Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis
Metadatos
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MDPI
Materia
Bacteria Biomineralization Magnetite Magnetotactic Magnetosome Nanocrystal Proteins TEM
Fecha
2021Referencia bibliográfica
Oestreicher, Z.; Valverde-Tercedor, C.; Mumper, E.; Pérez-Guzmán, L.; Casillas-Ituarte, N.N.; Jimenez-Lopez, C.; Bazylinski, D.A.; Lower, S.K.; Lower, B.H. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis. Crystals 2021, 11, 874. https://doi.org/10.3390/ cryst11080874
Patrocinador
National Science Foundation, grant number EAR-2038207; EAR-1423939; Ministerio de Economía y Competitividad, SPAIN and Fondo Europeo de Desarrollo Regional, FEDER grant numbers CGL2010-18274 and CGL2013-46612Resumen
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.