestine. Other microbes, such as Escherichia coli and Shigella, can colonize or cause disease even when small numbers of cells (10 to 100) are ingested. These resilient microbes are equipped with potent acid resistance systems able to withstand pH 2 challenge for at least 2 h (11, 31, 32, 52). In fact, E. coli possesses a level of acid resistance rivaling that of the gastric pathogen Helicobacter pylori (39, 45, 50, 59). It has now been shown that E. coli uses four inducible acid resistance systems to survive extreme acid environments. Acid resistance system 1 (AR1), also referred to as the oxidative or glucose-repressed system, is acid induced in stationary phase. Its expression requires the alternative sigma factor RpoS and the cyclic AMP receptor protein CRP (11). However, the structural components of AR1 as well as the mechanism by which it protects are still unknown. The second AR system, AR2, requires extracellular glutamate to work at pH 2.0 and is induced upon entry into stationary phase or by log-phase growth in acidic minimal medium (10). Known components of glutamate-dependent acid resistance include two isoforms of glutamate decarboxylase (GadA and GadB) and a putative glutamate:γ-aminobutyric acid (GABA) antiporter called GadC (11, 12, 19, 33, 46). The third system, AR3, is similar to AR2 except that AR3 only protects cells if extracellular arginine is present. AR3 is induced by low pH under anaerobic conditions and has only been demonstrated following growth in complex media. This arginine-dependent system is composed of the acid-inducible arginine decarboxylase AdiA and the AdiC antiporter, which exchanges extracellular arginine for the intracellular end product of decarboxylation, agmatine (11, 15, 22, 31). The last AR system was recently described as lysine dependent and probably involves the inducible lysine decarboxylase (22). Although it is clear that these systems protect E. coli during transient exposure to pH 2, how they actually function has been subject to speculation. It is believed that AR2 and AR3 protect the cell from acid stress by consuming intracellular protons during each decarboxylation reaction (11, 14). The siphoning off of intracellular protons was proposed to enhance pH homeostasis and allow the cell to maintain an internal pH compatible with viability. This model suggests that a specific internal pH may be crucial for survival during exposure to extreme acid stress. If the cell's internal pH fell below that point, it would succumb. The data obtained in the present study indicate that maintenance of a specific internal pH may not be paramount to cell survival. Survival may depend on E. coli taking an approach used by acidophiles, which is to reverse the electrical membrane potential (ΔΨ) in the presence of extreme low pH. Converting membrane potential from negative inside to positive inside can repel protons and mitigate the excess proton motive force (PMF) that can form.