Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli

Abstract

The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine alpha-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.

FIG. 1.

Dependence on amino acids for survival. Wild-type E. coli stationary-phase cells were subjected to acid shock at pH 2.5 in the presence of a 1 mM concentration of the amino acid indicated by the single-letter code (with “O” representing ornithine). Bars and error bars indicate the means ± ranges of two to three measurements. CON, control experiment with no added amino acid.

FIG. 2.

Arrangement of XAR genes in E. coli. Positions of genes are indicated according to the work of Blattner et al. (2). The glutamate XAR system contains additional genes located elsewhere in the chromosome (21, 31).

FIG. 3.

Effect of yjdE deletion on XAR. E. coli (wild-type [WT] or ΔyjdE [Δ]) cells were assayed for XAR in the absence of any amino acids (control [CON]; open bars) or in the presence of 1 mM arginine (black bars), glutamate (grey bars), or lysine (cross-hatched bars). The rescue experiments were conducted with ΔyjdE cells that had been transformed with pRI-adiC1 and induced with the indicated IPTG concentration. For glutamate experiments, cells were grown in Luria-Bertani agar plus 0.4% glucose medium before acid shock. Each bar represents the mean ± standard error of results from 3 to 14 independent experiments.

FIG. 4.

Effect of yjdE deletion on arginine-agmatine exchange during acid shock. Time courses of agmatine release to the extracellular medium were monitored for wild-type cells (open symbols), ΔyjdE cells (black symbols), or ΔyjdE cells transformed with the pRI-adiC1 rescue vector (grey symbols). The transport reactions were initiated by the addition of 1 mM arginine to the shock medium. Initial rates were determined by least-squares fitting (solid lines) and are 0.26 ± 0.02 and 0.005 ± 0.008 nmol/min for wild-type and ΔyjdE cells, respectively. In rescue experiments, cells were either uninduced (triangles) or induced with 1 mM IPTG (circles), giving initial rates of 0.05 ± 0.04 and 0.141 ± 0.002 nmol/min, respectively. Each data point represents the mean ± standard error of results from three to six independent experiments.

FIG. 5.

pH dependence of arginine-agmatine exchange. The initial rate of agmatine release was measured as a function of pH in the acid shock medium, as described for Fig. 4. Points represent means ± standard errors of results from triplicate time courses, except for the pH 2.8 data, which show the range of results from duplicate experiments.

FIG. 6.

Concentration dependence of arginine XAR. Wild-type cells were assayed for survival efficiency or the arginine-agmatine exchange rate. (A) For survival data, the solid curve is calculated with the Hill equation, with a half-maximal concentration of 100 μM and a slope factor of 3.2. (B) Initial transport rate data follow a Michaelis-Menten curve, with a half-maximal concentration of 83 μM. These curves are purely empirical and have no theoretical connotations.

FIG. 7.

Genome organization of adiC homologues in several bacterial species. The adiC homologues are marked by cross-hatched arrows; sequence identity to E. coli adiC is as indicated. The putative regulatory protein AdiY is marked by short black arrow, and adiA arginine decarboxylase gene homologues are represented as unfilled arrows. The question marks for P. aeruginosa represent a membrane protein of unknown function. Agm, agmatine.