J Bacteriol 181(11): 3525-3535

Control of Acid Resistance in <em>Escherichia coli</em>

Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688,^{and Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2}²

^{Corresponding author. Mailing address: Department of Microbiology and Immunology, University of South Alabama College of Medicine, Medical Sciences Building, Mobile, AL 36688. Phone: (334) 460-6323. Fax: (334) 460-7931. E-mail:}ude.lahtuosu.gcgnus@jretsof.

^{Present address: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105.}

^{Present address: Vaccine Design Group, Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6.}

Received 1999 Feb 3; Accepted 1999 Mar 26.

Abstract

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor ς^{encoded by}rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ς^{independent. Neither}gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ς^{-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus,}E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.

Abstract

Acid resistance (AR) is perceived to be an important property of Escherichia coli, enabling the organism to survive gastric acidity and volatile fatty acids produced as a result of fermentation in the intestine (8, 9, 12). The ability to resist these acid stresses is believed to be necessary for this organism to colonize and establish a commensal relationship with mammalian hosts. In addition, the low infectious dose associated with enterohemorrhagic E. coli serotype O157:H7 is attributed to its acid-resistant nature (1–4, 18).

Under fasting conditions, the median stomach pH of healthy volunteers is around 2.0 (27). Detailed studies of AR mechanisms in E. coli have exposed three systems that can protect cells against pH 2 to 2.5 (11, 13, 14). The first is a glucose-repressed system induced in Luria-Bertani broth (LB) that is dependent on the alternative sigma factor ς^{, encoded by the gene}rpoS. The other two clearly defined systems are induced following growth in LB containing 0.4% glucose (LBG) or brain heart infusion broth (BHI) containing 0.4% glucose (BHIG). One system requires glutamic acid during acid challenge to survive pH 2 and is thought to utilize an inducible glutamate decarboxylase, and the other requires arginine and an inducible arginine decarboxylase encoded by adiA. All three systems were identified in stationary-phase cells. How the oxidative system protects cells against acid stress is unknown. However, the two decarboxylase systems are believed to consume protons during the decarboxylation of glutamate or arginine. The end products, γ-aminobutyric acid (GABA, formed from glutamate decarboxylase [GAD]) and agmatine (formed from arginine decarboxylase), are then transported out of the cell in exchange for new substrate. This transport process is catalyzed by specific antiporter systems, GadC for glutamate and an unknown antiporter for arginine. The result is that protons leaking into the cell during acid stress are consumed and excreted from the cell, thereby preventing the internal pH from decreasing to lethal levels. While this appears to be a simple strategy, it is now clear that inducible amino acid-dependent AR requires more than a decarboxylase and an antiporter.

The GAD system encompasses three genes. Two of these genes, gadA and gadB, encode highly homologous glutamate decarboxylase isoforms (25). The third gene, gadC, encodes a putative glutamate:GABA antiporter. The gadB and gadC genes form what appears to be an operon in which gadB is the first gene. In this study, we examined whether one or both decarboxylase genes are expressed, how each is regulated in response to pH and growth conditions, and whether both isoforms contribute to AR. We also discovered that cyclic AMP (cAMP) receptor protein (CRP) and cAMP are required specifically for the oxidative glucose-repressed system, analyzed the apparent acid induction of this system, and performed experiments to determine if there is a fourth system of AR.

ACKNOWLEDGMENTS

We thank Alexey Atrazhev for help in purifying GAD, as well as M. Spector, M. Moreno, and S. Price for many helpful discussions. Technical assistance by Neelam Ahmad is also gratefully acknowledged.

This work was supported by grant 97-35201-4751 from the U.S. Department of Agriculture.

ACKNOWLEDGMENTS

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