Regulation of iron metabolism and magnetite formation in Magnetospirillum gryphiswaldense

Magnetotactic bacteria synthesize specific organelles, the magnetosomes, which are membrane-enveloped crystals of the magnetic mineral magnetite (Fe3O4). The biomineralization of magnetite involves the uptake and intracellular accumulation of large amounts of iron. However, it is not clear how iron uptake and biomineralization is regulated and balanced with the biochemical iron requirement and intracellular homeostasis. In this proposal, we plan to identify major routes for uptake and intracellular storage of iron, and we shall analyse their regulation and interconnection with magnetite biomineralization by an interdisciplinary and collaborative approach. Mutants which are deleted in genes for various putative iron uptake systems as well as for proteins from the Fur-like family impli-cated in global iron regulation will be analyzed with respect to growth, iron accumulation, and magne-tite biomineralization. Iron metabolites will be identified by quantitative in situ Mössbauer fingerprint techniques. In addition, isolation of magnetosomes, membranes and cytoplasmic fractions will provide samples for detailed structural analysis by Mössbauer spectroscopy, nuclear forward scattering(NFS), XAS spectroscopy, and nuclear inelastic scattering (NIS) in whole and fractionated cells. In particular, we want to employ the new P01-beam line at DESY (Hamburg) allowing NRS with very small sample volumes (1μl) and local resolution to approx. 50nm. Biochemical and genetic analysis will then be used to identify and functionally characterize an elusive membrane-bound iron storage compound that has been implicated in magnetite biomineralization by previous Mössbauer experiments. Our studies will for the first time reveal a detailed and comprehensive picture of iron metabolism in magnetotactic bacteria and its integration with magnetosome biosynthesis at the molecular and mechanistic level. The results are expected to not only improve our understanding of pathways for magnetite biomineralization, but will also facilitate the biogenic synthesis and application of magnetic nanoparticles.

Within this project we aimed to characterize the iron uptake routes for biochemical requirements and magnetosome formation. Deletion mutagenesis and careful characterization of the resulting iron uptake mutants revealed that iron for magnetosome formation is at least partially transported through the cytoplasm before accumulation in magnetosome vesicles. Additionally, the synthetic lethal effect of the co-deletion of the two cytoplasmic membrane iron transporters FeoB2 and Mgr0234 suggests the presence of distinct or only poorly connected iron pools for magnetosome formation and biochemical requirements. Next we analyzed candidate proteins required for the magnetosome-directed iron transport. While our data showed that MagA is not involved in magnetosome formation we obtained indirect evidence for an iron transporting activity by the CDF transporter MamM using sitedirected mutagenesis. We also showed that the second magnetosomal CDF protein MamB is required for magnetosome vesicle formation by an unknown mechanism. Furthermore, we showed that MamM directly interacts with MamB and thereby stabilizes it or protects it from degradation as in a mamM deletion mutant MamB levels were found to be drastically reduced. There exists a continuing dispute about the biogenesis of magnetosomes and of magnetite biosynthesis in magnetotactic bacteria (MTB). In particular, the existence, nature and location of possible mineral precursors in the pathway leading to the biosynthesis of magnetite is not clear. One of the possible precursors has been assumed to be bacterioferritin (Bfr). Here, isolation and characterization of bacterioferritin of Magnetospirillum gryphiswaldense (BfrMg) is described employing recombinant techniques. In contrast to typical representatives of the Bfr subgroup of ferritins, characterization of BfrMg disclosed a very atypical heterododecameric assembly of Bfr1Mg and Bfr2Mg subunits. Their amino acid sequences suggest two different functions. Subunit 1 harbors a ferroxidase function. Subunit 2 enables an intersubunit binding of heme. Untypically, BfrMg is membrane-associated. In contrast to phosphate rich amorphous minerals, generally observed in bacterial ferritins, the holoprotein shell of BfrMg harbors 180-360 Fe3+ ions bound as ferrihydrite similar to mammalian ferritins as shown by transmissiob Mössbauer spectroscopy(TMS). Moreover, in situ TMS of a ΔBfrMg mutant uncovered that the presence of ferrihydrite is coupled to the presence of BfrMg. Lack of BfrMg has no impact on magnetite biosynthesis in M. gryphiswaldense (TEM, TMS). As a consequence, ferrihydrite bound in BfrMg is not a precursor of magnetite biosynthesis. A function of BfrMg is found in DNA protection under conditions of oxidative stress.