Analysis of expression profiles of the S. meliloti rpoH1 mutant following an acidic pH shift in view of wild type results In order to elucidate the role of RpoH1 in transcription dynamics during pH stress response, the time-course transcriptomic analyses of the rpoH1 mutant upon acidic pH shift were compared to those of the wild type. For a most effective comparative analysis, K-means clustering was performed for the 210 genes selected through the filtering of the wild type data, but this time the clustering was carried out with their log2 expression data in the rpoH1 mutant arrays. This

approach enabled the identification of genes that, throughout the time-course, behaved in a similar fashion both in the rpoH1 mutant arrays and in the wild type, as well as the identification of genes that displayed SIS3 no differential expression in

the rpoH1 mutant arrays, even though they were Navitoclax differentially expressed, upon acidic pH shift, in the wild type. The dynamic gene expression profiles were also catalogued into six clusters for the rpoH1 mutant, separating groups of genes with the highest possible similarity. Clusters G and H comprise genes that were constantly upregulated over time, either with a very strong induction (M-value ≥ 2.5 for at least one time point) or a moderate one (M-value ≤ 2.5) (Figure 5). Among the strongly upregulated genes in cluster G were nex18 and lpiA, the exopolysaccharide biosynthesis genes exoV, exoH, exoN and the gene coding for the Cah carbonic anhydrase, which is also induced in response to phosphate starvation of S. meliloti [42]. Genes grouped in cluster H include many exo genes and the gene coding for a regulator of succynoglycan production chvI [43], as well as the gene 4-Hydroxytamoxifen cost encoding the translocation protein TolB. A few transiently upregulated Thiamine-diphosphate kinase genes were listed in cluster I, such as the gene coding for SerA dehydrogenase

(Figure 5A). Figure 5 Classification of expression profiles of S. meliloti rpoH1 mutant genes upon acidic pH shift in comparison to the wild type. Representative genes are listed below graphics. Uniquely classified groups (G-L) were obtained through K-means clustering of rpoH1 mutant microarray data. The graphics illustrate the expression profile based on the mean values; the X-axis represents time, whereas the Y-axis represents the log2 ratio of gene expression (detailed view of the axes is shown in Figure 6). Genes marked in bold present dissimilar expression profile in comparison to S. meliloti wild type and therefore fit into a different cluster in the wild type clustering results. Clusters J and K grouped genes that were downregulated throughout the time-course, with persistent and transient downregulation, respectively. Like in the wild type arrays, many flagellar genes were also downregulated in the mutant and grouped in cluster J. The phosphate transport system encoded in the phoCDET operon also grouped in this cluster. In E.