Pseudomonas aeruginosa Bacterioferritin Is Assembled from FtnA and BfrB Subunits with the Relative Proportions Dependent on the Environmental Oxygen Availability

Abstract

Ferritins are iron storage proteins assembled from 24 subunits into a spherical and hollow structure. The genomes of many bacteria harbor genes encoding two types of ferritin-like proteins, the bacterial ferritins (Ftn) and the bacterioferritins (Bfr), which bind heme. The genome of P. aeruginosa PAO1 (like the genomes of many bacteria) contains genes coding for two different types of ferritin-like molecules, ftnA (PA4235) and bfrB (PA3531). The reasons for requiring the presence of two distinct types of iron storage protein in bacterial cells have remained largely unexplained. Attempts to understand this issue in P. aeruginosa through the recombinant expression of the ftnA and bfrB genes in E. coli host cells, coupled to the biochemical and structural characterization of the recombinant 24-mer FtnA and 24-mer BfrB molecules, have shown that each of the recombinant molecules can form an Fe³⁺-mineral core. These observations led to the suggestion that 24-mer FtnA and 24-mer BfrB molecules coexist in P. aeruginosa cells where they share iron storage responsibilities. Herein, we demonstrate that P. aeruginosa utilizes a single heterooligomeric 24-mer Bfr assembled from FtnA and BfrB subunits. The relative content of the FtnA and BfrB subunits in Bfr depends on the O₂ availability during cell culture, such that Bfr isolated from aerobically cultured P. aeruginosa is assembled from a majority of BfrB subunits. In contrast, when the cells are cultured in O₂-limiting conditions, the proportion of FtnA subunits in the isolated Bfr increases significantly and can become the most abundant subunit. Despite the variability in the subunit composition of Bfr, the 24-mer assembly is consistently arranged from FtnA subunit dimers devoid of heme and BfrB subunit dimers each containing a heme molecule.

Keywords: aerobic culture; anaerobic culture; bacterioferritin; ferritin; iron metabolism; iron storage; microaerophilic culture.

Figure 1

Structural comparison of recombinantly expressed BfrB (PDB ID 5D8O) and FtnA (PDB ID 3R2K) proteins. (A) Superposed subunit dimers of BfrB (pale green) and FtnA (slate blue) viewed along the two-fold axis of symmetry; the heme in the BfrB subunit dimer is in magenta. (B) Zoomed-in view of the heme, which, in a BfrB subunit dimer, is axially coordinated by Met52 from each subunit (sulfur atoms in yellow). Met48 in the FtnA subunits is too far from the heme to form axial bonds with the heme iron.

Figure 2

Iron stored in ferritin-like molecules visualized in native polyacrylamide gels stained with Ferene S. BfrB_rec and FtnA_rec were used as electrophoretic mobility standards, loaded as a mixture or alone. Lysates of PAO1 and ΔftnA cells grown aerobically for 24 h in LB media supplemented with 30 µM Fe show an iron-stained band matching that of BfrB_rec, whereas lysates of ΔbfrB cells cultured similarly are characterized by the conspicuous absence of accumulated iron.

Figure 3

Bfr isolated from aerobically cultured P. aeruginosa is a mix of proteins consisting of 24-mer BfrB and 24-mer heterooligomeric Bfr constituted of FtnA and BfrB subunits. (A) Pure Bfr injected on a Source^TM 15Q column eluted in two peaks and a shoulder. (B) The extracted ion chromatogram (EIC) obtained by LC-MS analysis of protein eluted from the Source^TM 15Q in the first peak shows only BfrB (18,552 Da). (C,D) The EICs obtained from LC-MS analysis of protein eluted in the second peak and in the shoulder, respectively, show the presence of FtnA (17,940 Da) and BfrB subunits. The content of FtnA and BfrB subunits in Bfr was estimated from the corresponding integrated peak areas in the EIC, and the values are presented in Table 1.

Figure 4

Bfr isolated from P. aeruginosa cultured in 10% O₂ is a mix of 24-mer heterooligomeric protein constituted of FtnA and BfrB subunits of varying content. (A) Pure Bfr injected on a Source^TM 15Q column eluted in a single, broad peak. The extracted ion chromatograms (EICs) obtained by LC-MS analysis of protein eluted from the Source^TM 15Q column in the left (B), center (C), and right (D) portions of the broad peak show the presence of FtnA (17,940 Da) and BfrB (18,552 Da) subunits. The content of FtnA and BfrB in Bfr was estimated from the corresponding integrated peak areas in the EIC, and the values are presented in Table 1.

Figure 5

(A) Overlay of chromatographic traces from injecting a high-resolution anion exchange column (Source^TM 15Q) with pure bacterioferritin solutions isolated from P. aeruginosa PAO1 cells cultured aerobically (21% O₂ red), in 10% O₂ (blue), in 2% O₂ (green), and anaerobically (pink). The black trace corresponds to Bfr isolated from ΔftnA cells cultured in 21% O₂ and the orange trace corresponds to FtnA_rec. (B) Lysate solutions of PAO1, ΔftnA and ΔbfrB cells cultured under distinct O₂ levels in LB media supplemented with 30 μM Fe were separated in a native gel and stained with Ferene S.

Figure 6

The electronic absorption spectrum of Bfr isolated from P. aeruginosa indicates that heme is axially coordinated by two Met52 residues within BfrB inter-subunit dimers. Spectra shown correspond to Bfr isolated from cells cultured (A) aerobically, (B) micro aerobically in 2% O₂, and (C) anaerobically; the corresponding protein concentrations in mg/mL are 0.7, 0.9, and 0.6. The full spectra were obtained in a 0.2 cm path-length cuvette, and the traces in the insets were measured in a 1.0 cm path-length cuvette. The full spectra highlight the characteristic 418 nm Soret bands and the strong feature with absorption maximum ca. 300 nm, which originated from mineralized Fe³⁺ in the interior cavity. The bottom insets highlight the α- and β-bands (~560 and ~530 nm), and the top insets highlight the weak 740 nm band characteristic of bis-Met heme ligation in bacterioferritin.

Figure 7

In silico model showing one of the possible arrangements in which six FtnA inter-subunit dimers (wheat) and six BfrB inter-subunit dimers (green) assemble into a 24-mer Bfr. Heme (red) is contained between each BfrB inter-subunit dimer. (A,B) views of the heterooligomeric 24-mer Bfr viewed along a four-fold and a three-fold pore, respectively. (C,D) are close-up views of the corresponding three-fold and four-fold pores, which are nearly indistinguishable from the corresponding pores in the structures of FtnA_rec (PDB ID 3R2K) and BfrB_rec (PDB ID 5D8O) used in the model construction (see text). The distances across the four-fold and three-fold pore are 17 and 16 Å, respectively and are indicated by the arrows in (C,D).

Figure 8

Bfd binds Bfr at the surface of BfrB inter-subunit dimers. The association between Bfr and Bfd was studied with the aid of SPR. Reference and baseline-subtracted responses obtained from flowing Bfd solution over: (A) immobilized Bfr containing only BfrB subunits, isolated from cells cultured in 21% O₂ (see Table 1), (B) immobilized Bfr containing 45% FtnA isolated from cells cultured in 2% O₂, and (C) immobilized FtnA_rec. The concentrations of Bfd are 0 µM (black), 1.25 μM (red), 2.5 µM (green), 5 μM (blue), 10 µM (pink), 20 μM (cyan), and 40 μM (grey). The binding affinity was determined by steady-state analysis. (D,E) The steady state responses shown in (A,B), respectively, plotted as a function of Bfd concentration (black circles) and fitted to the binding model (solid line) described by equation 2. (C,F) Bfd does not bind to FtnA_rec.