Work Patterns of MamXY Proteins during Magnetosome Formation in Magnetospirillum gryphiswaldense MSR-1

Abstract

The bacterium Magnetospirillum gryphiswaldense MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The mamXY operon, part of the magnetosome island (MAI), includes the mamY, mamX, mamZ, and ftsZ-like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BS2G-d0/d4 reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute “hidden” interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size. IMPORTANCE mamXY operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.

A Compass To Boost Navigation: Cell Biology of Bacterial Magnetotaxis

“…Deletion of ftsZm does not cause a cell or magnetosome chain division phenotype under standard growth conditions. Instead, the mutant seems to have a biomineralization defect in the absence of the anaerobic electron acceptor nitrate (51,109), suggesting that the tubulin-like FtsZm is not a core constituent of the magnetoskeleton in contrast to the actin-like MamK, although its polymerization properties seem conserved (51,110). The second MipZ (midcell positioning of FtsZ) ( 108) protein (encoded outside the magnetosome island) was found to localize to the division plane as well, which stands in stark contrast to its canonical counterpart that forms pole-to-midcell concentration gradients that inhibit FtsZ polymerization (111).…”

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1

“…Antibiotics prepared in culture media were as follows: for E. coli, ampicillin at 100 μg/mL and kanamycin at 50 μg/mL; for MSR-1, kanamycin at 5 μg/mL and nalidixic acid at 5 μg/mL. The growth of MSR-1 (OD 565 ) and the response to a magnet field (Cmag) were detected according to the methods described previously [28].…”

Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes

“…Each of the nine strains was flask-cultured in BMGY/BMMY medium, and target protein expression was induced every 24 h [32]. Samples for total RNA extraction were taken at 48 h and 96 h. Trizol reagent (Tiangen Biotech Co., Beijing, China) was used to isolate total cellular RNA [34]. M-MLV reverse transcriptase (Promega, Madison, WI, USA) was used to reverse transcription of RNA into cDNA [34].…”

“…Protein was eluted with 3 mL buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl) containing successive concentrations of 50, 80, 100, 150, 200, and 250 mM imidazole. Protein concentration was quantified by Bradford assay [34], with bovine serum albumin (BSA) as standard.…”

“…To evaluate the expression trend of target gene involved in protein synthesis and secretion pathway of different strains, hierarchical clustering (HCL) was performed and a distance tree was generated using the multi-experiment viewer (MeV) program according to Wang et al(2013) [34]. A log 2 transformation of an expression fold change between control strain and sample strain was used for each expression element.…”