Fig 1.
MamO promotes the nucleation of magnetosome crystals.
(A) TEM micrograph of a wild-type AMB-1 cell. The electron-dense particles make up a magnetosome chain. (B) Cellular organization of the magnetosome compartment. MamO promotes the nucleation of magnetite within inner membrane invaginations. (C) Domain structure of the three biomineralization factors discussed in the text. “c” represents a CXXCH c-type cytochrome motif.
Fig 2.
Dissection of the MamO catalytic triad.
(A) Magnetic response of cultures with the indicated mamO alleles. The coefficient of magnetism is an optical density-based method assessing cells’ ability to turn in a magnetic field. Biological replicates represent independent cultures of each strain. Each measurement represents the average and standard deviation from three independent experiments. (B) Transmission electron microscopy (TEM) of whole AMB-1 cells with the indicated genetic backgrounds. (C) Magnetite crystals from cells with the indicated mamO alleles. (D) Crystal size distributions for the indicated mamO alleles as assessed by TEM. n > 200 for each strain.
Fig 3.
Genetic requirements for proteolytic processing of biomineralization factors.
Full-length proteins are marked with a circle and proteolytic fragments with a carat. mamEPD refers to the previously described allele with all three catalytic triad residues mutated to alanine. A nonspecific interaction with the anti-MamE antibody is marked with “NS.” (A) Proteolysis of MamE and MamP depends on both MamE and MamO, The MamE active site is required, but the MamO active site is dispensable. (B) Efficient proteolysis of MamE and MamP requires the TauE domain of MamO. In both the ΔOΔR9 strain and the ΔmamO strain (which contains limO), proteolysis of the two targets is minimal.
Fig 4.
Steric constraints on the MamO active site.
(A) Comparison of active site structures, showing MamO in the inactive conformation. Each residue’s chymotrypsin numbering position is in parentheses. Dashes represent hydrogen bonds contributed by the oxyanion hole. (B) Ramachandran plot showing favored (dark shades) and allowed (light shades) geometries for nonglycine residues (blue) and glycine (red). The configuration at residue 193 for a set of trypsin-like structures is plotted. Black squares: inactive conformation; white diamonds: active conformation; red circle: MamO. The active conformation is disallowed for MamO.
Fig 5.
W222 in MamO forces the bound peptide away from the active site.
(A) Fo−Fc omit map contoured at 3σ showing missing density for the peptide in the MamO crystal structure. (B-D) Comparison of peptide binding pockets in HtrA proteases. W222 in MamO blocks the normal exit path between loops LA and LD and pushes the peptide away from the active site. PDB codes for each panel are as follows B: 5HM9; C: 3PV3; D: 3NZI. Loops LA and LD are marked.
Fig 6.
Characterization of the MamO metal binding site.
(A) Anomalous map contoured at 5σ showing the placement of Ni2+ in MamO. (B) Ni2+ ion bound between loop LD and helix 2. (C) H148, H263, and three ordered water molecules participate in metal binding. (D) Transition metal ion Förster Resonance Energy Transfer (tmFRET) analysis of Ni2+ binding by MamO labeled at Q258C. Each measurement represents the average from four replicates. Error bars represent the standard deviation of the replicates. (E) Magnetic response of strains with disrupted metal coordination sites. Error bars represent the standard deviation from three cultures. Each measurement represents the average of three biological replicates. Error bars represent the standard deviation of the replicates. (F) TEM analysis showing that the mamOH148A and mamOH263A strains lack detectable minerals.
Fig 7.
Phylogenetic analysis of magnetotactic trypsin-like proteins.
(A) Phylogeny of the “Deg” branch created from the trimmed Trypsin-2 alignment. MamE and MamO clades and the three Escherichia coli HtrA proteases are marked. (B) Phylogeny of the MamE clade of HtrA proteases. Numbers represent the posterior probability determined by PhyloBayes. Circles represent the degree of support from 300 bootstrap replicates in RAxML. Black: >90% support; white: >80% support. MamE sequences are colored based on the class of the associated organism, and the catalytic triad residues are shown in parentheses after each name.
Fig 8.
MamO in magnetosome formation and evolution.
(A) A dual role in biomineralization. Distinct regions of the protein contribute to each activity separately. The protease domain promotes nucleation by binding iron and the TauE domain manipulates solute conditions that regulate MamE’s activity. (B) Specialization of the trypsin-like protease family in magnetotactic bacteria through gene duplication and subsequent neofunctionalization.