The cells were pelleted by centrifugation (6000 rpm, 28°C, 10 min), pellets were frozen in liquid nitrogen and stored at -80°C until RNA isolation.
Growth protocol
Azoarcus sp. wild type strain BH72 or nifLA mutant strain BHLAO were grown respectively in an oxygen-controlled bioreactor (Biostat B; B. Braun Melsungen AG, Melsungen, Germany) at 37°C with stirring at 6000 r.p.m, under microaerobic (0.6% oxygen concentration) nitrogen fixing conditions with 10 mM glutamate (poor nitrogen source)
Extracted molecule
total RNA
Extraction protocol
The cell pellets were re-suspended in a 1:1 mixture of pre-warmed phenol-chloroform-isoamylalcohol (25:24:1, pH 4.7) and NAES solution (50 mM sodium acetate, 10 mM EDTA, 1 % SDS, pH 5.1). This suspension was incubated for 5 min at 65°C in a water bath, followed by 10 min incubation on ice. For phase separation, the mixture was centrifuged for 10 min at 12°C and 8000 x g. The upper phase was transferred and mixed with the same volume of phenol-chloroform-isoamylalcohol. This phenol extraction was repeated three times, followed by an extraction with equal volume of chloroform isoamylalcohol (24:1). RNA precipitation was performed with the same volume of isopropanol for 45 min on ice. The RNA was pelleted by centrifugation (12900 x g, 10 min at 4°C) and salts were removed with 1 ml of Ethanol (70 %). The dried RNA was dissolved in 100 µl 1 x RNAsecure, frozen in liquid nitrogen and kept at -80°C until further processing. Contaminating DNA was removed from RNA preparations by DNase I using Qiagen columns (RNeasy Mini Kit, Qiagen, Hilden, Germany) according to manufacturer’s instructions. The DNase I treated RNA was eluted twice in 30 µL of elution buffer, pooled and finally stored in 1x RNA secure in -80°C.
Label
Cy3
Label protocol
For transcriptome microarray analysis, 20 µg of total RNA was reverse transcribed with BioScript RT in respective reaction buffer (Bioline) with random hexamers and 4:1 aminomodified-dUTP/dTTP nucleotide mix for 90 min at 42°C. For coupling of fluorescent dyes to the aminoallyl-labelled first strand cDNA, aliquots of Cy3-NHS or Cy5-NHS esters (GE Healthcare) were dissolved in 60 µl of first strand cDNA, mixed and incubated for 90 min at room temperature in the dark. Blocking of all remaining dyes (quenching) was necessary after labelling. This quenching was achieved by adding 4.5 µl hydroxylamine (4 M) to the sample solution, followed by an incubation for 15 min at room temperature. Subsequent cleaning up of labelled cDNA from remaining dyes was performed with the CyScribe GFX Purification Kit (GE Healthcare) according to manufacturer’s instructions. The Cy5- and Cy3-labelled cDNA that was used in one hybridization experiment were cleaned up together. The labelled cDNA (60 µl) was stored at -20°C until microarray hybridization (Hauberg et al. 2012).
Channel 2
Source name
Reference: pure culture of bacteria, wild type (nitrogen fixing condition)
The cells were pelleted by centrifugation (6000 rpm, 28°C, 10 min), pellets were frozen in liquid nitrogen and stored at -80°C until RNA isolation.
Growth protocol
Azoarcus sp. wild type strain BH72 or nifLA mutant strain BHLAO were grown respectively in an oxygen-controlled bioreactor (Biostat B; B. Braun Melsungen AG, Melsungen, Germany) at 37°C with stirring at 6000 r.p.m, under microaerobic (0.6% oxygen concentration) nitrogen fixing conditions with 10 mM glutamate (poor nitrogen source)
Extracted molecule
total RNA
Extraction protocol
The cell pellets were re-suspended in a 1:1 mixture of pre-warmed phenol-chloroform-isoamylalcohol (25:24:1, pH 4.7) and NAES solution (50 mM sodium acetate, 10 mM EDTA, 1 % SDS, pH 5.1). This suspension was incubated for 5 min at 65°C in a water bath, followed by 10 min incubation on ice. For phase separation, the mixture was centrifuged for 10 min at 12°C and 8000 x g. The upper phase was transferred and mixed with the same volume of phenol-chloroform-isoamylalcohol. This phenol extraction was repeated three times, followed by an extraction with equal volume of chloroform isoamylalcohol (24:1). RNA precipitation was performed with the same volume of isopropanol for 45 min on ice. The RNA was pelleted by centrifugation (12900 x g, 10 min at 4°C) and salts were removed with 1 ml of Ethanol (70 %). The dried RNA was dissolved in 100 µl 1 x RNAsecure, frozen in liquid nitrogen and kept at -80°C until further processing. Contaminating DNA was removed from RNA preparations by DNase I using Qiagen columns (RNeasy Mini Kit, Qiagen, Hilden, Germany) according to manufacturer’s instructions. The DNase I treated RNA was eluted twice in 30 µL of elution buffer, pooled and finally stored in 1x RNA secure in -80°C.
Label
Cy5
Label protocol
For transcriptome microarray analysis, 20 µg of total RNA was reverse transcribed with BioScript RT in respective reaction buffer (Bioline) with random hexamers and 4:1 aminomodified-dUTP/dTTP nucleotide mix for 90 min at 42°C. For coupling of fluorescent dyes to the aminoallyl-labelled first strand cDNA, aliquots of Cy3-NHS or Cy5-NHS esters (GE Healthcare) were dissolved in 60 µl of first strand cDNA, mixed and incubated for 90 min at room temperature in the dark. Blocking of all remaining dyes (quenching) was necessary after labelling. This quenching was achieved by adding 4.5 µl hydroxylamine (4 M) to the sample solution, followed by an incubation for 15 min at room temperature. Subsequent cleaning up of labelled cDNA from remaining dyes was performed with the CyScribe GFX Purification Kit (GE Healthcare) according to manufacturer’s instructions. The Cy5- and Cy3-labelled cDNA that was used in one hybridization experiment were cleaned up together. The labelled cDNA (60 µl) was stored at -20°C until microarray hybridization (Hauberg et al. 2012).
Hybridization protocol
The combined Cy3/Cy5-labelled targets were dried in a speed vac and further dissolved in 60 µl DIG Easy hybridization solution (Roche, Mannheim, Germany). 1 µl of sonicated salmon sperm (5 µg/µl) was further added. The transcriptome microarray spotted on epoxysilane-coated Nexterion Slide E (Schott) (CeBiTec, University Bielefeld) contained 70mer oligonucleotide probes for the 3,989 predicted protein-coding genes from Azoarcus sp. strain BH72 in four replicates (Hauberg 2012). Before hybridization, the targets were denatured at 65°C for 10 min. The hybridization of fluorescently labelled cDNA targets to oligonucleotide microarrays was performed at 42°C in a hybridization chamber (ArrayIt, Sunnyvale, CA, USA) in a water bath for 14 to 18 hours. followed by appropriate washing steps as recommended.
Scan protocol
The Cy3- and Cy5-fluorescence was scanned at 532 nm and 635 nm with the GenePix Scanner 4000A (Molecular Devices, Sunnyvale, CA, USA) with a pixel size of 10 µm.The image analysis was performed with the GenePix 4.1 program and the subsequent analyses were carried out with the open-source software Microarray Data Analysis System (MIDAS v2.19) of TM4 (Saeed et al. 2003, http://www.tm4.org/). After scanning, raw data images of the slides that contain information about gene expression levels were obtained by the GenePix software. These images were analyzed by identifying each spot on the array (with the help of an array layout template) with measurements of its fluorescence intensity and the corresponding background intensity.
Description
302728_rep3 Biological replicate 3 of 3
Data processing
With the file transformation tool Express Converter v.2.1 (http://www.tm4.org/utilities.html), data files generated from GenePix (.gpr) were converted to TM4 files (.mev) that could be uploaded to the MIDAS platform. Normalization with MIDAS (TM4 suite) was done in order to reduce variability by appropriately adjusting the data. In this study, the LOWESS (locally weighted scattered plot smoothing) normalization was performed using the block mode as set-up parameter as this mode allowed correction of systematic spatial variation between the grids of the array. The normalized data for each spot were exported to Excel and aligned to the corresponding Azoarcus sp. BH72 gene name. Overall, three independent experiments (biological experiments) were performed. Average expression fold were determined from normalized values of the three sets. Statistical analysis was performed using the intensity values in a one tail paired t-test. Genes with an expression factor ≥ 1.8 and P value ≤ 0.05 were considered as differentially expressed.