Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium

Abstract

Cysteine and its derivatives, including H₂S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H₂S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H₂S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H₂S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Keywords: Fur and SOS regulons; H2S; antibiotics; bacterial survival; cysteine homeostasis; glutathione; growth; respiration.

Figure 1

Mutations in cysteine synthesis and degradation (cysK, cysM, cyuA, iscS, malY, metC, mstA, tnaA), cystine import and cysteine export (tcyP, eamA, eamB, bcr, cydD), glutathione synthesis (gshA), and regulatory proteins (cyuR and cysB) influenced the intensity and kinetics of sulfide generation in the medium (a–d) and the accumulation of H₂S in the gas phase (e,f). (a–d) Readings of the sulfide electrode from the moment of inoculation of bacteria into a fresh LB medium. Representative results from one of at least three independent experiments are presented. (e,f) Data were obtained using lead acetate-soaked paper strips in the OD₆₀₀ range from 0.3 to 1.2 and are shown as the means and standard error (vertical bars) for every 30 min (e) or for a total of 2 h (f) of incubation. Statistical differences compared to the wild-type strain (p < 0.05) are noted with asterisk.

Figure 2

Effect of 3 and 10 μg/mL ciprofloxacin on sulfide production in cultures of the studied E. coli mutants. Sulfide electrode readings for wt (a), gshA (b), tcyP (c), mstA (d), tnaA (e), cysK (f), eamA (g), cyuA (h), and cysB (i) strains are shown. The arrows indicate the moment of adding the antibiotic. Representative results from one of at least three independent experiments are presented.

Figure 2

Effect of 3 and 10 μg/mL ciprofloxacin on sulfide production in cultures of the studied E. coli mutants. Sulfide electrode readings for wt (a), gshA (b), tcyP (c), mstA (d), tnaA (e), cysK (f), eamA (g), cyuA (h), and cysB (i) strains are shown. The arrows indicate the moment of adding the antibiotic. Representative results from one of at least three independent experiments are presented.

Figure 3

Effect of mutations in cysteine homeostasis on changes in intracellular (a–c) and extracellular (d–f) glutathione during normal growth (a,d) and when E. coli is treated with 25 μg/mL chloramphenicol (b,e) or 10 μg/mL ciprofloxacin (c,f). The arrows indicate the moment of adding antibiotics (at OD₆₀₀ 0.4). Values are the means and standard error (vertical bars) from at least three independent experiments.

Figure 4

Changes in the concentration of cysteine in the cells of the studied mutants during normal growth in LB medium (a) and under the influence of ciprofloxacin (b–e) and chloramphenicol (d). (b,c) Exposure of wt and gshA strains to CF (0.3–10 μg/mL) and Cam (25 μg/mL). (d) Cysteine level in cells 90 min after Cam treatment. (e) Cysteine level in cells 30 min after treatment with 10 μg/mL CF. The arrows indicate the moment of adding antibiotics (at OD₆₀₀ 0.4). Means and standard errors (vertical bars) from at least three independent experiments are presented. Statistical differences compared to the wild-type strain (p < 0.05) are noted with asterisk.

Figure 5

Effect of the studied mutations on the expression of iucC::lacZ (a), katG::lacZ (b) and sulA::lacZ (c–f) during the normal growth of E. coli in LB medium (a–c) and when treating with CF (d–f). (d) Expression of sulA::lacZ upon exposure of the parental strain to ciprofloxacin (0.03–3 μg/mL). (e,f) Expression of sulA::lacZ when the studied mutants were treated with 0.3 μg/mL CF. The arrows indicate the moment of adding CF (at OD₆₀₀ 0.4). Means and standard errors (vertical bars) from at least three independent experiments are presented.

Figure 5

Effect of the studied mutations on the expression of iucC::lacZ (a), katG::lacZ (b) and sulA::lacZ (c–f) during the normal growth of E. coli in LB medium (a–c) and when treating with CF (d–f). (d) Expression of sulA::lacZ upon exposure of the parental strain to ciprofloxacin (0.03–3 μg/mL). (e,f) Expression of sulA::lacZ when the studied mutants were treated with 0.3 μg/mL CF. The arrows indicate the moment of adding CF (at OD₆₀₀ 0.4). Means and standard errors (vertical bars) from at least three independent experiments are presented.

Figure 6

Effect of the studied mutations on the specific growth rate (a,b), oxygen consumption (c–e), and pH (f) during normal growth (a,c,d,f) and exposure to CF (b,e). (a) Value of µ at OD₆₀₀ 0.4. (b) Changes in specific growth rate upon exposure of the parental strain to ciprofloxacin (0.03–3 μg/mL). (c) Changes in dO₂ indicates the presence of a transient process. (d) Rate of dO₂ decrease in cultures of the studied mutants. (e) Changes in dO₂ upon exposure of the parental strain to 3 and 10 μg/mL CF. (f) pH changes during growth of the studied mutants in LB medium. (a,b,d) Means and standard errors (vertical bars) from at least three independent experiments are presented. Statistical differences compared to the wild-type strain (p < 0.05) are noted with asterisk. (c,e,f) Representative results of one of at least three independent experiments are presented. The arrows indicate the moment of adding CF.

Figure 7

Effect of the studied mutations on the lethal activity of ciprofloxacin. (a,c,e) Killing curves. (b,d,f) Killing rate. (a,b) Kt strain exposed to 0.03–10 µg/mL CF. (c,d) Effect of 0.3 μg/mL CF on the studied mutants. (e,f) Effect of 3 μg/mL CF on the studied mutants. Means and standard errors (vertical bars) from at least three independent experiments are presented. The arrows indicate the moment of adding CF.

Figure 7

Effect of the studied mutations on the lethal activity of ciprofloxacin. (a,c,e) Killing curves. (b,d,f) Killing rate. (a,b) Kt strain exposed to 0.03–10 µg/mL CF. (c,d) Effect of 0.3 μg/mL CF on the studied mutants. (e,f) Effect of 3 μg/mL CF on the studied mutants. Means and standard errors (vertical bars) from at least three independent experiments are presented. The arrows indicate the moment of adding CF.