Characterization of the Fe metalloproteome of a ubiquitous marine heterotroph, Pseudoalteromonas (BB2-AT2): multiple bacterioferritin copies enable significant Fe storage

Abstract

Fe is a critical nutrient to the marine biological pump, which is the process that exports photosynthetically fixed carbon in the upper ocean to the deep ocean. Fe limitation controls photosynthetic activity in major regions of the oceans, and the subsequent degradation of exported photosynthetic material is facilitated particularly by marine heterotrophic bacteria. Despite their importance in the carbon cycle and the scarcity of Fe in seawater, the Fe requirements, storage and cytosolic utilization of these marine heterotrophs has been less studied. Here, we characterized the Fe metallome of Pseudoalteromonas (BB2-AT2). We found that with two copies of bacterioferritin (Bfr), Pseudoalteromonas possesses substantial capacity for luxury uptake of Fe. Fe : C in the whole cell metallome was estimated (assuming C : P stoichiometry ∼51 : 1) to be between ∼83 μmol : mol Fe : C, ∼11 fold higher than prior marine bacteria surveys. Under these replete conditions, other major cytosolic Fe-associated proteins were observed including superoxide dismutase (SodA; with other metal SOD isoforms absent under Fe replete conditions) and catalase (KatG) involved in reactive oxygen stress mitigation and aconitase (AcnB), succinate dehydrogenase (FrdB) and cytochromes (QcrA and Cyt1) involved in respiration. With the aid of singular value decomposition (SVD), we were able to computationally attribute peaks within the metallome to specific metalloprotein contributors. A putative Fe complex TonB transporter associated with the closely related Alteromonas bacterium was found to be abundant within the Pacific Ocean mesopelagic environment. Despite the extreme scarcity of Fe in seawater, the marine heterotroph Pseudoalteromonas has expansive Fe storage capacity and utilization strategies, implying that within detritus and sinking particles environments, there is significant opportunity for Fe acquisition. Together these results imply an evolved dedication of marine Pseudoalteromonas to maintaining an Fe metalloproteome, likely due to its dependence on Fe-based respiratory metabolism.

Fig 1.

The metalloproteomics pipeline, where cells are lysed under native conditions (no detergent employed), and subjected to two dimensions of chromatography: anion exchange chromatography and size exclusion chromatography. The pipeline separates a single biological sample into 384 native samples suitable for protein, metal and enzyme assay analyses. Comparison of protein and metal distributions can elucidate major metal metalloprotein reservoirs within the soluble cellular lysate.

Fig 2.

Station locations for (A) METZYME (KM1128) and (B) ProteOMZ (FK160115) expeditions in the Central Pacific Ocean. Abundance (in total spectral counts) of Alteromonas TonB-dependent Fe complex outermembrane protein (KO2014) obtained from metaproteomic datasets collected on (C) METZYME (latitude on horizontal axis) and (D) ProteOMZ (distance from Station 4 on horizontal axis). Ocean sections were visualized using Ocean Data View and the Ocean Protein Portal.

Fig 3.

(A) Gene neighborhood diagram indicating two unique bacterioferritin (Bfr) proteins and their respective sequences (B) with highlighted portions indicating unique tryptic peptides that were observed in proteomic analysis of Bfr proteins. (C) Comparison of distribution of Fe within the purified cytosolic lysate of BB2-AT2 to that of two distinct Bfr proteins observed by proteomic analysis. (D) Comparison (with selection of exclusively the 450 mM anion exchange fraction, where these proteins were observed in highest abundance) with that of the distribution of Fe in the cytosol provides confidence of the Fe-containing nature of these proteins.

Fig 4.

The repertoire of reactive oxygen species mitigation proteins including, (A) comparison of the distributions of Fe and Mn within the purified cytosolic lysate of BB2-AT2 to that of reactive oxygen stress mitigation proteins, (B) superoxide dismutase (SodA) and (C) catalase/peroxidase (KatG) with comparison of the 450 mM anion exchange fraction (where these proteins were observed in highest abundance) of protein and the corresponding metals.

Fig 5.

(A) The distribution of aconitase (AcnB) observed within the cytosolic metalloproteome of BB2-AT2 and (B) comparison of this protein to the concentration of Fe within the 450 and 500 mM NaCl anion exchange fractions. (C) The distribution of the Fe-S cluster subunit of succinate dehydrogenase (FrdB) observed within the cytosolic metalloproteome of BB2-AT2 and (D) comparison of this protein to the concentration of Fe within the 450 and 500 mM NaCl anion exchange fractions.

Fig 6.

(A) Gene neighborhood diagram indicating three unique cytochrome proteins and (B) comparison of the distribution of ubiquinol-cytochrome c reductase (QcrA) along the 300 mM NaCl anion exchange fraction. (C) The distribution of cytochrome c1 (Cyt1) observed within the metalloproteome and its comparison along the 250 mM NaCl anion exchange fraction.

Fig 7.

Comparison of cell disruption methods within the 450 mM NaCl anion exchange fraction of the Fe metalloproteome of BB2-AT2, including the comparison of AcnB and Bfr₀₆₆₈ with (A) sonication, (B) bead beating and (C) freeze grinding.

Fig 8.

Comparison of the truncated Fe distribution obtained by ICP-MS analysis (A) and the SVD-constructed data of this Fe distribution (B) indicating a good relationship between the data and model of all Fe metalloproteins within BB2-AT2, demonstrating the utility of modeling the major features of a metalloproteomic dataset.