For microarray analysis, when the cells were in mid-log phase (~95 h), eight volumes (400 ml) of bacterial culture were condensed to one volume (50 ml) by centrifugation (10, 000×g) for 10 min at 4°C in a 5804R centrifuge (Eppendorf, Wesbury, N.Y.) and re-suspended with sterilized fresh medium when the cells were in mid-log phase. Samples (cellular density is about 5.86×108 cells/ml) were collected from three independent experiments grown under 0% (non stress condition) and 1% (stress condition) Lix984n for 0 (control), 5, 20, 40, 80 min respectively, and centrifuged at 10, 000×g for 10 min at 4°C in a 5804R centrifuge (Eppendorf, Wesbury, N.Y.). The resulting twenty-seven pellets were frozen immediately in liquid nitrogen and stored at -80°C before RNA extraction.
Growth protocol
Acidithiobacillus ferrooxidans ATCC 23270 obtained from American Type Culture Collection (ATCC) was cultivated at 30°C in medium containing 0.5 g/L MgSO4•7H2O, 3.0 g/L (NH4)2SO4, 0.5 g/L K2HPO4, 0.1 g/L KCl and 0.01g/L Ca(NO3)2. The medium (pH =2.0) was sterilized at 120°C for 20 min and added elemental sulfur to 10.0 g/L.
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA from A. ferrooxidans grown in the presence and absence of Lix984n was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. RNA samples were treated with RNase-free DNaseI (QIAGEN, Carlsbad, USA) to digest residual chromosomal DNA and then purified with an RNeasy kit (Qiagen, Valencia, USA). Total cellular RNA was quantified at OD260 and OD280 with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Three replicates of purified RNA for each sample mixed and the mixed RNA served as the template for labeling.
Label
Cy3
Label protocol
RNA samples from A. ferrooxidans in non stress and stress conditions were labeled with Cy3-labeled dUTP during cDNA production. 10μg of total cellular RNA was mixed with 10μg of random primer at 70°C for 10 min, then immediately chilled by ice for 2 min. The labeling reaction mixture, which contains 5×reverse transcription buffer; 5 mM MgCl2; 5 mM each of dATP, dTTP, and dGTP; 2.5 mM dCTP (New England Biolabs, Beverly, Mass); 1 mM Cy3 dUTP (Amersham Pharmacia Biotech, Piscataway, USA); and 40 units of RNAase OUT (Invitrogen), was added to the RNA primer solution and then incubated at 25°C for 5 min. Superscript II reverse transcriptase (200 units, Invitrogen) was added to the solution and the mixture was incubated at 25°C for 10 min and then 42°C for 2 h. The labeled cDNA probes was treated with 1 mM NaOH to remove residual RNA, and purified with a Qiagen PCR purification column, then concentrated in speedvac at 45°C for 30 min.
Hybridization protocol
The purified and fluorescently labeled probes were dissolved in 30μl of hybridization buffer containing 50% (v/v) formamide, 3×SSC (containing 0.45 mM NaCl and 0.045 mM sodium citrate), 20μg of unlabeled herring sperm DNA (Promega, USA), and 0.3% (v/v) sodium dodecyl sulfate (SDS). The hybridization mixture was heated at 98°C for 2 min, applied directly onto the microarray slide, then covered with a glass coverslip. After that the microarray was placed into a hybridization chamber (Corning, USA), and immersed into the preheated water bath (45°C) immediately for 10-12 h. After hybridization, each microarray slide was taken out, and washed sequentially in 1×SSC-0.2%SDS for 5 min, 0.1×SSC-0.2% SDS for 10 min and 0.1×SSC for 30 s. Drying of the microarray slide was necessary before data scanning.
Scan protocol
The microarrays were scanned with a GenePix 4000B Array Scanner (AXON instruments, Inc, U.S.A).The emitted fluorescent signal was detected by a photomultiplier tube (PMT) at 570 nm (Cy3). The scanned images were saved as 16-bit TIFF files, and each spot was quantified by using GenePix Pro 6.0 software (AXON instruments, Inc, USA).
Description
Each gene was spotted on the arrays in three replicates.
Data processing
After hybridization, microarrays were scanned with a ScanArray 4100 Microarray Analysis System (AXON instruments, Inc, USA).The emitted fluorescent signal was detected by a photomultiplier tube (PMT) at 532 nm. After background subtraction, signal intensities for each spot were quantified using GenePix Pro version 6.0 (AXON instruments, Inc, USA). The SNRs from three replicate data sets were then averaged to represent the SNR for a particular probe. A commonly accepted criterion for the minimum signal (threshold) that can be accurately quantified is SNR ≥ 2. Spots that appeared to be lower than the threshold value were removed from the data set. The data sheet from GenePix Pro was then exported to Excel for further processing. Spot intensities and median values for each spot were normalized by average intensity of each slide to account for any difference in total intensity between the scanned images. The processed stress and non stress signal intensities for each spot were used for calculating the Log transformed expression ratio (log2 stress / non stress) (data provided as a supplementary GSE28538.txt file). Genes with a |log2 ratio| larger than 1 (|ratio| > 2) are considered differentially expressed.
