| Magnetotactic bacteria (MTB) are widespread prokaryotes in nature environment that synthesize a unique organelle called the magnetosome, which are usually nano-sized chain-like magnetic iron crystals, composed of Fe3O4or Fe3S4. In consideration of the characteristics and application prospects of magnetosome, researchers have been focusing on the hot topics about improving the culture level of MTB. However, few axenic culture of MTB have been acquired because of their rigorous requirements on dissolved oxygen and nutrient elements. In addition, the cultivation levels of axenic MTB cultures are far from the production demand. The freshwater Magnetospirillum gryphiswaldense MSR-1and marine magnetic spirillum QH-2are chose as target strains, and some researches are performed on the relation between the changes in cellular physiology and magnetosome formation. The optimized methods to culture freshwater and marine magneto-spirillum are suggested on the basis of research results. Besides, the target strains have magnetosome-formation-associated conserved mamXY operon, on which most genes’functions have been elucidated except for mamX gene. Studies on the function features of mamX and mamXY operon in controlling the mature process of magnetosome are conducted.The freshwater M. gryphiswaldense MSR-1has been domesticated artificially for many years, hence it can have submerged culture on the fermentor. In the process of MSR-1submerged culture, It’s found that:8-20h is the stage when a large quantity of magnetosomes are formed and the dissolved oxygen should be kept between2-20ppb.18-20h is a crucial turning point for cellular physiological metabolism while the carbon source consumption speeds up, some extra fed medium should be added by manual feeding strategy to preserve the regular cell growth. From20h to26h, the concentration of carbon source lactate should be maintained between0.016-0.1g/L, which is helpful for cell growth and magnetosome formation.20-40h is the log phase of cell growth and the phase for magnetosome chain formation and maturing, when should has a dissolved oxygen concentration as20-40ppb to ensure the rapid cell growth. Thus the changes in cellular physiological characteristics are revealed, and the process route for improving MTB culture is proposed.The QH-2strain is the only one marine magneto-spirillum that has axenic culture. In this study, the QH-2cells are cultured using shake cultivation successfully and the original medium is optimized. For QH-2cell, the optimal carbon source is tested to be succinate, optimal nitrogen source being sodium chloride, optimal iron source being ferric citrate, and optimal temperature being25-28℃. The cell culture level is evidently improved under optimized conditions:the OD600value can be over0.6after shake cultivation for48h in liquid medium, about four times higher than before; the generation time is6h,10hours less than before. These results lay foundation for QH-2’s further domestication. Evidently, when the origin of magneto-spirillum differs, the culture condition differs. So it’s the best choice to analyze the cellular physiological characteristics firstly and then find purposeful detail cultivation methods. In order to complete the function illumination of mamλY operon, a deletion mutant and a complemented strain for mamX are constructed. The mutant has normal growth, while synthesizes irregular superparamagnetic Fe3O4particles. Analysis of MamX protein sequence showing that it contains conserved heme-binding motif called Magnetochrome, similar to that of cytochrome c. The MamX protein can bind heme in vitro. Thus MamX is suspected to be involved in the electron transport inside magnetosome membrane, or in the oxidized/reduced process of iron, during magnetosome maturation. The qPCR tests show the transcription rules of the all four genes mamY, mamX, mamZ and ftsZ-like of mamXY operon:in the mamX mutant, mamY and ftsZ-like are up-regulated, while mamZ is down-regulated. The bacterial two hybrid assay shows evident interactions between the four MamXY proteins. Hence a protein association network is drew by STRING tool, indicating FtsZ-like as node. The four proteins are speculated to form protein complex and promote the magnetosome maturation. These results complement the interpretation of mamXY operon’s function, and providing supplementary evidences for further revealing the biomineralization mechanism in magnetotactic bacteria. |
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