The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter

Abstract

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.

FIG. 1.

Genetic organization of a DNA region of the S. meliloti chromosome containing the fur gene and the sitABCD operon. The DNA region of the S. meliloti genome containing the fur gene, the sitABCD operon, and the two neighboring genes, smc02511 and smc02505, of unknown function, is shown. The putative Fur box sequence as identified by an HMM-based search is indicated. The deletions Δ1 and Δ2 were used to construct mutations in the genes fur and sitA, respectively. The fur deletion Δ1 ranges from bases 140 to 400 of the fur gene, and the S. meliloti strain carrying the deletion Δ1 was designated Rm1021Δfur. The sitA deletion Δ2 ranges from bases 312 to 896 of the sitA gene, and the strain carrying the deletion Δ2 was named Rm1021ΔsitA. In addition, the 760-bp BamHI/XhoI fragment containing most probably the sitA promoter was used to construct the promoter test vector pTCC1 for sitA promoter studies.

FIG. 2.

Growth of the S. meliloti wild type and the S. meliloti sitA mutant strain with dependence on Mn(II) and/or Fe(II) supplementation of the medium. Overnight cultures of the S. meliloti wild-type strain Rm1021 (A) and the S. meliloti sitA mutant Rm1021ΔsitA (B) were washed and diluted in fresh VMM* containing 30 μM Fe(II) (▪), 60 μM Fe(II) (□), 1 μM Mn(II) (▴), or 30 μM Fe(II) + 1 μM Mn(II) (▵). Growth was monitored via optical density at 580 nm (o.d.₅₈₀). The results are derived from five independent replicates, and the error bars indicate the standard deviations.

FIG. 3.

Effects of the iron chelator Con on the growth of the S. meliloti wild-type strain and the S. meliloti sitA mutant strain. Strains Rm1021 (▪) and Rm1021ΔsitA (▴) were grown in TY medium containing 240 mg of Con/liter (A), 240 mg of Con/liter and 25 μM Fe(II) (B), or 240 mg of Con/liter and 25 μM Mn(II) (C). The depicted results were obtained from five independent replicates, and the error bars indicate the standard deviations. o.d.₅₈₀, optical density at 580 nm.

FIG. 4.

Growth of the S. meliloti wild-type strain Rm1021 and the fur mutant strain Rm1021Δfur under high and low iron concentrations. Overnight cultures of Rm1021 (▪) and Rm1021Δfur (▴) were washed and diluted into fresh VMM* containing 30 μM Fe(II) or no added iron sources and incubated at 30°C for 180 h. The growth was monitored by measuring the optical density at 580 nm (o.d.₅₈₀). The error bars indicate the standard deviations, which were calculated from six independent experiments.

FIG. 5.

Effects of different metal sources on the expression of the S. meliloti sitA promoter. A promoter test plasmid containing a sitA promoter-gusA fusion (pTCC1) was introduced into the S. meliloti wild-type strain Rm1021 (white bars), the S. meliloti fur mutant strain Rm1021Δfur (black bars), and the fur mutant strain Rm1021Δfur complemented with plasmid pTCB1 carrying an intact fur gene (grey bars). The strains were grown in VMM* under the following conditions: (A) no added iron, (B) addition of Fe(III), (C) addition of Fe(II) as the sole iron source. Additionally, Mn(II) was added as indicated. The different strains were grown to an optical density of 0.6 before the assays for glucuronidase activity were carried out. The β-glucuronidase levels are expressed in Miller units, and the error bars indicate the standard deviations. In each case the depicted results were derived from six independent cultures.

FIG. 6.

Scatter plot for the microarray-based analysis of S. meliloti genes affected by deletion of the fur gene. The scatter plot shows the logarithmic mean signal ratios (M values) versus. the logarithmic mean signal intensities (A values) obtained for comparison of the S. meliloti wild-type strain Rm1021 to the fur mutant Rm1021Δfur by microarray hybridization. Indicated are the differentially expressed genes that are involved in metal acquisition. Genes were only regarded as differentially expressed when the M values were ≥1.0 or ≤−1.0, the A values were ≥9.0, and the P values ≤0.05.

FIG. 7.

Visualization of S. meliloti sitA promoter activity in M. sativa nodules. Nodules of M. sativa were harvested, thin sectioned, and stained with the dye X-Gluc. (A) M. sativa nodule induced by the S. meliloti wild-type strain carrying pJP2. (B) M. sativa nodule induced by the S. meliloti fur mutant carrying sitA promoter-gusA fusion plasmid pTCC1. (C) M. sativa nodule induced by the S. meliloti wild-type strain carrying pTCC1. Abbreviations: M, meristem; I, infection zone; N, nitrogen fixation zone.