Genetic Dissection of the mamAB and mms6 Operons Reveals a Gene Set Essential for Magnetosome Biogenesis in Magnetospirillum gryphiswaldense

Abstract

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...

Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals

“…The results of biochemical analyses of gene mutants and proteins have indicated that the complex process of magnetosome biosynthesis is routed through multiple steps by using various magnetosome proteins (11-13, 15, 39). A number of proteins involved in the process are derived from invagination of the cytoplasmic membrane, whereas some may be assembled on the magnetosome surface or inserted into the membrane during magnetosome maturation (12,31). The complex magnetosome formation process is considered to require the spatial regulation of magnetosome proteins.…”

“…Thus, the formation of magnetosomes confers a magnetic moment to the cells and allows them to migrate along oxygen gradients in an aquatic environment under the influence of the Earth's geomagnetic field (7). Based on recent molecular analyses in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1, Ͼ30 proteins (magnetosome proteins) have been suggested to be involved in the process of magnetosome formation (11)(12)(13). Moreover, the genes encoding magnetosome proteins are conserved in the genomes of magnetotactic bacteria and appear in the form of a genomic island-like structure (11,12,14) that is specifically referred to as the magnetosome island (MAI).…”

“…Based on recent molecular analyses in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1, Ͼ30 proteins (magnetosome proteins) have been suggested to be involved in the process of magnetosome formation (11-13). Moreover, the genes encoding magnetosome proteins are conserved in the genomes of magnetotactic bacteria and appear in the form of a genomic island-like structure (11,12,14) that is specifically referred to as the magnetosome island (MAI). Through the functions of magnetosome proteins, magnetite formation occurs by means of a process involving multiple steps, including membrane invagination (15, 16), vesicle formation (17, 18), assembly of vesicles into a chain structure (17, …”

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

“…In MSR, deletion of the entire mamXY operon that encodes various functions in redox and transport of iron also caused a mixed phenotype, characterized by smaller, regular-shaped magnetite crystals that were flanked by irregular-shaped tiny flake-like hematite crystals. Unlike deletions of all other magnetosome genes, which consistently resulted in crystal size reductions of various degrees, the loss of mms36 and mms48 (within the mms6 operon) intriguingly caused the synthesis of fewer but larger magnetite crystals (18). Plasmid expression of the entire mamGFDC operon in the wild type (WT) also resulted in the formation of slightly enlarged (by 14.7%) magnetite particles (2).…”

“…In the native host MSR, the large mamAB operon alone is sufficient to sustain rudimentary magnetite biomineralization in the absence of all other magnetosome genes (16). From the 17 mamAB operon genes, a subset of only a few (mamE, mamM, mamO, mamQ, and mamB) is essential for magnetite biomineralization, while in single-deletion mutants of the other genes, biomineralization was impaired to different degrees (18). The smaller mamGFDC, mms6, and mamXY operons are not essential but have accessory roles in controlling the biomineralization of properly sized and shaped crystals (2,16,19).…”