Allochromatium vinosum DSM 180T wildtype cells were cultivated photolithoautotrophically in batch culture at 30°C under anaerobic conditions and continuous illumination in completely filled screw-capped culture bottles containing modified Pfennig´s medium (“0” medium without sulfide). Sulfide (4 mM), thiosulfate (10 mM), sulfite (5 mM) or sulfur (50 mM) were added to the cultures as sulfur sources. Cells of A. vinosum, grown photoorganoheterotrophically on malate (RCV medium) for three days were used as an inoculum for growth experiments. The culture volume of the precultures was 500 ml. Inoculum cells were harvested by centrifugation (10 min, 2680 x g) and washed once in “0” medium. For DNA microarray experiments cells were cultivated in a volume of 100 ml RCV or “0” medium supplemented with the respective sulfur source. These cultures were inoculated with stationary phase cells taken from 100 ml preculture.
Extracted molecule
total RNA
Extraction protocol
Total RNA of A. vinosum was isolated from cells grown photoorganoheterotrophically on 22 mM malate or photolithoautotrophically on 50 mM sulfur, 4 mM sulfide, 10 mM thiosulfate or 5 mM sulfite in 100 ml culture bottles. Briefly, cells of two culture aliquots (35 ml each) culture were harvested (12800 x g, 10 min, 4°C), each pellet was resuspended in 350 µl RLT buffer (Qiagen, Hilden, Germany) containing 10 mM DTT and disrupted by vortexing (Ivoclar Vivadent Silamat S 6, Ivoclar Vivadent AG, Schaan, Lichtenstein) using 0,1 mm Zirconia/Silica beads (Roth, Karlsruhe, Germany). Cell debris and glass beads were sedimented (15000 x g, 2 min, 4°C) and the supernatant was mixed with 500 µl phenol (1 min) followed by the addition of 500 µl chloroform and a further mixing step. After centrifugation (10000 x g, 10 min, 4°C) RNA of the supernatant was precipitated by the addition of 1/10 volume 3 M sodium acetate (pH 6) and two volumes of ice-cold ethanol. Precipitated RNA was collected by centrifugation (10000 x g, 10 min, 4°C) and the pellets generated from 70 ml culture were resuspended in 100 µl RNAse free water. The RNA was further purified using the RNAeasy Total RNA kit (Qiagen, Hilden, Germany) according to the manufactures instructions. RNA concentration was measured photometrically with an Analytic Jena Specord 210 spectrophotometer.
Label
Cy5
Label protocol
For synthesis of fluorescently-labeled cDNA with the fluorescent nucleotide analogues Cy3-dCTP and Cy5-dCTP (GE Healthcare, Freiburg, Germany) 25 μg of RNA and 500 ng of random hexamer primers were used. The reaction mixture (30 µl) contained 3 μl 1 mM Cy3-dCTP or Cy5-dCTP, 3 μl 0.1 M DTT, 6 μl 5x first strand buffer (Invitrogen, Karlsruhe, Germany), 0.6 μl dNTP-mix (25 mM each of dATP, dUTP, and dGTP and 10 mM dCTP) and 2 μl Superscript II reverse transcriptase (Invitrogen, Darmstadt, Germany). The mixture was incubated for 10 min at room temperature and 110 min at 42 °C, stopped by addition of 10 μl 0.1 N NaOH, incubated for 10 min at 70 °C, and then neutralized by addition of 10 μl 0.1 N HCl. cDNA samples were purified by washing three times with water on a Microcon column (Millipore, YM-30).
Allochromatium vinosum DSM 180T wildtype cells were cultivated photolithoautotrophically in batch culture at 30°C under anaerobic conditions and continuous illumination in completely filled screw-capped culture bottles containing modified Pfennig´s medium (“0” medium without sulfide). Sulfide (4 mM), thiosulfate (10 mM), sulfite (5 mM) or sulfur (50 mM) were added to the cultures as sulfur sources. Cells of A. vinosum, grown photoorganoheterotrophically on malate (RCV medium) for three days were used as an inoculum for growth experiments. The culture volume of the precultures was 500 ml. Inoculum cells were harvested by centrifugation (10 min, 2680 x g) and washed once in “0” medium. For DNA microarray experiments cells were cultivated in a volume of 100 ml RCV or “0” medium supplemented with the respective sulfur source. These cultures were inoculated with stationary phase cells taken from 100 ml preculture.
Extracted molecule
total RNA
Extraction protocol
Total RNA of A. vinosum was isolated from cells grown photoorganoheterotrophically on 22 mM malate or photolithoautotrophically on 50 mM sulfur, 4 mM sulfide, 10 mM thiosulfate or 5 mM sulfite in 100 ml culture bottles. Briefly, cells of two culture aliquots (35 ml each) culture were harvested (12800 x g, 10 min, 4°C), each pellet was resuspended in 350 µl RLT buffer (Qiagen, Hilden, Germany) containing 10 mM DTT and disrupted by vortexing (Ivoclar Vivadent Silamat S 6, Ivoclar Vivadent AG, Schaan, Lichtenstein) using 0,1 mm Zirconia/Silica beads (Roth, Karlsruhe, Germany). Cell debris and glass beads were sedimented (15000 x g, 2 min, 4°C) and the supernatant was mixed with 500 µl phenol (1 min) followed by the addition of 500 µl chloroform and a further mixing step. After centrifugation (10000 x g, 10 min, 4°C) RNA of the supernatant was precipitated by the addition of 1/10 volume 3 M sodium acetate (pH 6) and two volumes of ice-cold ethanol. Precipitated RNA was collected by centrifugation (10000 x g, 10 min, 4°C) and the pellets generated from 70 ml culture were resuspended in 100 µl RNAse free water. The RNA was further purified using the RNAeasy Total RNA kit (Qiagen, Hilden, Germany) according to the manufactures instructions. RNA concentration was measured photometrically with an Analytic Jena Specord 210 spectrophotometer.
Label
Cy3
Label protocol
For synthesis of fluorescently-labeled cDNA with the fluorescent nucleotide analogues Cy3-dCTP and Cy5-dCTP (GE Healthcare, Freiburg, Germany) 25 μg of RNA and 500 ng of random hexamer primers were used. The reaction mixture (30 µl) contained 3 μl 1 mM Cy3-dCTP or Cy5-dCTP, 3 μl 0.1 M DTT, 6 μl 5x first strand buffer (Invitrogen, Karlsruhe, Germany), 0.6 μl dNTP-mix (25 mM each of dATP, dUTP, and dGTP and 10 mM dCTP) and 2 μl Superscript II reverse transcriptase (Invitrogen, Darmstadt, Germany). The mixture was incubated for 10 min at room temperature and 110 min at 42 °C, stopped by addition of 10 μl 0.1 N NaOH, incubated for 10 min at 70 °C, and then neutralized by addition of 10 μl 0.1 N HCl. cDNA samples were purified by washing three times with water on a Microcon column (Millipore, YM-30).
Hybridization protocol
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
Scan protocol
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
Data processing
For background correction of spot intensities, ratio calculation and ratio normalization, GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
Genome-wide transcriptional profiling of the purple sulfur bacterium Allochromatium vinosum DSM 180T during growth on different reduced sulfur compounds [Set2]
Genome-wide transcriptional profiling of the purple sulfur bacterium Allochromatium vinosum DSM 180T during growth on different reduced sulfur compounds
Data table header descriptions
ID_REF
VALUE
lowess normalized log2 ratio (sulfur compound / RCV control)
