Control of magnetite nanocrystal morphology in magnetotactic bacteria by regulation of mms7 gene expression

Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology.


Construction of mms7 gene-inducible expression vectors
For construction of pUMPmms7tORM7, the gene encoding the tetracycline repressor of E. coli (TetR) and an msp3 promoter (Pmsp3) was amplified as a template using the primers Msp3_F_EcoRI and TetR_R_EcoRI and pUMtOR 1 . These amplified fragments were digested and ligated in pUMG 2 , generating the pUMR. The sequences 17 bp upstream of the putative 35, and the 10 promoter consensus sequences of Pmms7, which is located upstream of the mamGFDC operon, were replaced with tetracycline operator elements 3 . This Pmms7 derivative, named Pmms7 tetO , was amplified using the primers Pmms7(TetO)_F and Pmms7(TetO)_R_SspI, with the artificial synthetic DNA (TaKaRa) as a template. The PCR-amplified fragment of Pmms7(TetO) was cloned into the SspI site in pUMR, generating the pUMPmms7tOR. The mms7 sequence was also amplified using the primers mms7_F and mms7_R, cloned into the SspI site in pUMPmms7tOR, generating pUMPmms7tORM7. For construction of pUMtORM7, the PCR-amplified mms7 sequence was cloned into pUMtOR. Plasmids were transformed into wild-type M. magneticum AMB-1 by electroporation and a colony formation experiment was performed as previously described 2 .

Quantitative real-time PCR
Total RNA was extracted from wild-type and mms7 gene-inducible strains cultured at middle logarithmic growth phase, using Trizol reagent (Life Technologies). Genomic DNA was removed from total RNA by PureLink® DNase Set (Life Technologies). RNA quality and quantity were checked by Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip Kit. Total RNA (1 μg) was used to synthesize cDNA with a 1st strand Synthesis Kit (TaKaRa).
qRT-PCR was performed in a ViiA™7 Real-Time PCR System (Applied Biosystems), with Fast SYBR® Green Master Mix (Applied Biosystems) according to the manufacture instructions. Primer sets using qRT-PCR are shown in Table S1. cDNA samples of 1 μL were used as templates in 20 μL of PCR reagents.

Magnetosome protein profile analysis by Tricine SDS-PAGE
Isolation of the magnetosome, cytoplasm-periplasm, and cell membrane was performed 4 . The purified magnetosomes were boiled in 1% SDS solution to extract proteins from the surface of the magnetite crystals. Tricine SDS-PAGE was performed according to the method described by Schagger 5 . Gels were stained with Bio-Safe Coomassie G-250 (Bio-Rad). Expression levels of Mms7 and Mms13 were measured with ImageQuant TL software (GE Healthcare) and compared between the wild-type strain and the mms7-inducible strain cultured with 500 ng/mL ATc.

Western blotting analysis
For the determination of Mms7 protein expression in the gene-inducible strain, the ΔSID25 Δmms7 harboring pUMtORM7his, which expresses His-tag-fused Mms7, was constructed. The magnetosome protein fraction was extracted from this strain and analyzed by western blotting. The fraction was separated by Tricine SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Target proteins were detected using His-tag with alkaline phosphatase (AP)-conjugated antipolyhistidine tag antibody (1:2000 dilution in 1% PBST), purchased from Sigma Aldrich Inc.
5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT,Sigma) was used as the AP colorimetric substrate for visualization.  Data represent the mean ± standard deviation. The shape factor was calculated as the minor axis divided by the major axis (minor/major axis). At least 184 crystals were measured for each strain. Minor axis a in the crystals