Rapid evolution of a bacterial iron acquisition system

Abstract

Under iron limitation, bacteria scavenge ferric (Fe³⁺ ) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram-negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain-of-function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe²⁺ ) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non-selective uptake components that do not require mutation to accommodate new iron sources.

FIGURE 1. Dominant gain-of-function mutations in siderophore outer membrane transporter genes rescue growth on the synthetic iron chelator EDDHA

Cells were serial diluted, and 5 μl aliquots were spotted onto plates 20 μM FeCl₃ (A), 100 μM EDDHA (B), 100 μM EDDHA plus 20 μM FeCl₃ (C), 20 μM DTPA (D), 20 μM DTPA and 100 μM EDDHA (E) or 20 μM DTPA with 20 μM FeCl₃ (F). The strains entR(L272P), entR(T437P) and fegA(L497Q) represent the isogenic siderophore transporter mutants reconstructed in the wild type background. Strains entR(L272P)+, entR(T437P)+ and fegA(L497Q)+ denote the partial diploids harboring the wild type copy and the designated mutated copy of the transporter, integrated in the wild type genome.

FIGURE 2. Requirement of tonB2 for growth on natural siderophores or EDDHA

Serial dilution and spotting on plates of modified GSY media containing 10 μM heme (A), 100 μM EDDHA (B), 4 μM ferrichrome (C), 4 μM enterobactin (D) or 25 μM desferrioxamine (E). The tonB2 and entR(L272P) tonB2 strains have an in-frame deletion in the tonB2 (blr3908) gene in the wild type and entR(L272P) background respectively. The fegA, entR and fhuE strains are defective in the respective ferric-siderophore transporters.

FIGURE 3. Uptake of siderophore-mediated ⁵⁹Fe³⁺

Uptake of ⁵⁹Fe³⁺- siderophores was measured in the cells of the wild type, tonB2 and feoB strains along with the respective siderophore transporter mutant entR, fegA or fhuE. Cells were suspended in uptake buffer containing 4 μM enterobactin (A), 4 μM ferrichrome (B) or 25 μM desferrioxamine (C). At time 0, 25 nM ⁵⁹Fe³⁺ was added to the cell suspension, and aliquots were subsequently collected at 0, 10, 50 minutes to measure counts. Each time point is the average of three biological replicates ± S.D. (error bars) after subtracting the 0 minute reading.

FIGURE 4. Gain-of-function mutations in the heme transporter gene hmuR suppress the growth and Fe³⁺ uptake phenotypes of an fhuE mutant on desferrioxamine

(A) Growth of mutants on desferrioxamine. Cells were serially diluted, and 5 μl aliquots were spotted onto plates containing 20 μM FeCl₃ (a), 25 μM desferrioxamine (b) or 25 μM desferrioxamine plus 20 μM FeCl₃ (c). Strains with a + sign denote partial diploids with both the wild type and the mutated hmuR allele integrated into the genome. (B) ⁵⁹Fe³⁺- desferrioxamine uptake. Cells of the wild type (open square), fhuE (closed square), fhuE hmuR(W351R) (open triangle), fhuE hmuR(Δ182-193) (open circle) and fhuE hmuR (Δ403-425) (closed circle) strains were suspended in uptake buffer containing 25 μM desferrioxamine (a) or 20 mM sodium-citrate (b). At time 0, 50 nM ⁵⁹Fe³⁺ was added to the cell suspension, 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).

FIGURE 5. FeoB is essential for the utilization of natural and synthetic iron chelates

(A) Growth on natural xenosiderophores. Serial dilution and spotting of cells grown in liquid GSY media with 100nM heme was performed on modified GSY media plates containing 10 μM heme (a), no added iron (b), 20 μM FeCl₃ (c), 4 μM ferrichrome (d), 4 μM enterobactin (e) or 25 μM desferrioxamine (f). The fegA, entR and fhuE strains have defects in the ferricrhome, enterobactin and desferrioxamine transporter respectively. (B) Growth on EDDHA. Cells were grown in liquid GSY media with 100 nM heme, serially diluted and spotted on modified GSY media containing 10 μM heme (a), 100 μM EDDHA (b) or 100 μM EDDHA plus 10 μM heme (c). The entR(L272P) feoB strain has in-frame deletion in the feoB gene in the entR(L272P) background.

FIGURE 6. B. japonicum is resistant to albomycin compared with E. coli cells

Cells were serially diluted, and 5 μl aliquots were spotted onto plates in the presence or absence of 2 μM albomycin. (A) E. coli cells. (B) B. japonicum strain 110 (Bj) or B. japonicum cells harboring the E. coli ferrichrome transport genes fhuCDB

FIGURE 7. Comparison of ferric-chelate acquisition systems between E. coli and B. japonicum

(A) In E.coli, outer membrane transporters FecA, FepA, FhuE, FhuA import Fe³⁺ chelates of citrate, enterobactin, desferrioxamine and ferrichrome, respectively. Translocation of ferric-chelates across the outer membrane is dependent on TonB (auxiliary proteins ExbB and ExbD are not shown). (B) In B. japonicum, intact heme is transported into the cytoplasm through the TonB-dependent outer membrane heme transporter, HmuR. The outer membrane xenosiderophore transporters FhuE, FegA, and EntR import Fe³⁺ chelates of desferrioxamine, ferrichrome and enterobactin, respectively. These transporters depend on TonB2. In the periplasm, Fe³⁺-siderophore is likely reduced to Fe²⁺ iron, potentially causing its release from the chelator. A single inner membrane transporter, FeoB, is responsible for importing Fe²⁺ iron into the cytoplasm.