Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli

Abstract

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

Figure 1. General organization of the EfeUOB and FepABC systems.

This scheme represents both the genetic (left part of the figure) and envelope organization (right part of the figure) of the S. aureus fepABC and E. coli efeUOB. Orthologous proteins are shown with the same pictogram and the same color. Both operons are regulated by iron loaded Fur repressor. Fur repressor is represented by an arrow with a black circle for iron; FepB and EfeB by a hexagone; FepC and EfeU by a hollow barrel; EfeO by a N-terminal square corresponding to the cupredoxin domain (C) and a C-terminal hexagone corresponding to the M75 peptidase domain; FepA which has only a M75 peptidase domain by a hexagone; heme is represented by a ring and a black circle for iron; HasR, the outer membrane receptor for heme is represented by a barrel; OM for outer membrane; PG for peptidoglycan; IM for inner membrane. Their putative function and cellular localization are shown in the right part of the figure.

Figure 2. FepB-6His characterization.

A and C: UV-visible spectra of apo-FepB (gray line) and FepB-hemin or FepB-PPIX complex of 2.2 mM (black line). B and D: Spectroscopic measure of hemin and PPIX binding to apo-FepB-6His. Aliquots of hemin or PPIX solutions were successively added to 0.5 ml of apo-FepB-6His (2.22 µM). Absorbance at the Soret bands (404 nm and 407 nm for hemin and PPIX respectively) were reported versus porphyrin concentration corrected for absorbance of free porphyrin at the same concentration. Experiments were done 5 times giving similar curves.

Figure 3. Heme iron acquistion plates.

Growth around hemoglobin-containing wells on Petri dishes seeded with strain SET 538 carrying the indicated plasmids was photographed after 48 h incubation at 37°C. Wells were filled with 50 µl hemoglobin at the following concentrations in µM. Top left: 50; top right: 10; bottom left: 5 and bottom right: 1.

Figure 4. FepB production and cellular localization in strain SET 538 carrying various plasmids.

A: FepB immunodetection in total cell pellets: SET 538 strains harboring pMD1-fepABC, pMD1-fepAB or pMD1-fepB were grown at 37°C in M63 gly supplemented with xyl at two concentrations, 0.2 and 2% as indicated on the figure. Cells were harvested when they reached an OD₆₀₀ = 1. M: FepB precursor proteins purified as inclusion bodies from strain JP313 pBAD24-fepB (the determined amino-terminal sequence was MTNYE); T1: total cell extract of strain SET 538 pMD1-fepABC; T2: total cell extract of strain SET 538 pMD1-fepAB; T3: total cell extract of strain SET 538 pMD1-fepB. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. B: FepB immunodetection in cytoplasmic and periplasmic fractions: SET 538 strains harboring pMD1-fepABC, pMD1-fepAB or pMD1-fepB were grown in M63 gly supplemented with xyl at 2% for the first two strains and 0.2% concentration for the last strain. Cells were harvested when they reached OD₆₀₀ = 1. C1: cytoplasmic fraction of strain SET 538 pMD1-fepABC; C2: cytoplasmic fraction of strain SET 538 pMD1-fepAB; C3: cytoplasmic fraction of strain SET 538 pMD1-fepB. P1: periplasmic fraction of strain SET 538 pMD1-fepABC; P2: periplasmic fraction of strain SET 538 pMD1-fepAB; P3: periplasmic fraction of strain SET 538 pMD1-fepB. FepB is not immunodetected in P1, P2 or P3 periplasmic fractions. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. C: MBP immunodetection in cytoplasmic and periplasmic fractions: The same cytoplasmic (C1, C2, C3) and periplasmic fractions (P1, P2, P3) were immunodetected with anti-MBP antibodies. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. MBP was not immunodetected in C1, C2 or C3 cytoplasmic fractions, but in P1, P2 and P3 periplasmic fractions of the 3 strains.