The Bradyrhizobium japonicum Ferrous Iron Transporter FeoAB Is Required for Ferric Iron Utilization in Free Living Aerobic Cells and for Symbiosis

Abstract

The bacterium Bradyrhizobium japonicum USDA110 does not synthesize siderophores for iron utilization in aerobic environments, and the mechanism of iron uptake within symbiotic soybean root nodules is unknown. An mbfA bfr double mutant defective in iron export and storage activities cannot grow aerobically in very high iron medium. Here, we found that this phenotype was suppressed by loss of function mutations in the feoAB operon encoding ferrous (Fe(2+)) iron uptake proteins. Expression of the feoAB operon genes was elevated under iron limitation, but mutants defective in either gene were unable to grow aerobically over a wide external ferric (Fe(3+)) iron (FeCl3) concentration range. Thus, FeoAB accommodates iron acquisition under iron limited and iron replete conditions. Incorporation of radiolabel from either (55)Fe(2+) or (59)Fe(3+) into cells was severely defective in the feoA and feoB strains, suggesting Fe(3+) reduction to Fe(2+) prior to traversal across the cytoplasmic membrane by FeoAB. The feoA or feoB deletion strains elicited small, ineffective nodules on soybean roots, containing few bacteria and lacking nitrogen fixation activity. A feoA(E40K) mutant contained partial iron uptake activity in culture that supported normal growth and established an effective symbiosis. The feoA(E40K) strain had partial iron uptake activity in situ within nodules and in isolated cells, indicating that FeoAB is the iron transporter in symbiosis. We conclude that FeoAB supports iron acquisition under limited conditions of soil and in the iron-rich environment of a symbiotic nodule.

Keywords: BRADYRHIZOBIUM; bacteria; iron; nitrogen fixation; symbiosis; transport metal.

FIGURE 1.

Growth of mutants harboring feoA or feoB suppressor alleles or deletions in the wild type background. Cells of mutant strains harboring feoB(Y454term), feoA(E40K) ΔfeoA, and ΔfeoB grown in liquid cultures supplemented with 100 nm heme were serial diluted 10⁻¹–10⁻⁵-fold, and 10 μl was spotted on plates containing either 1.65 mm FeSO₄ (A) or 50 μm FeSO₄ (B). Each panel represents a single plate, and the image was separated for clarity of presentation.

FIGURE 2.

Requirement of feoA and feoB for aerobic growth with ferric iron in the growth medium over a wide concentration range of iron. Growth medium was inoculated with 1 × 10⁶ cells/ml of the wild type (green, closed circles), feoB (magenta, open circles), feoA (blue, closed diamonds), feoB(Y454term) (black, crosses), and feoA(E40K) (red, open triangles) strains. Strains were grown in minimal medium supplemented with either no added iron (A), 5 μm FeCl₃ (B), 50 μm FeCl₃ (C), 500 μm FeCl₃ (D), or 0.5 μm heme (E). Aliquots were taken at the indicated time points, and the optical density was measured at 540 nm (A₅₄₀).

FIGURE 3.

Requirement of feoA and feoB for Fe²⁺ and Fe³⁺ uptake by B. japonicum. A, cells of the wild type (closed circles), feoB (open circles), feoA (closed diamonds), and feoA(E40K) (open triangles) strains were grown in modified GSY medium with 100 nm heme. At time 0, 25 nm ⁵⁵Fe²⁺ was added to the assay medium, and aliquots were subsequently taken at various time points and counted. Each time point is the average of three biological replicate samples ± S.D. (error bars). B, Lineweaver-Burk plot of initial velocity⁻¹ (10 min) of uptake of ⁵⁵Fe²⁺ by wild type cells as a function of the starting iron concentration⁻¹, ranging from 1.25 to 25 nm substrate. C, cells of the wild type (closed circles), feoB (open circles), feoA (closed diamonds), and feoA(E40K) (open triangles) strains were grown in modified GSY medium with 100 nm heme. At time 0, 50 nm ⁵⁹Fe³⁺ was added to the assay medium, and aliquots were subsequently taken at various time points and counted. Each time point is the average of triplicate samples ± S.D. (error bars). D, Lineweaver-Burk plot of initial velocity⁻¹ (10 min) of uptake of ⁵⁹Fe³⁺ by wild type cells as a function of the starting iron concentration⁻¹, ranging from 25 to 1000 nm substrate.

FIGURE 4.

Regulation of feoA and feoB by iron. The steady-state transcript levels of feoA and feoB in the wild type strain grown in modified GSY medium with either no added iron, 10 μm FeCl₃, 20 μm FeCl₃, or 100 μm FeCl₃ were analyzed by qualitative real time PCR. The data are expressed as relative starting quantities (SQ) of respective mRNAs normalized to the housekeeping gene gapA and are presented as averages of three replicates ± S.D. (error bars).

FIGURE 5.

Aberrant regulation of iron-dependent gene expression. A, Western blot analysis of Irr was performed on wild type strain grown in modified GSY medium supplement with no added iron (-Fe) and on wild type, feoB, feoA, feoB(Y454), and feoA(E40K) strains grown in modified GSY medium with 100 nm heme. The protein was detected using anti-Irr antibodies. GroEL was used as a control for a protein not regulated by iron, and it was detected using anti-GroEL antibodies. 15 μg of protein were loaded per lane. B, steady-state transcript levels of fhuE the wild type strain grown in modified GSY medium supplemented with no added iron (-Fe) and the wild type, feoB, feoA, feoB(Y454), and feoA(E40K) strains grown in modified GSY medium with 100 nm heme were analyzed by qualitative real time PCR. The data are expressed as relative starting quantities (SQ) of respective mRNAs normalized to the gene recA and are presented as averages of three biological replicates ± S.D. (error bars).

FIGURE 6.

feoA and feoB are essential for symbiosis with soybean. A, roots of 28-day-old soybean plants, along with nodules incited by either wild type, feoB, feoA, and feoA(E40K) strains. All pictures were taken with lens fixed at the same distance and magnification. B, table listing the symbiotic properties of soybean nodules incited by the wild type, feoB, feoA, and feoA(E40K) strains. The data presented are averages of data obtained from 10 plants from each strain ± S.D.

FIGURE 7.

Bacterial iron transport in nodules in situ and in isolated bacteroids. A, roots with attached nodules incited by either the wild type strain (closed circles) or feoA(E40K) strain (open triangles) were incubated with 5 μm ⁵⁵Fe²⁺ at time 0. At each time point, root with attached nodules was harvested, and the radiolabel from isolated bacteroids was counted. B, bacteroids were isolated from nodules incited by either the wild type strain (closed circles) or feoA(E40K) strain (open triangles) and were resuspended in assay medium. At time 0, 25 nm ⁵⁵Fe²⁺ was added to the assay medium, and aliquots were subsequently taken at various time points and counted. C, wild type strain grown in modified GSY medium supplemented with 100 nm heme (closed circles) or bacteroids isolated from nodules incited by the wild type strain (open squares) were suspended in assay medium. At time 0, 1 μm ⁵⁹Fe³⁺ was added to the assay medium, and aliquots were subsequently taken at various time points and counted. Each time point is the average of three biological replicates ± S.D. (error bars).