0 and pH 5.75. All sigma factor mutants grew slightly more poorly than wild type cells at both pH 7.0 and pH 5.75, with the exception of the rpoH1 mutant, whose growth was severely impaired at pH 5.75 (Figure 1). Restoration of the wild type growth phenotype was observed for the rpoH1 mutant carrying a recombinant plasmid with the intact rpoH1 gene, confirming that the lack of growth was solely caused by the rpoH1 mutation (Additional file 1). The results indicate that the RpoH1 sigma factor is therefore essential for growth at acidic pH. Figure 1 Growth curves of S. meliloti 1021 wild type strain and mutant strains for sigma factor genes at neutral and acidic pH. S. meliloti
1021 (open Trametinib price circles) and mutant strains for sigma factor genes rpoE1 (filled squares), rpoE2 (filled triangles), rpoE5 (open triangles), fecI (filled circles) and rpoH1 (open squares) GDC-0980 cost were grown in VMM medium at 30°C at either pH 7.0 (A) or pH 5.75 (B). Each panel shows the data from three representative experiments. The error bars indicate the standard deviation
calculated from three independent cultures. Transcription profiling of the rpoH1 mutant versus wild type at neutral pH reveals RpoH1 involvement only in the regulation of the rhizobactin operon Among all the sigma factors analyzed, the rpoH1 mutant showed the most peculiar phenotype in the growth tests, presenting no growth at low pH values. This mutant was therefore
selected for transcription profiling experiments. With the intent of examining the differential expression of genes in the sigma factor rpoH1 deletion mutant in comparison to the wild type, both S. meliloti wild type strain 1021 and rpoH1 mutant were cultivated at pH 7.0 and harvested for microarray analysis after reaching an optical density of 0.8 at 580 nm. Only genes with a twofold difference in spot intensities on the microarray slides (M-value of ≥ 1 or ≤ -1) were considered. Surprisingly, at neutral pH, the rhizobactin biosynthesis operon was nearly exclusively observed among the significant differentially expressed genes Thiamine-diphosphate kinase (Figure 2). Rhizobactin is an iron siderophore, that is, a low molecular weight ligand that binds to ferric iron with high affinity . All genes for the rhizobactin biosynthesis operon, rhbABCDEF, were upregulated, as well as the rhizobactin transporter gene rhtA. The gene for the rhizobactin activator rhrA, however, was downregulated in the mutant. The unexpected but dramatic increase in siderophore production by the rpoH1 deletion mutant in comparison to the S. meliloti wild type was additionally confirmed by Chrome azurol S (CAS) assay, which is a chemical test for the detection of siderophore production based on the removal of ferric iron from a pigmented complex by a competing ligand such as a siderophore  (Additional file 2).