Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia coli

Abstract

When cystine is added to Escherichia coli, the bacterium becomes remarkably sensitive to hydrogen peroxide. This effect is due to enlarged intracellular pools of cysteine, which can drive Fenton chemistry. Genetic analysis linked the sensitivity to YdjN, a secondary transporter that along with the FliY-YecSC ABC system is responsible for cystine uptake. FliY-YecSC has a nanomolar Km and is essential for import of trace cystine, whereas YdjN has a micromolar Km and is the predominant importer when cystine is more abundant. Oddly, both systems are strongly induced by the CysB response to sulfur scarcity. The FliY-YecSC system can import a variety of biomolecules, including diaminopimelate; it is therefore vulnerable to competitive inhibition, presumably warranting YdjN induction under low-sulfur conditions. But the consequence is that if micromolar cystine then becomes available, the abundant YdjN massively overimports it, at >30 times the total sulfur demand of the cell. The imported cystine is rapidly reduced to cysteine in a glutathione-dependent process. This action avoids the hazard of disulfide stress, but it precludes feedback inhibition of YdjN by cystine. We conjecture that YdjN possesses no cysteine allosteric site because the isostructural amino acid serine might inappropriately bind in its place. Instead, the cell partially resolves the overaccumulation of cysteine by immediately excreting it, completing a futile import/reduction/export cycle that consumes a large amount of cellular energy. These unique, wasteful, and dangerous features of cystine metabolism are reproduced by other bacteria. We propose to rename ydjN as tcyP and fliY-yecSC as tcyJLN.

Importance: In general, intracellular metabolite pools are kept at steady, nontoxic levels by a sophisticated combination of transcriptional and allosteric controls. Surprisingly, in E. coli allosteric control is utterly absent from the primary importer of cystine. This flaw allows massive overimport of cystine, which causes acute vulnerability to oxidative stress and is remedied only by wasteful cysteine efflux. The lack of import control may be rationalized by the unusual properties of cysteine itself. This phenomenon justifies the existence of countervailing cysteine export systems, whose purpose is otherwise hard to understand. It also highlights an unexpected link between sulfur metabolism and oxidative damage. Although this investigation focused upon E. coli, experiments confirmed that similar phenomena occur in other species.

FIG 1

The addition of cystine immediately sensitizes sulfate-grown cells to H₂O₂. Exponentially growing E. coli strains were cultured for 5 generations in medium either containing sulfate alone or supplemented with cystine. They were then diluted into the same medium with or without cystine addition for 3 min, and then 2.5 mM H₂O₂ was added. At the indicated time points, the H₂O₂ exposure was terminated by the addition of catalase, and cell viability was determined by plating on LB medium (A) or defined medium (B). (A) Representative time courses. (B) Survival at 5-min time point, with standard deviations from three independent experiments. WT, wild type, AB1157; ΔgshA, JTG10; ΔydjN, KCI1137; ΔfliY, KCI1142; ΔydjN ΔfliY, KCI1160; ΔydjN ΔfliY + p(ydjN), KCI1281.

FIG 2

Cystine is imported by the FliY-YecSC and YdjN systems. Cultures were grown in sulfate or cystine medium. Chloramphenicol was added at 37°C for 5 min. [¹⁴C]cystine cocktail was then added, and intracellular radioactivity was measured at the indicated time points. The OD₆₀₀ of cells in the reaction mixtures was 0.2. (A and B) Representative time courses. (C) Import rates (counts per minute per OD unit per second) normalized to the rate for sulfate-grown wild-type cells, with error bars (standard errors of the means) from three independent experiments. WT, wild type, AB1157; ΔydjN, KCI1137; ΔfliY, KCI1142; ΔydjN ΔfliY, KCI1160.

FIG 3

E. coli requires either YdjN or FliY-YecSC to utilize cystine. Strains were inoculated into sulfur-free medium that was supplemented with sulfate, cystine, or no sulfur compound. Growth was monitored by optical density. The data are representative of more than three independent experiments. Open symbols, sulfate provided; closed symbols, cystine provided; ×, wild-type strain with no sulfur source. Circles, wild-type (wt) strain (AB1157); triangles, ΔfliY strain (KCI1142); squares, ΔydjN strain (KCI1133); diamonds, ΔfliY ΔydjN strain (KCI1160).

FIG 4

Genomic neighborhoods of genes encoding the cystine importers YdjN and FliY-YecSC. 5′ RACE was used to identify transcriptional start sites for ydjN, fliY, dcyD, and yecS in a wild-type strain (MG1655) grown in sulfate medium. Precise start sites are indicated in Fig. S1 in the supplemental material. Northern analyses indicate that ydjN and fliY transcripts are predominantly monocistronic. No end was found within 500 bases upstream of yecS; hence, transcription most likely is from the dcyD promoter. RT-PCR results suggest that a small amount of read-through occurs from fliY to the dcyD-yecS-yecC operon (see the text).

FIG 5

Regulation of ydjN transcription. (A) Northern blot prepared from the RNA of wild-type (MG1655) cells reveals a single 1.4-kb transcript that is visible from sulfate- but not cystine-grown cells. (B) Relative transcript levels in MG1655 analyzed by RT-PCR. (C) Expression of a ydjN′-lacZ transcriptional fusion that was incorporated at the lambda attachment site. The native ydjN allele remained intact. Strains contained either the wild-type cysB⁺ allele (KCI1230), a constitutively active cysB* allele (32) expressed from a plasmid (KCI1232), or a ΔcysB null allele (KCI1236). Medium contained cystine, sulfate, or djenkolate. (Djenkolate activates the CysB regulon but, unlike sulfate, supports growth of cysB null mutants.)

FIG 6

Regulation of fliY-dcyD-yecSC transcription. (A) Northern blot of fliY with mRNA prepared from wild-type (MG1655) cells reveals a single 0.8-kb transcript. (The asterisks mark nonspecific binding to rRNA.) (B) Relative transcript levels analyzed by RT-PCR. Yields are normalized to the same transcript in cystine medium. (C) Expression of fliY::lacZ and yecS::lacZ transcriptional fusions in their native loci. Strains were KCI1450, KCI1470, and KCI1452.

FIG 7

The rate-limiting step for import of micromolar cystine by FliY-YecSC is passage through the YecSC complex. Uptake of [¹⁴C]cystine was measured for wild-type (WT; AB1157), ΔydjN (KCI1137), and ΔydjN P_tet-yecSC (KCI1392) cells after growth in sulfate medium. Although fliY transcription is induced ∼7-fold when cells are grown in sulfate medium, the rate of cystine import remains as low as in cystine-grown cells unless yecSC transcription is elevated using a Tet-driven promoter (3 μg/ml tetracycline). (A) Representative time course. (B) Import rates (counts per minute per OD unit per second) from three independent experiments, with error bars (standard errors of the means).

FIG 8

Transport kinetics of the FliY-YecSC and YdjN systems. (A) Transport by YdjN. KCI1142 (ΔfliY) was suspended to an OD₆₀₀ of 0.5 in fresh sulfate medium and incubated with chloramphenicol for 5 min at RT. [¹⁴C]cystine was added, and at certain time points, samples were removed for counting. Data points represent initial linear rates; distinct symbols represent data from three independent experiments. Half-maximum transport occurred at ∼2 μM cystine. (B) Transport by FliY-YecSC. KCI1137 (ΔydjN) was suspended to an OD₆₀₀ of 0.2 in fresh sulfate medium and incubated with chloramphenicol for 5 min at 37°C. Prewarmed [¹⁴C]cystine was added, and at certain time points samples were removed for counting. Data points represent initial linear rates; distinct symbols represent data from four independent experiments. Half-maximum transport occurred at ∼30 nM cystine.

FIG 9

The FliY-YecSC system is essential for competitive growth at nanomolar cystine concentrations. Wild-type (KCI826) and ΔfliY (KCI1726) strains were mixed at equivalent titers and inoculated at very low cell density into sulfur-free medium supplemented with 0 to 40 nM or 40 μM cystine. Cell outgrowth was monitored by colony formation. The wild-type strain grew well (A), but the fliY mutant was able to grow only when 40 μM cystine was provided (B). The data are representative of three independent experiments.

FIG 10

Cystine import through YdjN drives rapid thiol excretion. (A) Thiol release was measured from sulfate-grown wild-type cells (dotted circles), cystine-grown cells (open circles), and sulfate-grown cells immediately after the addition of 0.1 mM cystine (filled circles). (B) Thiol release was measured upon the addition of 0.1 mM cystine to sulfate-grown cells. Strains were wild type (MG1655; circles), ΔfliY mutant (KCI1252; triangles), ΔydjN mutant (KCI1248), and ΔgshA mutant (KCI1220). (C) Mean thiol excretion rates after cystine addition, from three independent experiments with standard errors of the means. WT, wild type.

FIG 11

Glutathione is necessary for rapid cystine import. ΔfliY (KCI1142), ΔfliY ΔgshA (KCI1210), ΔydjN (KCI1254), and ΔydjN ΔgshA (KCI1516) strains grown in sulfate medium were suspended to an OD₆₀₀ of 0.2 in the same medium. The cells were incubated for 5 min with chloramphenicol at 37°C, and then [¹⁴C]cystine cocktail was added. Mean rates normalized to gshA⁺ strains, with error bars (standard errors of the means) from three independent experiments.

FIG 12

Cystine metabolism in Escherichia coli. Extracellular cystine enters the periplasm through porins and then is imported into the cytoplasm by either the YdjN or FliY-YecSC system. Cystine reduction to cysteine is glutathione dependent and probably occurs either during YdjN transport or subsequently in a glutaredoxin-dependent reaction. The intracellular pool of cysteine is ∼0.2 mM in sulfate-grown cells (4) but surges to millimolar levels when cystine becomes available. Excess cysteine is excreted through unidentified exporters. Ribosomal profiling studies suggest that the relative ratio of importer-protein synthesis in sulfate-grown cells is ∼8 YdjN per ∼24:1:1 FliY-YecSC (33). The larger flux is through YdjN when cystine is ample but through FliY-YecSC when it is scarce.