Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches.

The synthetic production of monodisperse single magnetic domain nanoparticles at ambient temperature is challenging. In nature, magnetosomes--membrane-bound magnetic nanocrystals with unprecedented magnetic properties--can be biomineralized by magnetotactic bacteria. However, these microbes are difficult to handle. Expression of the underlying biosynthetic pathway from these fastidious microorganisms within other organisms could therefore greatly expand their nanotechnological and biomedical applications. So far, this has been hindered by the structural and genetic complexity of the magnetosome organelle and insufficient knowledge of the biosynthetic functions involved. Here, we show that the ability to biomineralize highly ordered magnetic nanostructures can be transferred to a foreign recipient. Expression of a minimal set of genes from the magnetotactic bacterium Magnetospirillum gryphiswaldense resulted in magnetosome biosynthesis within the photosynthetic model organism Rhodospirillum rubrum. Our findings will enable the sustainable production of tailored magnetic nanostructures in biotechnologically relevant hosts and represent a step towards the endogenous magnetization of various organisms by synthetic biology.

c Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletions resulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those in the wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon contains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by single deletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to those of their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at least rudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for formation of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic iron oxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edge structure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M. gryphiswaldense. Magnetotactic bacteria (MTB) orient along Earth's magnetic field lines to navigate to their growth-favoring microoxic habitats within stratified aquatic sediments (1). This behavior is enabled by the synthesis of ferrimagnetic intracellular organelles termed magnetosomes (2). In the alphaproteobacterium Magnetospirillum gryphiswaldense and related MTB, magnetosomes consist of crystals of the magnetic iron oxide magnetite (Fe 3 O 4 ) enclosed by the magnetosome membrane (MM), which contains a specific set of about 30 proteins (3, 4). The biosynthesis of magnetosomes is a complex process that comprises the (i) invagination of vesicles from the inner membrane (5, 6), (ii) sorting of magnetosome proteins to the MM (7), (iii) iron transport and crystallization of magnetite crystals (8), (iv) crystal maturation (7), and (v) assembly as well as positioning of mature crystals into a linear chain along a filamentous cytoskeletal structure (6, 9). Each step is under strict genetic control, and responsible genes were found to be located mostly within a genomic magnetosome island (MAI) (10, 11), comprising the mms6 operon (mms6 op ), mamGFDC operon (mamGFDC op ), mamAB operon (mamAB op ), and mamXY operon (mamXY op ) (10-12). These operons were found to be highly conserved also in the closely related Magnetospiri...

Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ϳ35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches. IMPORTANCEBefore our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.