Transcriptome of Nitrosomonas europaea with a disrupted nitrite reductase (nirK) gene

Global gene expression was compared between Nitrosomonas europaea wild-type and a nitrite reductase-deficient mutant using a genomic microarray. Keywords: wild-type to mutant global transcriptome comparison The nirK mutant was generated from the wild-type by integrating a kanamycin resistance gene cassette into the center of the nirK ORF via homologous recombination from an engineered plasmid introduced into N. europaea by conjugation. Batch cultures of wild-type and nirK mutant Nitrosomonas europaea (ATCC 19718) were grown in mineral medium to late exponential phase. Total cellular RNA was extracted from cells harvested from multiple cultures for each hybridization experiment (AquaPure RNA Isolation; Biorad), treated with RNase-free DNase I (Ambion), and purified (RNeasy Mini Kit; Qiagen). Fluorescently labeled cDNA libraries from total RNA were generated by incorporation of Cy3- or Cy5-dUTP by reverse transcriptase (SuperScript III; Invitrogen). Labeled cDNA was treated with NaOH (1N, 65 °C, 30 min), neutralized with Tris-Cl (1M, pH 7.6), column purified (Qiagen), and concentrated by vacuum centrifugation. Two sets of triplicate reactions were performed in which the fluorescent dyes were reversed during cDNA synthesis to minimize dye-specific effects that could influence analysis of hybridization signals to the microarrays. Labeled cDNA libraries (0.6-0.9 ug with 20-30% label incorporation frequency) were hybridized to N. europaea genomic microarrays in buffered solution (5x SSC, 0.1% SDS, 50% formamide, 0.1 mg/ml salmon sperm DNA) in a chamber (Corning) under Hybrislips (Sigma-Alrich) at 50 C for 12 to 16 h. The slides were washed with 2x SSC, 0.1% SDS for 5 min at 42 C; 0.1x SSC, 0.1% SDS for 10 min at 42 C; and 4 times with 0.1x SSC for 1 min at room temperature. Scanned images (Scan Microarray Express, Perkin Elmer) were analyzed using ImaGene version 5.6 (Biodiscovery). Spot grids were manually fitted to microarray images and averaged signal intensity and local background was calculated for each microarray element. The quality of the hybridization signals were assessed for each microarray by the numbers of detected genes plus low and consistent background levels. Spots with poor signal quality, irregular shape, and/or high background were flagged and removed from each data set. Remaining data were transferred to Excel and Access 2001 (Microsoft Corp. Redmond, WA) and GeneSpring version 6.0 (Silicon Genetics). Normalization was done by scaling total fluorescence measured for both Cy3 and Cy5 to an equivalent averaged intensity across each microarray. Pearson correlation coefficients were calculated for 12 spots for each gene (duplicate spots across 6 microarrays), and genes showing statistically significant and greater than 2 fold differences in Cy3/Cy5 hybridizations from at least 5 spots were considered differentially regulated.