For gene complementation assays, all strains were transformed with the pBAD24 (Guzman et al., 1995) vector containing the necessary gene. The plasmids and oligonucleotide sequences used in this study are listed Cyclopamine chemical structure in Supporting Information, Table S1 and were obtained from Integrated DNA Technologies. For the construction of pBADcusS plasmid, the pBADcusS-F and the pBADcusS-R primers were used to amplify the cusS gene from E. coli W3110 genomic DNA. The PCR product was digested with HindIII/EcoRI restriction enzymes and ligated into the HindIII/EcoRI sites
in vector pBAD24. All plasmids were purified and sequenced for accuracy. Overnight cultures were grown aerobically in MLB to an OD600 nm of 2.05–2.10 and then diluted 1 : 200 into MM9 medium containing 100 μg mL−1 ampicillin. Growth was continued until the OD reached 0.6–0.8, and then, the cells were induced with 0.2% arabinose. To study the effect of increasing copper on growth of wild-type and mutant E. coli BW25113 strains in liquid medium, selleck chemicals llc 30 min
after induction with arabinose, the cultures were diluted again 1 : 200 in MM9 medium containing various concentrations of CuSO4 and incubated at 37 °C under anaerobic conditions. Cell growth was measured after 15 h, and cell densities were normalized before plotting as a function of copper concentrations. To study the effect of AgNO3, wild-type and mutant E. coli BW25113 strains were grown as mentioned earlier. The cell density was allowed to reach OD600 nm 0.9–1.0. The cultures were diluted in sterile phosphate-buffered saline of pH 7.4 (PBS) 1 : 200 and spotted on MM9 agar plates containing different concentrations of AgNO3. The plates were incubated aerobically at 37 °C for 20 h Celastrol in the dark. The MIC values were determined as the minimum concentration of AgNO3 at which no growth was observed. To determine the metal accumulation in cells, wild-type
E. coli, E. coli ΔcusS, and E. coli ΔcusS/pBADcusS were grown as described earlier. After induction of genes on the pBAD24 vectors with L-arabinose, 7.5 μM CuSO4 was added to the medium, and cell aliquots were taken at 0, 2, and 4 h after addition of copper. All cultures were normalized to 3 × 108 cells mL−1 and centrifuged to obtain the cell pellet. The pellets were washed three times with MM9 containing 1 mM EDTA and dried at 75 °C for 3 h. 50 μL nitric acid (10% v/v) was added to the pellet, and the samples were incubated at 75 °C for 30 min. The copper concentrations in the sample were measured using inductively coupled plasma mass spectrometry (ICP-MS) on a Elan DRC II instrument (Perkin Elmer). The instrument was initially monitored for background noise and metal contaminants and then calibrated using an ICP multielement stock solution (AccuStandard) prepared in 1% nitric acid. Samples were also diluted with 1% nitric acid until the signal was within the calibration range. All glassware used for this experiment was washed with 10% nitric acid.