Isolation of pre-magnetic magnetosome membrane vesicles by synthetic affinity tags

Magnetosomes are sensory organelles which are formed by magnetotactic bacteria and consist of magnetite crystals (~ 40 nm) enveloped by a proteinaceous lipid-bilayer membrane. In the alphaprotebacterium Magnetospirillum gryphiswaldense magnetosomes are formed by a genetically encoded pathway, which starts with the invagination of empty magnetosome membrane vesicles (eMMs) from the cytoplasmic membrane, probably simultaneous with the targeting of ca. 30 MM-specific proteins, and followed by iron uptake and biomineralization of the magnetite core [1, 2]. While purification and analysis of mature magnetite-containing magnetosomes (magMM) by sedimentation and magnetic separation is relatively straightforward, isolation of pre-magnetic eMMs has proven to be challenging due to their low abundance and the lack of magnetic and density tags. Thus, little is known about the early stages of magnetosome biosynthesis represented by eMMs.

In this study, we assessed if eMMs can be purified using genetically encoded affinity tags. To this end, a multi-adaptor construct was assembled, which comprises multiple tags, molecular adaptors and fluorescence reporters. A MBP/His6-Tag hybrid protein was fused to the abundant magnetosome protein MamG as an eMM anchor, while an eGFP-SpyTag-Twin-StrepTag protein [3] was fused to the MamC protein. Upon expression of the construct in mutant strains of M. gryphiswaldense producing eMMs, chromatography of cell extracts using complementary affinity columns yielded conspicuous spherical membranous structures, which were absent in similar preparations from mutant strains expressing the same construct but incapable of eMM formation. Analysis of these vesicles by various imaging and biochemical methods revealed a similar size and appearance, but somewhat distinct protein composition as compared to magMMs.

In summary, our findings seem to indicate that pre-magnetic eMMs vesicles can be isolated. In future approaches, this may set the stage for their use as novel engineerable bacterial membrane vesicles with potential for various applications.

[1] Uebe R., Schüler D., Magnetosome biogenesis in magnetotactic bacteria, Nat. Rev. Microbiol. 2016, 14, 621

[2] Raschdorf O. et al., Genetic and ultrastructural analysis reveals the key players and initial steps of bacterial magnetosome membrane biogenesis, PLoS Genet. 2016, 12, e1006101

[3] Mickoleit F. et al., A versatile magnetic nanoplatform for plug-and-play functionalization, ACS Nano 2024, 18, 27974