UPREGULATION OF MAGNETOSOME PROTEINS IN MAGNETOSPIRILLUM MAGNETICUM (AMB-1) TO MODULATE MAGNETITE CRYSTAL CHARACTERISTICS

Abstract

Potential applications for magnetite crystals include bioremediation, magnetic resonance imaging, and tumour suppression. These require highly pure, and sometimes, specific morphologies. Remarkably, Magnetotactic Bacteria (MTB) produce such crystals at the nanoscale inside organelles called magnetosomes, while retaining ferrimagnetism. The morphology of these biogenic crystals varies because of genetic variation in bacterial biomineralization. This suggests the possibility of genetically engineering MTB to produce high-quality nanocrystals with custom-tailored properties. Mms6, Mms36, Mms48, and MmsF are some key proteins modulating MTB magnetite morphometrics. Current studies use “on/off” approaches to protein overexpression. In contrast, we conducted an incremental IPTG induction assay, varying over-expression of these proteins from four very-low copy plasmids constructed via isothermal DNA assembly and introduced into the MTB Magnetospirillum magneticum (AMB-1, Aerobic Magnetotactic Bacteria) by conjugation. This last step was improved by modifying current plating protocols to accelerate MTB colony formation down from 1 week to 32 hours. Proteins were fused to the fluorescent protein mNeonGreen (mNG), to correlate local protein concentrations with crystals' size, shape, and number using correlative light (fluorescence) and electron microscopy (CLEM). We found three of the constructs significantly affected crystal size, with Mms6-mNG being a positive influencer, while MmsF-mNG and Mms48-mNG were negative influencers. Notably, optimal intermediate induction regimes were observed to cause maximal effects. To characterize these protein interactions with the magnetosome, we performed fluorescence recovery after photobleaching (FRAP) experiments. To this end, we developed a new data acquisition and analysis protocol allowing quantitative measurements of diffusion coefficients for fast proteins in helical-shaped cells like spirilla. The diffusion coefficient in AMB-1 (to our knowledge, the first report for spirilla), was very similar to E. coli. Overall, this work is a first step towards a better characterization of the role and mechanisms of action for several important magnetosome proteins in controlling magnetite crystal morphometrics when over-expressed.