Set-up of a pharmaceutical cell bank of Magnetospirillum gryphiswaldense MSR1 magnetotactic bacteria producing highly pure magnetosomes.

We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.108 cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.

Nitric oxide sensor NsrR is the key direct regulator of magnetosome formation and nitrogen metabolism in Magnetospirillum.

Nitric oxide (NO) plays an essential role as signaling molecule in regulation of eukaryotic biomineralization, but its role in prokaryotic biomineralization is unknown. Magnetospirillum gryphiswaldense MSR-1, a model strain for studies of prokaryotic biomineralization, has the unique ability to form magnetosomes (magnetic organelles). We demonstrate here that magnetosome biomineralization in MSR-1 requires the presence of NsrRMg (an NO sensor) and a certain level of NO. MSR-1 synthesizes endogenous NO via nitrification-denitrification pathway to activate magnetosome formation. NsrRMg was identified as a global transcriptional regulator that acts as a direct activator of magnetosome gene cluster (MGC) and nitrification genes but as a repressor of denitrification genes. Specific levels of NO modulate DNA-binding ability of NsrRMg to various target promoters, leading to enhancing expression of MGC genes, derepressing denitrification genes, and repressing nitrification genes. These regulatory functions help maintain appropriate endogenous NO level. This study identifies for the first time the key transcriptional regulator of major MGC genes, clarifies the molecular mechanisms underlying NsrR-mediated NO signal transduction in magnetosome formation, and provides a basis for a proposed model of the role of NO in the evolutionary origin of prokaryotic biomineralization processes.

Exploring the host range for genetic transfer of magnetic organelle biosynthesis.

Magnetosomes produced by magnetotactic bacteria have great potential for application in biotechnology and medicine due to their unique physicochemical properties and high biocompatibility. Attempts to transfer the genes for magnetosome biosynthesis into non-magnetic organisms have had mixed results. Here we report on a systematic study to identify key components needed for magnetosome biosynthesis after gene transfer. We transfer magnetosome genes to 25 proteobacterial hosts, generating seven new magnetosome-producing strains. We characterize the recombinant magnetosomes produced by these strains and demonstrate that denitrification and anaerobic photosynthesis are linked to the ability to synthesize magnetosomes upon the gene transfer. In addition, we show that the number of magnetosomes synthesized by a foreign host negatively correlates with the guanine-cytosine content difference between the host and the gene donor. Our findings have profound implications for the generation of magnetized living cells and the potential for transgenic biogenic magnetic nanoparticle production.

Experimental analysis of diverse actin-like proteins from various magnetotactic bacteria by functional expression in Magnetospirillum gryphiswaldense.

IMPORTANCE: To efficiently navigate within the geomagnetic field, magnetotactic bacteria (MTB) align their magnetosome organelles into chains, which are organized by the actin-like MamK protein. Although MamK is the most highly conserved magnetosome protein common to all MTB, its analysis has been confined to a small subgroup owing to the inaccessibility of most MTB. Our study takes advantage of a genetically tractable host where expression of diverse MamK orthologs together with a resurrected MamK LUCA and uncharacterized actin-like Mad28 proteins from deep-branching MTB resulted in gradual restoration of magnetosome chains in various mutants. Our results further indicate the existence of species-specific MamK interactors and shed light on the evolutionary relationships of one of the key proteins associated with bacterial magnetotaxis.

Lipid membrane modulated control of magnetic nanoparticles within bacterial systems.

Bacterial magnetosomes synthesized by the magnetotactic bacterium Magnetospirillum magneticum are suitable for biomedical and biotechnological applications because of their high level of chemical purity of mineral with well-defined morphological features and a biocompatible lipid bilayer coating. However, utilizations of native magnetosomes are not sufficient for maximum effectiveness in many applications as the appropriate particle size differs. In this study, a method to control magnetosome particle size is developed for integration into targeted technological applications. The size and morphology of magnetosome crystals are highly regulated by the complex interactions of magnetosome synthesis-related genes; however, these interactions have not been fully elucidated. In contrast, previous studies have shown a positive correlation between vesicle and crystal sizes. Therefore, control of the magnetosome vesicle size is tuned by modifying the membrane lipid composition. Exogenous phospholipid synthesis pathways have been genetically introduced into M. magneticum. The experimental results show that these phospholipids altered the properties of the magnetosome membrane vesicles, which yielded larger magnetite crystal sizes. The genetic engineering approach presented in this study is shown to be useful for controlling magnetite crystal size without involving complex interactions of magnetosome synthesis-related genes.

Magnetospirillum sulfuroxidans sp. nov., capable of sulfur-dependent lithoautotrophy and a taxonomic reevaluation of the order Rhodospirillales.

A spiral-shaped, highly motile bacterium was isolated from freshwater sulfidic sediment. Strain J10T is a facultative autotroph utilizing sulfide, thiosulfate, and sulfur as the electron donors in microoxic conditions. Despite high 16S rRNA gene sequence sequence identity to Magnetospirillum gryphiswaldense MSR-1 T (99.6 %), digital DNA-DNA hybridisation homology and average nucleotide identity between the two strains was of the different species level (25 % and 83 %, respectively). Strain J10T is not magnetotactic. The DNA G + C content of strain J10T is 61.9 %. The predominant phospholipid ester-linked fatty acids are C18:1ω7, C16:1ω7, and C16:0. Strain J10T (=DSM 23205 T = VKM B-3486 T) is the first strain of the genus Magnetospirillum showing lithoautotrophic growth and is proposed here as a novel species, Magnetospirillum sulfuroxidans sp. nov. In addition, we propose to establish a framework for distinguishing genera and families within the order Rhodospirillales based on phylogenomic analysis using the threshold values for average amino acid identity at Ì´ 72 % for genera and Ì´ 60 % for families. According to this, we propose to divide the existing genus Magnetospirillum into three genera: Magnetospirillum, Paramagnetospirillum, and Phaeospirillum, constituting a separate family Magnetospirillaceae fam. nov. in the order Rhodospirillales. Furthermore, phylogenomic data suggest that this order should accomodate six more new family level groups including Magnetospiraceae fam. nov., Magnetovibrionaceae fam. nov., Dongiaceae fam. nov., Niveispirillaceae fam. nov., Fodinicurvataceae fam. nov., and Oceanibaculaceae fam. nov.

Silent gene clusters encode magnetic organelle biosynthesis in a non-magnetotactic phototrophic bacterium.

Horizontal gene transfer is a powerful source of innovations in prokaryotes that can affect almost any cellular system, including microbial organelles. The formation of magnetosomes, one of the most sophisticated microbial mineral-containing organelles synthesized by magnetotactic bacteria for magnetic navigation in the environment, was also shown to be a horizontally transferrable trait. However, the mechanisms determining the fate of such genes in new hosts are not well understood, since non-adaptive gene acquisitions are typically rapidly lost and become unavailable for observation. This likely explains why gene clusters encoding magnetosome biosynthesis have never been observed in non-magnetotactic bacteria. Here, we report the first discovery of a horizontally inherited dormant gene clusters encoding biosynthesis of magnetosomes in a non-magnetotactic phototrophic bacterium Rhodovastum atsumiense. We show that these clusters were inactivated through transcriptional silencing and antisense RNA regulation, but retain functionality, as several genes were able to complement the orthologous deletions in a remotely related magnetotactic bacterium. The laboratory transfer of foreign magnetosome genes to R. atsumiense was found to endow the strain with magnetosome biosynthesis, but strong negative selection led to rapid loss of this trait upon subcultivation, highlighting the trait instability in this organism. Our results provide insight into the horizontal dissemination of gene clusters encoding complex prokaryotic organelles and illuminate the potential mechanisms of their genomic preservation in a dormant state.

Identifying magnetosome-associated genes in the extended CtrA regulon in Magnetospirillum magneticum AMB-1 using a combinational approach.

Magnetotactic bacteria (MTB) are worth studying because of magnetosome biomineralization. Magnetosome biogenesis in MTB is controlled by multiple genes known as magnetosome-associated genes. Recent advances in bioinformatics provide a unique opportunity for studying functions of magnetosome-associated genes and networks that they are involved in. Furthermore, various types of bioinformatics analyses can also help identify genes associated with magnetosome biogenesis. To predict novel magnetosome-associated genes in the extended CtrA regulon, we analyzed expression data of Magnetospirillum magneticum AMB-1 in the GSE35625 dataset in NCBI GEO. We identified 10 potential magnetosome-associated genes using a combinational approach of differential expression analysis, Gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analysis, protein-protein interaction network analysis and weighted gene co-expression network analysis. Meanwhile, we also discovered and compared two co-expression modules that most known magnetosome-associated genes belong to. Our comparison indicated the importance of energy on regulating co-expression module structures for magnetosome biogenesis. At the last stage of our research, we predicted at least four real magnetosome-associated genes out of 10 potential genes, based on a comparison of evolutionary trees between known and potential magnetosome-associated genes. Because of the discovery of common subtrees that the stressed species are enriched in, we proposed a hypothesis that multiple types of environmental stress can trigger magnetosome evolution in different waters, and therefore its evolution can recur at different times in various locations on earth. Overall, our research provides useful information for identifying new MTB species and understanding magnetosome biogenesis.

McaA and McaB control the dynamic positioning of a bacterial magnetic organelle.

Magnetotactic bacteria are a diverse group of microorganisms that use intracellular chains of ferrimagnetic nanocrystals, produced within magnetosome organelles, to align and navigate along the geomagnetic field. Several conserved genes for magnetosome formation have been described, but the mechanisms leading to distinct species-specific magnetosome chain configurations remain unclear. Here, we show that the fragmented nature of magnetosome chains in Magnetospirillum magneticum AMB-1 is controlled by genes mcaA and mcaB. McaA recognizes the positive curvature of the inner cell membrane, while McaB localizes to magnetosomes. Along with the MamK actin-like cytoskeleton, McaA and McaB create space for addition of new magnetosomes in between pre-existing magnetosomes. Phylogenetic analyses suggest that McaA and McaB homologs are widespread among magnetotactic bacteria and may represent an ancient strategy for magnetosome positioning.

Magnetic Particle Imaging of Magnetotactic Bacteria as Living Contrast Agents Is Improved by Altering Magnetosome Arrangement.

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as imaging agents to differentiate between normal and diseased tissue or track cell movement. Magnetic particle imaging (MPI) detects the magnetic properties of SPIONs, providing quantitative and sensitive image data. MPI performance depends on the size, structure, and composition of nanoparticles. Magnetotactic bacteria produce magnetosomes with properties similar to those of synthetic nanoparticles, and these can be modified by mutating biosynthetic genes. The use of Magnetospirillum gryphiswaldense, MSR-1 with a mamJ deletion, containing clustered magnetosomes instead of typical linear chains, resulted in improved MPI signal and resolution. Bioluminescent MSR-1 with the mamJ deletion were administered into tumor-bearing and healthy mice. In vivo bioluminescence imaging revealed the viability of MSR-1, and MPI detected signals in livers and tumors. The development of living contrast agents offers opportunities for imaging and therapy with multimodality imaging guiding development of these agents by tracking the location, viability, and resulting biological effects.

A Magnetosome-Based Platform for Flow Biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein "cargo" can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space-time yield of 49.2 mmol L-1 h-1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.

Immunological Responses to Transgene-Modified Neural Stem Cells After Transplantation.

Neural stem cell (NSC) therapy is a promising therapeutic strategy for stroke. Researchers have frequently carried out genetic modification or gene editing of stem cells to improve survival or therapeutic function. However, NSC transplantation carries the risk of immune rejection, and genetic modification or gene-editing might further increase this risk. For instance, recent studies have reported on manipulating the stem cell genome and transplantation via the insertion of an exogenous gene derived from magnetotactic bacteria. However, whether transgene-modified stem cells are capable of inducing immunological reactions has not been explored. Although NSCs rarely express the major histocompatibility complex (MHC), they can still cause some immunological issues. To investigate whether transgene-modified NSCs aggravate immunological responses, we detected the changes in peripheral immune organs and intracerebral astrocytes, glial cells, and MHC-I and MHC-II molecules after the injection of GFP-labeled or mms6-GFP-labeled NSCs in a rat model. Xenogeneic human embryonic kidney (HEK-293T) cells were grafted as a positive control group. Our results indicated that xenogeneic cell transplantation resulted in a strong peripheral splenic response, increased astrocytes, enhanced microglial responses, and upregulation of MHC-I and MHC-II expression on the third day of transplantation. But they decreased obviously except Iba-1 positive cells and MHC-II expression. When injection of both mms6-GFP-labeled NSCs and GFP-labeled NSCs also induced similar responses as HEK-293T cells on the third days, but MHC-I and MHC-II expression decreased 3 weeks after transplantation. In addition, mms6 transgene-modified NSCs did not produce peripheral splenic response responses as well as astrocytes, microglial cells, MHC-I and MHC-II positive cells responses when compared with non-modified NSCs. The present study provides preliminary evidence that transgenic modification does not aggravate immunological responses in NSC transplantation.

High-Yield Production, Characterization, and Functionalization of Recombinant Magnetosomes in the Synthetic Bacterium Rhodospirillum rubrum "magneticum".

Recently, the photosynthetic Rhodospirillum rubrum has been endowed with the ability of magnetosome biosynthesis by transfer and expression of biosynthetic gene clusters from the magnetotactic bacterium Magnetospirillum gryphiswaldense. However, the growth conditions for efficient magnetite biomineralization in the synthetic R. rubrum "magneticum", as well as the particles themselves (i.e., structure and composition), have so far not been fully characterized. In this study, different cultivation strategies, particularly the influence of temperature and light intensity, are systematically investigated to achieve optimal magnetosome biosynthesis. Reduced temperatures ≤16 °C and gradual increase in light intensities favor magnetite biomineralization at high rates, suggesting that magnetosome formation might utilize cellular processes, cofactors, and/or pathways that are linked to photosynthetic growth. Magnetosome yields of up to 13.6 mg magnetite per liter cell culture are obtained upon photoheterotrophic large-scale cultivation. Furthermore, it is shown that even more complex, i.e., oligomeric, catalytically active functional moieties like enzyme proteins can be efficiently expressed on the magnetosome surface, thereby enabling the in vivo functionalization by genetic engineering. In summary, it is demonstrated that the synthetic R. rubrum "magneticum" is a suitable host for high-yield magnetosome biosynthesis and the sustainable production of genetically engineered, bioconjugated magnetosomes.

Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system.

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions.

BACKGROUND: Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. RESULTS: We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. CONCLUSION: We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1.

BACKGROUND: Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. RESULTS: Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%. CONCLUSIONS: The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

OxyR-Like Improves Cell Hydrogen Peroxide Tolerance by Participating in Monocyte Chemotaxis and Oxidative Phosphorylation Regulation in Magnetospirillum Gryphiswaldense MSR-1.

The formation of magnetosomes inside magnetotactic bacteria is a complex process strictly controlled by the intracellular metabolic regulatory system. A series of transcriptional regulators are involved in the biosynthesis of the magnetosome, including OxyR-Like protein, which is indispensable for the maturation of magnetosomes in Magnetospirillum Gryphiswaldense MSR-1. In this study, a new function of the OxyR-Like protein that helps cells resist reactive oxygen species (ROS) was identified. A comparison of expression profile data between wild-type MSR-1 and an oxyR-Like defective mutant demonstrated that seven genes encoding chemotaxis proteins were down-regulated in the latter. On the contrary, the expression levels of numerous genes encoding proteins that are critical for cellular aerobic respiration were up-regulated. Thus, OxyR-Like enhanced the resistance of cells to ROS by increasing their environmental perception and maintaining their oxidative phosphorylation at a reasonable level to avoid the excessive production of endogenous ROS. These results increase our knowledge of the OxyR-Like regulatory network and establish a relationship between the antioxidant metabolic pathway and magnetosome biomineralization in MSR-1.

OxyR controls magnetosome formation by regulating magnetosome island (MAI) genes, iron metabolism, and redox state.

Magnetospirillum gryphiswaldense MSR-1 uses chains of magnetosomes, membrane-enveloped magnetite (Fe(II)Fe(III)2O4) nanocrystals, to align along magnetic field. The process of magnetosome biomineralization requires a precise biological control of redox conditions to maintain a balanced amounts of ferric and ferrous iron. Here, we identified functions of the global regulator OxyR (MGMSRv2_4250, OxyR-4250) in MSR-1 during magnetosome formation. OxyR deletion mutant ΔoxyR-4250 displayed reduced magnetic response, and increased levels of intracellular ROS (reactive oxygen species). OxyR-4250 protein upregulated expression of six antioxidant genes (ahpC1, ahpC2, katE, katG, sodB, trxA), four iron metabolism-related regulator genes (fur, irrA, irrB, irrC), a bacterioferritin gene (bfr), and a DNA protection gene (dps). OxyR-4250 was shown, for the first time, to directly regulate magnetosome island (MAI) genes mamGFDC, mamXY, and feoAB1 operons. Taken together, our findings indicate that OxyR-4250 helps maintain a proper redox environment for magnetosome formation by eliminating excess ROS, regulating iron homeostasis and participating in regulation of Fe2+/Fe3+ ratio within the magnetosome vesicle through regulating MAI genes.

Spatiotemporal Organization of Chemotaxis Pathways in Magnetospirillum gryphiswaldense.

Magnetospirillum gryphiswaldense employs iron-rich nanoparticles for magnetic navigation within environmental redox gradients. This behavior termed magneto-aerotaxis was previously shown to rely on the sensory pathway CheOp1, but the precise localization of CheOp1-related chemoreceptor arrays during the cell cycle and its possible interconnection with three other chemotaxis pathways have remained unstudied. Here, we analyzed the localization of chemoreceptor-associated adaptor protein CheW1 and histidine kinase CheA1 by superresolution microscopy in a spatiotemporal manner. CheW1 localized in dynamic clusters that undergo occasional segregation and fusion events at lateral sites of both cell poles. Newly formed smaller clusters originating at midcell before completion of cytokinesis were found to grow in size during the cell cycle. Bipolar CheA1 localization and formation of aerotactic swim halos were affected depending on the fluorescent protein tag, indicating that CheA1 localization is important for aerotaxis. Furthermore, polar CheW1 localization was independent of cheOp2 to cheOp4 but lost in the absence of cheOp1 or cheA1 Results were corroborated by the detection of a direct protein interaction between CheA1 and CheW1 and by the observation that cheOp2- and cheOp3-encoded CheW paralogs localized in spatially distinct smaller clusters at the cell boundary. Although the findings of a minor aerotaxis-related CheOp4 phenotype and weak protein interactions between CheOp1 and CheOp4 by two-hybrid analysis implied that CheW1 and CheW4 might be part of the same chemoreceptor array, CheW4 was localized in spatially distinct polar-lateral arrays independent of CheOp1, suggesting that CheOp1 and CheOp4 are also not connected at the molecular level.IMPORTANCE Magnetotactic bacteria (MTB) use the geomagnetic field for navigation in aquatic redox gradients. However, the highly complex signal transduction networks in these environmental microbes are poorly understood. Here, we analyzed the localization of selected chemotaxis proteins to spatially and temporally resolve chemotaxis array localization in Magnetospirillum gryphiswaldense Our findings suggest that bipolar localization of chemotaxis arrays related to the key signaling pathway CheOp1 is important for aerotaxis and that CheOp1 signaling units assemble independent of the three other chemotaxis pathways present in M. gryphiswaldense Overall, our results provide deeper insights into the complex organization of signaling pathways in MTB and add to the general understanding of environmental bacteria possessing multiple chemotaxis pathways.