Statistical significance of these data was analyzed using anova with multiple comparisons. Escherichia coli cells were grown in MM9 as described earlier. At an OD600 nm of 0.6–0.8, 0.2% l-arabinose was added to the cells, and when the OD600 nm reached 0.9–10, 5 μM AgNO3 was added. Cell aliquots were taken 0.25, 2.25, and 4.25 h post silver stress, were Ganetespib normalized to 3 × 108 cells mL−1, and were frozen. RNA was extracted by resuspending 3 × 108 cells in 300 μL TRIZOL reagent, phase separated using chloroform, and total RNA was precipitated using isopropanol followed by centrifugation. The RNA pellet was resuspended in nuclease-free water
(Bioexpress). Quality and purity of RNA preparations were assessed by electrophoresis and via spectrophotometric determination of the ratio of absorbance at 260/280 nm. Total-RNA extracted from the previous step was treated with RNase-free DNaseI (Fermentas). First-strand cDNA was prepared from 2 μg of total RNA using the Superscript III cDNA synthesis kit (Quanta Biosciences). The cDNA was then diluted with SYBR green qPCR master mix. Reactions MDV3100 supplier were amplified using the Applied Biosystems 7300 Real-time PCR system. Each cDNA sample was assessed in triplicate using 16S-rRNA gene as an internal control. Thermal cycle conditions consisted
of an initial denaturation step at 95 °C for 60 s followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Fluorescence was measured at the beginning of each annealing/extension step. Amplicon size was also determined using electrophoresis on an agarose gel (1% w/v). The quantity of cDNA measured by real-time PCR was normalized to the abundance
of 16S cDNA. Primers used for qRT-PCR are listed in Table S1. To check the specificity of each primer, the predicted amplicon melting temperature was confirmed via dissociation curve analysis. Relative expression from the cusC gene was calculated using the ΔΔCt quantification method (Livak & Schmittgen, 2001), and values are averages of three independent experiments. Statistical significance of these data was analyzed using anova with multiple comparisons. The role of copper ions in bacterial growth ADP ribosylation factor and survival is well documented. Owing to the toxic nature of copper ions, bacteria such as E. coli and Salmonella have molecular systems that tightly control the copper concentration within the cells (Pontel & Soncini, 2009). In E. coli, the Cus system was first identified as a silver resistance system and was shown to be inducible by copper ions as well. Upon further investigation, it was revealed that the chemiosmotic CusCFBA system in E. coli confers anaerobic copper and silver resistance and is regulated by the CusR/CusS two-component system. The sensor kinase CusS and the response regulator CusR are activated by copper (Munson et al., 2000) and silver (Franke et al., 2001) ions, and these proteins are required for regulation of the cusCFBA operon. To define the role of CusS in copper resistance in E.
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