【作者】 王小柯；

【导师】 吴龙飞； 宋涛； 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2007， 博士

【摘要】 趋磁细菌(Magnetotactic bacteria)细胞内含有磁小体,磁颗粒为单磁畴的铁氧/硫化物(Fe3O4或Fe3S4)晶体,每个磁颗粒由生物膜包裹,沿细胞的长轴排列形成磁小体链。在自然环境中,地磁场作用于磁小体链产生磁力矩,使趋磁细菌沿磁力线游动,表现出趋磁特性。磁颗粒大小均匀,50~100nm,是极好的纳米永磁材料,在材料、生物医学、电子、光学、磁学、能量存贮和电化学领域具有巨大的潜在应用。磁小体膜由细胞内膜凹陷形成,磁小体膜上至少含有48种特有蛋白,其中至少13种与磁小体形成密切相关。magA、mms6、mamA和mms13基因在磁小体形成过程中Fe3+摄取、磁晶体形成、磁小体囊泡活化和磁小体形成等重要环节起不可缺少的作用。因此,这四个基因是具代表性的磁小体形成相关基因。磁小体具有感应地磁场定向趋磁运动的功能,外部磁场变化对其形成应存在影响。为研究磁场对磁小体形成的影响,从细胞和分子水平探讨外部磁场影响磁小体形成的机制,以趋磁螺菌AMB-1为对象,研究了磁场对磁小体形成及其相关基因表达的影响。为研究地磁场、外部磁场强度、频率、方向变化和AMB-1菌体内部磁小体磁场与磁小体形成的关系,选择了补偿式零磁空间(<500nT)、强恒磁场(0.2T)、低频脉冲磁场(50Hz 1mT)和低频正弦磁场(50Hz 1mT)分别处理不同初始状态的趋磁螺菌AMB-1,应用OD600和Cmag测量、透射电镜观察和qRT-PCR技术,研究磁场对磁小体形成及相关基因mamA、mms13、mms6和magA表达的影响。结果表明:补偿式零磁空间(<500nT)能延迟指数期AMB-1磁小体的形成,当初始菌种为含有磁小体的AMB-1时,零磁空间对指数期AMB-1菌体的mms13基因有上调表达作用,同时下调mms6基因表达,而当初始菌种为不含磁小体的AMB-1时,则只上调指数期菌体的mms13基因表达。零磁空间下,菌体内磁小体链单元数较地磁对照少,只含有1~2个链单元。含磁小体菌体的平均磁颗粒数也低于地磁场10%;接种含磁小体种子时,菌体内磁小体链单元中部磁小体颗粒大而链两端逐渐减小,接种不含磁小体的种子,磁小体链单元磁颗粒均匀,表明菌体内原有的单磁畴晶体对邻近磁晶体的形成有诱导作用。强恒磁场(0.2T)可抑制趋磁螺菌AMB-1菌体生长,但促进其磁小体形成,上调指数期菌体的mms13基因的表达;而对mamA、mms6和magA基因表达无影响。强恒磁场虽然能使磁小体颗粒增大,含磁小体菌的平均磁颗粒数较地磁场增加29%,但导致磁小体排列不整齐,可能是内部磁小体与外部强磁场相互作用影响了相邻磁小体的形成和排列;脉冲磁场(50Hz 1mT)可促进趋磁螺细菌AMB-1磁小体形成。当初始菌种含有磁小体时,脉冲磁场促进指数期菌体magA基因的表达,而当初始菌种不含磁小体时,则促进指数期菌体magA和mamA基因的表达。脉冲磁场虽然导致指数期含磁小体菌体的平均磁颗粒数较地磁场增加25%,但使磁颗粒大小不均匀,磁小体链增长,可能是邻近磁小体的相互诱导聚集作用受到了脉冲磁场干扰。正弦磁场( 50Hz 1mT)抑制趋磁螺细菌AMB-1菌体增殖,促进其磁小体形成。当初始菌种含有磁小体时,正弦磁场促进指数期菌体mms6基因的表达,而当初始菌种不含磁小体时,则促进指数期菌体magA、mms6和mamA基因的表达。正弦磁场虽然导致指数期含磁小体菌体的平均磁颗粒数高于地磁场11%;磁小体颗粒总体上仍沿细胞长轴线性排列。但相邻磁颗粒排列不整齐,形成的短链走向不一致。可能是正弦磁场不断变化的磁场强度和磁场方向导致新生磁小体磁极转换,影响磁小体链的排列。实验结果为从细胞和分子水平研究外部磁场影响磁小体形成的机制,应用磁场干预磁小体形成,提高磁小体产率的研究提供了实验依据,也有助于进一步了解磁场的生物学效应。更多还原

【Abstract】 Magnetic bacteria synthesize intracellular magnetosomes, which of nano-sized crystals of magnetic iron minerals inside membrane vesicles. Magnetosomes aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients, along the Earth’s magnetic field lines, a behavior referred to as magnetotaxis. The superior crystalline and magnetic properties of magnetosomes have drawn attention for their potential use in bioscience, medicine and related disciplines and geobiology. Magnetosomes membrane is derived from the cytoplasmic membrane. Forty-eight proteins are identified as magnetosome specific proteins in Magnetospirillum magneticum AMB-1, and at least 13 proteins are potentially involved in formation of magnetosome. MagA,mms6,mamA and mms13 are the genes involved in iron uptake, priming and trafficking of budding vesicles,magnetite biomineralization and formation of magnetosome.Magnetosomes contain single domain magnetite crystals. Magnetic field may affect the formation of magnetosome. To investigate effects of magnetic field on magnetosome formation in Magnetospirillum magneticum AMB-1 <500nT magnetic free field space, 0.2 T constant-strength magnetic field, 50Hz-1mT pulse magnetic field and 50Hz-1mT sinusoidal magnetic field were applied to cellular cultures. Magnetic and non-magnetic pre-cultures were prepared by controlling growth conditions. They were inoculated into various growth media and incubated under different magnetic fields or geomagnetic field. Magnetism of cells was measured by using spectrophotometer coupled with applied magnetic fields and the values were described as Cmag. Magnetosome in cells were inspected and counted by transmission electron microscopy (TEM) observation. The expression of mamA, mms13, mms6 and magA was analyzed by qRT-PCR.The results showed that the magnetic free field space up-regulated the mms13 expression and down-regulated the mms6 in the cultures inoculated with magnetic cells. Only mms13 expression was up-regulated in the cultures inoculated with non-magnetic cells. Magnetic free field space seemed to postpone magnetosome formation, compared to geomagnetic field. In addition, the amount of the magnetosome chain and the average amount of magnetosome in M. magneticum AMB-1 contained magnetosome appeared decreased when magnetic free field space was applied to the cultures. The size of magnetic crystals at the middle of the magnetosome chain was big, and it was smaller gradually at the two ends of the magnetosome chain. It was likely that the existed magnetic crystals in AMB-1 could induce the formation of the new neighboring magnetosme under this condition. The magnetic crystals were homogeneous in the cultures inoculated with non-magnetic cells, suggesting that magnetite precipitation begins simultaneously from the same location within magnetosome vesicles. It also supports the hypothesis that existing magnetic crystal may influence formation of neighboring crystals.Comparing to geomagnetic field, constant-strength magnetic field impair cellular growth, but seemed to enhance magnetosome formation and up-regulated mms13 expression. The homogeneity of the magnetosome morphology was decreased but the size of the magnetosome and the average amount of magnetosome in M. magneticum AMB-1 contained magnetosome were increased. It is likely that the interaction of the magnetic field created by magnetosome in AMB-1 and the imposed magnetic field could affect the size and arrangement of the neighboring magnetosome.The pulse magnetic field up-regulated magA expression in the cultures inoculated with magnetic cells, and magA, mamA expression in the cultures inoculated with non-magnetic cells. Comparison with geomagnetic field, pulse magnetic field did not affect cellular growth, but seemed to enhance magnetosome formation. The length of the magnetosome chain appeared increased and homogeneity of the magnetosome morphology was decreased when pulse magnetic field was applied to the cultures. It is likely that magnetite precipitation induced by the neighboring magnetosome was affected by pulse magnetic field.Sinusoidal magnetic field up-regulated mms6 expression in the cultures inoculated with magnetic cells, and magA, mms6 and mamA expression in the cultures inoculated with non-magnetic cells. Sinusoidal magnetic field impaired cellular growth, but seemed to enhance magnetosome formation, compared to geomagnetic field. In addition, the homogeneity of the magnetosome morphology and linearity of magnetosome chains were impaired, but the average amount of magnetosome in M. magneticum AMB-1 contained magnetosome appeared increased when sinusoidal magnetic field applied to the cultures. It is likely that variable intensity and alternating orientation of sinusoidal magnetic field result in magnetic pole conversion in the new forming magnetosome, which affect the arrangement of the magnetosome.These results would contribute to further studies on the molecular mechanism of the effects of magnetic fields on formation of magnetosome, biotechnological application studies in magnetotactic bacteria, and the further understandings of the biological effects of magnetic fields.更多还原