Abstract
Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.
Highlights
Magnetotactic bacteria (MTB) are a group of prokaryotes, which biomineralize magnetic crystals of magnetite (Fe3O4) and/or greigite (Fe3S4) surrounded by a lipid-bilayer, called the magnetosome membrane (MM) [1,2,3,4]
Cloning the polymerase chain reaction (PCR) product into the pET160/GW/D-TOPO vector yielded a heterologous expressed protein, which preferentially accumulated in E. coli cells as inclusion bodies and had to be solubilized and purified using urea
Peptide 1 was from the N-terminal end of Mms13 and peptides 2–4 were from the C-terminal end of Mms13 (Figure 2c). These results show that the mature Mms13 protein in M. magneticum AMB-1 contains at the very least amino acid residues 33–145 (Figure 2c)
Summary
Magnetotactic bacteria (MTB) are a group of prokaryotes, which biomineralize magnetic crystals of magnetite (Fe3O4) and/or greigite (Fe3S4) surrounded by a lipid-bilayer, called the magnetosome membrane (MM) [1,2,3,4]. Previous studies have shown that the MM contains proteins required for the biogenesis of intracellular magnetosomes and that the MM appears to be derived from the cytoplasmic membrane [9,12,13,15,16,17,18,19]. Some of these MM proteins are found in the cytoplasmic membrane and fatty acid analysis suggests that the MM is derived from the cytoplasmic membrane [13,16,19]. In Magnetospirillum magneticum AMB-1, genomic and proteomic analysis have identified almost 100 magnetosome specific proteins, and at least a dozen of these proteins are believed to be involved in magnetosome biomineralization [3,4,16,22]
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