Ionic silver stock solutions were prepared from AgNO3 powder (Fisher Scientific Bioblock) as previously described (Saulou, Jamme et al., 2013) and stored in darkness. The final tested concentrations (5.0, 6.5, and 8.5 µM) were obtained by diluting the corresponding AgNO3 stock solution directly into the M9 growth medium. Control experiment was simultaneously performed by adding deionised water into the M9 broth. In both cases, a stabilization period of 24 h (at 37 °C and 120 rpm) was carried out before cell inoculation.
Growth protocol
The laboratory strain E. coli K12 MG1655 was grown for 24 h at 37 °C under continuous shaking (120 rpm), in 200 mL M9 medium (Merck Prolabo) complemented with 2.5 g/L of anhydrous glucose, and inoculated at 108 CFU/mL (corresponding to an optical density at 595 nm [OD595 nm] of 0.12). The inoculum for this culture was prepared in 40 mL M9 broth inoculated with approximately 1.5x107 CFU/mL (corresponding to an OD595 nm of 0.12) and incubated overnight under the same conditions (37 °C, 120 rpm). This inoculum was obtained by adding 1 mL of stock culture of the bacterial cells in 5 mL of Luria Bertani broth (Biokar Diagnotics, PRS Panreac) in a 15-mL half-opened BD FalconTM tube, followed by 8 h of incubation at 37 °C and 120 rpm.
Extracted molecule
total RNA
Extraction protocol
At 3 h of culture, 60 mL of culture (at OD595 nm = 0.73 ± 0.07, 0.63 ± 0.02, 0.12 ± 0.01 and 0.11 ± 0.02 for cultures subjected to 0, 5.0, 6.5 and 8.5 µM AgNO3, respectively) were sampled and centrifuged (4000 rpm, 5 min, 4 °C), then the pellet was frozen in liquid nitrogen. The pellet was resuspended with 1 mL of TE buffer (Tris-HCl 10 mM, pH 8, EDTA 1 mM, 1 mg lysozyme) and incubated 5 min at room temperature. Total RNA was extracted with a RNeasy midi kit (Qiagen), including the DNase I treatment described in the manufacturer’s instructions. Total RNA quantity and integrity were checked by Nanodrop® and Agilent BioAnalyzer, respectively.
Label
Cy3
Label protocol
According to manufacturer instructions, 1 µg of cDNA was labeled using one color DNA labeling kit
Hybridization protocol
According to manufacturer instructions, 2 µg of labeled cDNA were hybridized onto E. coli K-12 gene expression arrays (Nimblegen, Roche) during 17 h at 42 °C.
Scan protocol
Arrays were later washed and scanned using MS200 Microarray Scanner (Nimblegen, Roche). Pictures were analyzed with DEVA 1.2.1 software.
Description
This sample is the culture with 0 µM of AgNO3. It is the second of three biological replicates used in this experiment, each from separate cultures.
Data processing
Raw probe intensities ( .pair files three replicates for each growth condition with respectively 0, 5.0, 6.5 and 8.5 µM of AgNO3 in the medium) was processed and analyzed with R computing environment using the affy and limma package of Bioconductor. Raw data were submitted to a RMA-base background correction [Irizarry et al, 2003, Biostatistics 4(2): 249-264]. After background correction, intra-replicate quantil normalization was performed for each growth condition. A set of probes in the background for which the ranks were roughly invariant across all the twelve arrays was selected. The median value of the invariant probeset intensities on each array was used as a scaling factor for normalization between growth conditions. After normalization, the expression of a gene was calculated by a RMA-summarization procedure within each growth condition.
