Magnetosome-specific expression of chimeric proteins in Magnetospirillum gryphiswaldense for applications in cell biology and biotechnology

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.