Role of the regulatory gene rirA in the transcriptional response of Sinorhizobium meliloti to iron limitation

Abstract

A regulatory network of Sinorhizobium meliloti genes involved in adaptation to iron-limiting conditions and the involvement of the rhizobial iron regulator gene (rirA) were analyzed by mutation and microarray analyses. A constructed S. meliloti rirA mutant exhibited growth defects and enhanced H2O2 sensitivity in the presence of iron, but symbiotic nitrogen fixation was not affected. To identify iron-responsive and RirA-regulated S. meliloti genes, a transcriptome approach using whole-genome microarrays was used. Altogether, 45 genes were found to be jointly derepressed by mutation of rirA and under different iron-limited conditions. As expected, a number of genes involved in iron transport (e.g., hmuPSTU, shmR, rhbABCDEF, rhtX, and rhtA) and also genes with predicted functions in energy metabolism (e.g., fixN3, fixP3, and qxtAB) and exopolysaccharide production (e.g., exoY and exoN) were found in this group of genes. In addition, the iron deficiency response of S. meliloti also involved rirA-independent expression changes, including repression of the S. meliloti flagellar regulon. Finally, the RirA modulon also includes genes that are not iron responsive, including a gene cluster putatively involved in Fe-S cluster formation (sufA, sufS, sufD, sufC, and sufB).

FIG. 1.

Effects of the rirA mutation on growth rates of S. meliloti strains. Overnight cultures of S. meliloti wild-type strain Rm1021 (WT) and rirA mutant Rm1021-TR2 (ΔrirA) were washed and diluted in fresh minimal medium containing 37 μM FeCl₃ (a), 0.37 μM FeCl₃ (b), or 10 μM hemin (c) as the sole iron source. Samples were taken, and their optical densities at 580 nm (OD₅₈₀) were determined. The error bars indicate the standard deviations calculated from six independent cultures.

FIG. 2.

Scatter plots of the microarray-based analysis of S. meliloti gene expression affected by iron limitation and the rirA mutation. The plots show the logarithmic mean signal ratio (M-value) versus the logarithmic mean signal intensity (A-value) obtained by comparison of the transcriptomes of S. meliloti rirA mutant Rm1021-TR2 (ΔrirA) and S. meliloti wild-type strain Rm1021 (WT) (a), S. meliloti wild-type cells grown in iron-limited VMM and cells grown in iron-sufficient VMM (b), and S. meliloti wild-type cells incubated in iron-limited TY medium and cells grown in iron-sufficient TY medium (c). A number of genes with the greatest changes in mRNA abundance are indicated. The functions of the genes indicated are shown in Tables 2 and 3.

FIG. 3.

Venn mapping of S. meliloti genes with significantly altered expression obtained from three microarray experiments. The amounts of significantly induced genes (left) and repressed genes (right) were derived from the microarray-based global profiling of iron-responsive and RirA-regulated gene expression in S. meliloti. Of special interest were genes that were substantially up- or downregulated under both low- and high-iron conditions but not in the rirA mutant (subset A), during all three microarray experiments (subset B), and only in the rirA mutant compared to the wild type (WT) (subset C). The Venn diagrams are based on the microarray results shown in Table S1 in the supplemental material.

FIG. 4.

Clustered S. meliloti genes whose expression is affected by iron availability and by the rirA mutation. (a and b) Genetic maps of the rhizobactin 1021 synthesis/uptake and putative hemin uptake clusters (a) and a selection of putative energy metabolism gene clusters which are induced in the rirA mutant and under iron-limited conditions (b). (c) Gene cluster putatively involved in Fe-S cluster formation which is induced only in the S. meliloti rirA mutant. The numbers below the genes indicate the log₂ expression ratios of the genes derived from the microarray-based transcriptome analysis. Genes that were significantly induced in at least one experiment are indicated by solid arrows. The values in parentheses are expression ratios that were below the threshold used in this study.