For each time point the transcribed and labelled RNA of the pH 5.75 grown culture was hybridised together with the differently labelled RNA of the pH 7.0 reference culture to the Sm6kOligo microarray. The whole procedure was performed in three biological replicates to ensure the validity of the microarray data. The microarray images were analysed using the Imagene Software and EMMA [26] (For microarray data see: http://www.ebi.ac.uk/microarray-as/ae/). As expected, the microarray analysis for the six selleckchem successive time points revealed a high number of genes with different expression characteristics over the tested period. In order to identify
genes that presumably play a significant role in the cellular Fludarabine cost response to acidic pH the following filtering criteria were applied. Only genes with a log2 fold PRIMA-1MET mouse difference in spot intensities on the microarray slides (M value) of ≥ 2 or ≤ -2 were considered. Because we were
also interested in genes that were only transiently active, this limit of significance had to be achieved for at least one time point during the time series. In addition, it was of importance for clustering that each gene was represented with an evaluable expression value (R ≥ 1.5 for both channels) for at least 5 out of the 6 time points. 230 genes fulfilled these filtering criteria. To estimate the number of false positive genes after filtering the false discovery rate (FDR) control was applied for all expression data of these 230 genes. The FDR control revealed a proportion Rutecarpine of less than 1% false positives. Additionally, the tendency of the microarray results was confirmed by qRT-PCR for two of the obtained genes (lpiA and phoC) (data not shown). Since the S. meliloti genome is composed of three replicons with distinctive functional features [8] the distribution of the 230 genes
fulfilling the filtering criteria was determined. The percentage of differentially expressed genes of the total number of genes was 3.95% for the chromosome, 2.48% for pSymA, and 4.20% for pSymB. Therefore, compared to the chromosome genes located on pSymB were slightly over represented whereas genes of pSymA were noticeably under represented in the time course experiment. A possible explanation is that pSymA carries mostly symbiosis related genes which are not responding, whereas pSymB and the chromosome contain housekeeping genes. The slight over representation of pSymB might be based on the up-regulation of exopolysaccharide biosynthesis genes (see below). In the next step, a clustering of these genes was performed according to their expressional characteristics over time. By hierarchical clustering, a separation into eight different clusters was estimated.