Effects of copper-phenanthroline on pentachlorophenol-induced adaptation and cell death of Escherichia coli

Abstract

Objective: To evaluate the effects of copper-phenanthroline (CuOP) on pentachlorophenol (PCP)-induced adaptation and cell death of Escherichia coli.

Methods: Bacterial growth and adaptation to PCP were monitored spectrophotometrically at 600 nm. Inactivation of bacterial cells was determined from colony count on agar dishes. Cellular ATP content and accumulation of PCP were assessed by chemiluminescence and HPLC analysis respectively. The formation of PCP-Cu-OP complex was shown by UV-visible spectra.

Results: Escherichia coli (E. coli) could adapt to PCP, a wood preservative and insecticide used in agriculture. The adaptation of E. coli to PCP prevented its death to the synergistic cytotoxicity of CuOP plus PCP and declined cellular accumulation and uncoupling of oxidative phosphorylation of PCP. Furthermore, CuOP and PCP neither produced reactive oxygen species (ROS) nor had a synergistic effect on uncoupling of oxidative phosphorylation in E. coli. The synergistic cytotoxicity of CuOP and PCP in E. coli might be due to the formation of lipophilic PCP-Cu-OP complex.

Conclusion: Our data suggested that adaptation of E. coli to PCP decreased the synergistic effects of CuOP and PCP on prokaryotic cell death due to the formation of lipophilic PCP-Cu-OP complex, but it had no effect on the uncoupling of oxidative phosphorylation and production of reactive oxygen species in E. coli.

FIG. 1

Adaptation of E. coli to PCP. A: uninduced cells treated with or without 0.4 mmoL/L PCP; B: PCP-induced cells. After growth for 3 hours in minimal medium with 0.2 mmoL/L PCP, E. coli cells were washed three times with minimal medium and grown in the same medium containing different concentrations of PCP. E. coli growth was monitored spectrophotometerically at 600 nm every 30 min.

FIG. 2

Effects of adaptation on cytotoxicity of CuOP and/or PCP. All the reaction mixtures contained 4.8×10⁶ E. coli cells/mL, glucose (0.5% w/v), and MgSO₄ (1 mmol/L) in HEPES buffer (10 mmol/L, pH7.4). A: Survival rate of uninduced or PCP-induced E. coli treated with 1.4 mmol/L PCP. B: Survival rate of E. coli treated with 1.5 µmol/L CuOP plus 0.3 mmol/L PCP. *P<0.01 (Student’s t-test), uninduced vs. PCP-induced cells.

FIG. 3

Effects of adaptation on PCP- or CuOP-declined cellular ATP contents. A: The incubation mixture contained 3×10⁹ E. coli cells/mL of uninduced or PCP-induced cells in phosphate buffer (1 mmol/L, pH7.4), glucose (0.5%) and MgSO₄ (1 mmol/L), treated with 0.4 mmol/L PCP. The ATP levels in PCP-induced cells was significantly different from that in uninduced cells, *P<0.05, **P <0.01 (Student’s t-test). B: 3×10⁹ E. coli cells/mL were treated for 30 minutes with 100 µmol/L, 15 µmol/L or 1.5 µmol/L CuOP. There were no significant differences between uninduced and PCP-induced cells in the effects of CuOP on ATP contents. Cellular ATP was determined by chemiluminescence method and expressed as the percent of initial light intensity value.

FIG. 4

Effects of CuOP and/or PCP on cellular ATP levels. Uninduced E. coli B cells were exposed to different concentrations of CuOP and/or PCP for 30 minutes. Cellular ATP levels were measured as described in Materials and Methods. CuOP had no synergetic effect on the decline of cellular ATP content by PCP.