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.

The Complex Transcriptional Landscape of Magnetosome Gene Clusters in Magnetospirillum gryphiswaldense

Magnetosomes have emerged as a model system to study prokaryotic organelles and a source of biocompatible magnetic nanoparticles for various biomedical applications. However, the lack of knowledge about the transcriptional organization of magnetosome gene clusters has severely impeded the engineering, manipulation, and transfer of this highly complex biosynthetic pathway into other organisms.

OxyR-Like Improve Cell's Hydrogen Peroxide Tolerance via Participate in Monocyte Chemotaxis and Oxidative Phosphorylation Regulation in Magnetospirillum Gryphiswaldense MSR-1

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Multifunktionale bakterielle Nanomagnete für Biotechnologie und Medizin

Bacterial magnetosomes represent magnetic core-shell nanoparticles biomineralized by magnetotactic bacteria like Magnetospirillum gryphiswaldense. The establishment of fermentation regimes for high-yield particle production, standardized isolation procedures as well as the development of a genetic toolkit for the generation of “tailored” particles might soon pave the way for the application of engineered magnetosomes in the biomedical and biotechnological field.

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.