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Lang, Claus (2009): Magnetosome-specific expression of chimeric proteins in Magnetospirillum gryphiswaldense for applications in cell biology and biotechnology. Dissertation, LMU München: Faculty of Biology
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Abstract

Magnetosomes are magnetic nanoparticles that are formed by magnetotactic bacteria (MTB) by a complex, genetically controlled biomineralization process. Magnetosomes from the model organism Magnetospirillum gryphiswaldense consist of single-magnetic-domain sized nanocrystals of chemically pure magnetite, which are formed intracellularly within specialized membranous compartments. The natural coating by the biological membrane and the defined physico-chemical properties designate magnetosomes as a biogenic material with high bio- and nanotechnological potential. In addition, there is a great interest in the cell biology of magnetosome formation in MTB. The development of these true bacterial organelles involves the invagination of distinctly sized membrane vesicles and the assembly of magnetosome vesicles in chain-like arrangements along novel cytoskeletal structures. The first part of this thesis focussed on the development of genetic tools for the functionalization and expression of modified magnetosome proteins. The identification of proteins that are specifically and efficiently inserted into the magnetosome membrane (MM) was facilitated by analysis of green fluorescent protein (GFP) fusions of different magnetosome membrane proteins (MMP). After optimization of cultivation conditions for the utilization of GFP in MTB, it has been demonstrated that fusions of the proteins MamC, MamF and MamG are specifically targeted to the MM. In particular, the MamC-GFP fusion protein was stably integrated and highly abundant in the MM. Therefore, MamC represents an ideal anchor protein for the immobilization of functional proteins in the MM. To address the question, if a specific signal sequence determines the magnetosome specific targeting of MamC-GFP, the localization of truncated MamC derivatives was studied. These experiments have shown that, except for the last nine C-terminal amino acids, the entire sequence is required for the correct targeting and membrane insertion of MamC. Stability of MamC-GFP is greatly reduced if larger parts are missing or if the N-terminus is deleted. MamC-GFP localized at the expected position of the magnetosome chain irrespective of cultivation conditions that impeded magnetite formation. This shows that MMP targeting, magnetosome vesicle formation and magnetosome chain assembly are not dependent on the prevalence of magnetite inducing conditions or the presence of magnetite crystals. In contrast, the localization of MamC-GFP was altered in the magnetic mamK as well as in the non-magnetic MSR-1B, mamB, mamM, mamJKL mutants in comparison to the wild type. This indicates that the interaction with specific proteins in the magnetosome vesicle is required for the correct localization of MamC. The spotted MamC-GFP signals in the mamJ mutant, which are congruent with the position of magnetosomes in this strain, indicate that MamJ is not required for the magnetosome-specific targeting of MamC-GFP. It has also been demonstrated that the native MamC protein and other proteins encoded by the mamGFDC operon are not required for the magnetosome-directed targeting of MamC, as the localization patterns of MamC-GFP in the mamC and mamGFDC mutants were similar to the localization of MamC-GFP in the wild type and congruent with the position of the magnetosomes. The comparison of different promoters from E. coli and M. gryphiswaldense by fluorometry and flow cytometry with a GFP-reporter system revealed that the magnetosomal promoter, PmamDC, is highly efficient in M. gryphiswaldense. The applicability of this promoter for the functionalization of magnetosomes has been demonstrated by expression of a fusion protein of MamC and the antibody binding ‘ZZ’ protein in the MM to generate antibody-binding magnetosomes. In addition, the E. coli Ptet promoter has been identified as the first inducible promoter for regulated gene expression in MTB. The expression was tightly regulated in the absence of an inducer and a ten-fold increase of the proportion of fluorescent cells was observed in the presence of the inducer anhydrotetracycline. Therefore, the Ptet promoter is an important addition to the M. gryphiswaldense genetic toolbox. In the second part of this thesis, magnetosomes were tested for their use in biomedical and biotechnological applications. To this end, large scale procedures for the purification of intact magnetosomes were developed. In collaboration with the groups of Prof. Dr. C. M. Niemeyer (Universität Dortmund) and Dr. R. Wacker (Chimera Biotec), streptavidin-biotin chemistry was employed to develop a modular system for the production of DNA- and antibody-coated magnetosomes. The modified magnetosomes were used in DNA- and protein detection systems, and an automatable magnetosome-based Magneto-Immuno-PCR procedure was developed for the sensitive detection of antigens. With collaborators from the groups of Dr. T. Hieronymus (RWTH Aachen) and Dr. I. Hilger (Universität Jena), it has been shown that magnetosomes can be used as specific magnetic resonance imaging (MRI) contrast agents for phagocytotic cells such as macrophages and dendritic cells to study cell migration. Fluorescently labelled magnetosomes were successfully used as bimodal contrast agents for the visualization of labelled cells by MRI and fluorescence imaging.