Control of acid resistance in Escherichia coli

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:gamma-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 sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS 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 sigmaS-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.

FIG. 1

Suicide mutagenesis using pRR10-250v vector. (A) Plasmid pCF305 (internal fragment of gadC inserted into pRR10-250v) was integrated into the chromosome of strain EF393 by homologous recombination at the gadC gene. The positions of the different primers used either to generate internal fragments or to verify the final insertion are shown. The sequences of these oligonucleotides are given in Table 2. The integration site was verified by PCR with primer pairs 43 and 101 (gadC), which produced a fragment of the predicted size, 1.4 kb. (B) The procedure was the same as in panel A, except that plasmid pCF312 containing an internal fragment of gadA was used. The integration site was verified by PCR with primer pairs 43 and 121 (gadA), which produced a fragment of the predicted size, 1.2 kb. Insertions that amplify with oligonucleotides 43 and 121 were distinguished as being within gadA or gadB by using oligonucleotides 121 and 106. If the insertion was in gadA, a PCR product was obtained which represented normal gadBC. If the insertion was in gadB, no PCR product was formed. Heavy arrows indicate the direction of transcription.

FIG. 2

Genetic dissection of AR. Various mutants of E. coli K-12 were tested for the three AR systems. The test for the oxidative system (open bars) involved overnight growth in LB-MES (pH 5.5) followed by dilution to 1 × 10⁶ to 3 × 10⁶ CFU per ml in EG (pH 2.5). Survival (viable counts) was measured after 2 h at 37°C. The glutamate (shaded bars) and arginine (hatched bars) systems required overnight LBG (glutamate system) or BHIG (arginine system) cultures, which were diluted to 1 × 10⁶ to 3 × 10⁶ CFU/ml in EG (pH 2.5) containing 1.5 mM glutamate and 0.6 mM arginine, respectively. Percent survival is calculated as the number of CFU per milliliter remaining after the acid treatment divided by the initial CFU per milliliter at time zero. Initial cell densities ranged from 1 × 10⁶ to 3 × 10⁶ CFU/ml. Experiments were repeated two or three times. Variations were within 50% of the stated value.

FIG. 3

Western blot analysis of GadA versus GadB expression. (A) Exponential-phase cells (OD₆₀₀ = 0.5) were grown in EG (pH 7 or 5.5). GAD isoforms (A and/or B) present in each strain are indicated. (B) Exponential-phase (OD₆₀₀ = 0.4) and stationary-phase (18 h) cells were grown in LB-MOPS (pH 8). (C) Cells were grown to early stationary phase (OD₆₀₀ = 1.8) in unbuffered LB either with or without 0.4% glucose as a means of changing the pH. Final pH without glucose was approximately pH 7. The final pH with glucose was approximately pH 5. Equivalent amounts of protein were loaded in each lane and probed with anti-GAD antibody.

FIG. 4

Effect of glutamine on AR. (A) Cells (EK227 [hatched bars] and EF333 [gadC; shaded bars]) were grown overnight in either LB-MES (pH 5.5) or LB-MOPS (pH 8) as indicated and then diluted 1:1,000 into various pH 2.5 media, also as indicated. Survival was measured after 2 h. (B) EF333 (gadC) was grown overnight in LB-MES (pH 5.5) and diluted 1:1,000 into pH 2.5 EG containing various amino acids. Survival was measured at 2 h (hatched bars) and 4 h (shaded bars).

FIG. 5

Demonstration of an activator of oxidative AR in yeast extract. Cells were grown overnight (18 h) in the media and at the pHs indicated. Treatments included transferring cells to fresh LB (pH 8). In addition, Casamino Acids (CAA; 0.5%), yeast extract (YE; 0.5%), glutamate (Glt; 5.9 mM), or glutamine (Gln; 9.6 mM) was added directly to overnight cultures immediately prior to acid challenge (less than 1-min exposure). Acid challenge involved diluting cells to 1 × 10⁶ to 3 × 10⁶ CFU/ml in EG (pH 2.5). The results shown indicate survival for 2 h at pH 2.5. The gadC, crp, and rpoS mutants used were EF491, EF529, and EF362, respectively.

FIG. 6

Demonstration of an inhibitor of oxidative AR produced at pH 8. Cells were grown overnight (18 h) in the media and at the pHs indicated. Treatments included transferring cells to fresh EG (pH 7). In addition, yeast extract (YE; 0.5%) was added directly to overnight cultures immediately prior to acid challenge (less than 1-min exposure). Acid challenge involved diluting cells to 1 × 10⁶ to 3 × 10⁶ CFU/ml in EG (pH 2.5). Results shown indicate survival for 2 h at pH 2.5.

FIG. 7

Effect of gad mutations on AR at pH 2 in LB. Cells were grown to stationary phase in the media indicated (see the legend to Fig. 1). The final pH before acid challenge is also shown. Cells were diluted to approximately 10⁶ CFU/ml in acid challenge media. The results presented indicate survival after 1 h at pH 2 in LB.