趋磁细菌AMB-1培养条件优化及磁小体变化过程研究
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摘要
趋磁细菌(Magnetotactic bacteria)的研究是国际微生物学研究热点之一。趋磁细菌体内含有纳米单磁畴的氧化铁/硫化铁(Fe3O4或Fe3S4)晶体,称为磁小体。由于趋磁细菌营养条件要求苛刻,在环境中需要微好氧条件,且营养类型属于化能自养,使得培养趋磁细菌时常遇到问题。
本研究首先通过正交试验优化趋磁细菌AMB-1菌株培养条件,在培养条件铁源为奎尼酸铁0.02 mmol/L,装瓶量75% ,pH值6.7,温度25℃时,AMB-1 OD600达到0.440(1.166×109 cells/ml)。同时运用磁收集传代法,使带有磁小体的AMB-1细胞比例占95%以上(Cmag值稳定在1.9-2.0)。
在AMB-1具有较好的生物量,同时又具有较好的含磁小体细胞比例后,研究磁小体的变化过程。通过透射电镜观察磁小体变化过程,发现培养24 h细菌体内已有较小晶体形成(平均27 nm,n=188)且沿长轴分布;48 h晶体长大(平均43 nm,n=203)且形成分段链沿长轴排列;72 h晶体进一步成熟(平均50 nm,n=191)仍以分段链沿长轴排列;随后细菌逐渐衰亡磁小体变小,168 h可见部分自溶细菌中仍有磁小体链(平均37 nm,n=186);192 h细菌自溶磁小体链(平均33 nm,n=184)分散到环境中。
通过透射电镜在细胞水平上研究趋磁细菌细胞分裂时发现,磁小体在细菌分裂时采用两种分离方式:一种为磁小体分配到两个子细胞;另一种为磁小体只分配到一个子细胞。无磁小体的子细胞,在随后的生长过程又分为两种情况:一种为细胞逐渐产生磁小体,另一种为不再产生磁小体。这种现象的发现,解释了随着传代次数的增多,细菌磁性有所下降的原因(Cmag值降低)。
在对趋磁细菌磁小体合成机制的研究中,常使用基因敲除的办法获得缺陷型,并与野生型对比进行研究。但是,利用基因敲除获得缺陷型不仅操作繁琐并且所得缺陷型不稳定。本研究利用特殊的磁富集传代法,先将带有磁小体的菌体收集并连续传代,筛选获得了高磁菌株;利用这种方法,收集不含磁小体的菌体并连续传代,筛选获得了无磁菌株。
趋磁细菌磁小体在医疗、环保等领域具有广阔应用价值,但是目前由于趋磁细菌难以大规模培养,并且磁小体纯化存在成本高等原因,将磁小体真正实际应用尚有一段距离。通过研究磁小体在趋磁细菌中的变化过程发现,AMB-1菌株在培养192 h后自溶,并且磁小体随着细胞的破碎释放到环境中去。
Magnetotactic bacteria research is hot spot in microbiology field. Magnetotactic bacteria have nano single-domain magnetite/greigite (Fe3O4/Fe3S4), which are called magnetosome. There are problems in culturing magnetotactic bacteria bacause they need chemo-autolithotrophic and microaerobic growth conditions.
Growth conditions of Magnetospirillum magneticum AMB-1 were optimized by orthogonal experimental design. When culture conditions were chosen with ferric quinine 0.02 mmol/L, 75% volume, pH 6.7 and 25℃, OD600 values reached 0.440 (1.166×109 cells/ml). AMB-1 cells were all collected by magnet before inoculation. After magnetic enrichment and cultivation for several times, Cmag values of AMB-1 reached 1.9-2.0 steadily.
The process of magnetosome assembling was studied after optimization, that AMB-1 had high production with many magnetosomes. During cultivating process, magnetosomes were characterized by Transmission Electron Microscope (TEM). As a result, smaller crystals (average 27 nm, n=188) were observed in bacteria after 24 h incubation. Over 48 h, the size of crystals increased (43 nm, n=203) and MS formed several chains arranging along major axes. After 72 h, crystals maturated (50 nm, n=191) and after 168 h, part-autolyzing bacteria with magnetosomes can be detected. Over 192 h, the bacteria autolyzed and magnetosomes were released to the media.
There were tow ways of magnetosome chain separating modes in dividing AMB-1 cells. One is symmetric separation into the daughter cells. The other is asymmetric separation and only one daughter cell was heritor of the magnetosomes. Part of non-magnetosome daughter cells might produce magnetosomes later. These phenomena explained why magnetism of magnetotactic bacteria decreased with more multiplications (Cmag value decreased).
Method of deleting some kind of genes was usually used in research of magnetosomes synthesis. Comparing wild strain with gene mutation strain can find out the function of the gene. However, this method need many steps and the strain of mutation can not inherit stably. Special method of magnetic cells collected to inoculation was used to screen out strains with and without magnetosomes. Such kind of strains was obtained, and they could inherit stably.
Magnetosomes have applying valve in medical treatment, environmental protecting etc. However, it is a long way to put magnetosomes to application. Because magnetotactic bacteria are difficult to culture, and costs of pure magnetosomes are high. During the investigating process of magnetosome assembling, magnetosomes were set free after 192 h culture.
本研究首先通过正交试验优化趋磁细菌AMB-1菌株培养条件,在培养条件铁源为奎尼酸铁0.02 mmol/L,装瓶量75% ,pH值6.7,温度25℃时,AMB-1 OD600达到0.440(1.166×109 cells/ml)。同时运用磁收集传代法,使带有磁小体的AMB-1细胞比例占95%以上(Cmag值稳定在1.9-2.0)。
在AMB-1具有较好的生物量,同时又具有较好的含磁小体细胞比例后,研究磁小体的变化过程。通过透射电镜观察磁小体变化过程,发现培养24 h细菌体内已有较小晶体形成(平均27 nm,n=188)且沿长轴分布;48 h晶体长大(平均43 nm,n=203)且形成分段链沿长轴排列;72 h晶体进一步成熟(平均50 nm,n=191)仍以分段链沿长轴排列;随后细菌逐渐衰亡磁小体变小,168 h可见部分自溶细菌中仍有磁小体链(平均37 nm,n=186);192 h细菌自溶磁小体链(平均33 nm,n=184)分散到环境中。
通过透射电镜在细胞水平上研究趋磁细菌细胞分裂时发现,磁小体在细菌分裂时采用两种分离方式:一种为磁小体分配到两个子细胞;另一种为磁小体只分配到一个子细胞。无磁小体的子细胞,在随后的生长过程又分为两种情况:一种为细胞逐渐产生磁小体,另一种为不再产生磁小体。这种现象的发现,解释了随着传代次数的增多,细菌磁性有所下降的原因(Cmag值降低)。
在对趋磁细菌磁小体合成机制的研究中,常使用基因敲除的办法获得缺陷型,并与野生型对比进行研究。但是,利用基因敲除获得缺陷型不仅操作繁琐并且所得缺陷型不稳定。本研究利用特殊的磁富集传代法,先将带有磁小体的菌体收集并连续传代,筛选获得了高磁菌株;利用这种方法,收集不含磁小体的菌体并连续传代,筛选获得了无磁菌株。
趋磁细菌磁小体在医疗、环保等领域具有广阔应用价值,但是目前由于趋磁细菌难以大规模培养,并且磁小体纯化存在成本高等原因,将磁小体真正实际应用尚有一段距离。通过研究磁小体在趋磁细菌中的变化过程发现,AMB-1菌株在培养192 h后自溶,并且磁小体随着细胞的破碎释放到环境中去。
Magnetotactic bacteria research is hot spot in microbiology field. Magnetotactic bacteria have nano single-domain magnetite/greigite (Fe3O4/Fe3S4), which are called magnetosome. There are problems in culturing magnetotactic bacteria bacause they need chemo-autolithotrophic and microaerobic growth conditions.
Growth conditions of Magnetospirillum magneticum AMB-1 were optimized by orthogonal experimental design. When culture conditions were chosen with ferric quinine 0.02 mmol/L, 75% volume, pH 6.7 and 25℃, OD600 values reached 0.440 (1.166×109 cells/ml). AMB-1 cells were all collected by magnet before inoculation. After magnetic enrichment and cultivation for several times, Cmag values of AMB-1 reached 1.9-2.0 steadily.
The process of magnetosome assembling was studied after optimization, that AMB-1 had high production with many magnetosomes. During cultivating process, magnetosomes were characterized by Transmission Electron Microscope (TEM). As a result, smaller crystals (average 27 nm, n=188) were observed in bacteria after 24 h incubation. Over 48 h, the size of crystals increased (43 nm, n=203) and MS formed several chains arranging along major axes. After 72 h, crystals maturated (50 nm, n=191) and after 168 h, part-autolyzing bacteria with magnetosomes can be detected. Over 192 h, the bacteria autolyzed and magnetosomes were released to the media.
There were tow ways of magnetosome chain separating modes in dividing AMB-1 cells. One is symmetric separation into the daughter cells. The other is asymmetric separation and only one daughter cell was heritor of the magnetosomes. Part of non-magnetosome daughter cells might produce magnetosomes later. These phenomena explained why magnetism of magnetotactic bacteria decreased with more multiplications (Cmag value decreased).
Method of deleting some kind of genes was usually used in research of magnetosomes synthesis. Comparing wild strain with gene mutation strain can find out the function of the gene. However, this method need many steps and the strain of mutation can not inherit stably. Special method of magnetic cells collected to inoculation was used to screen out strains with and without magnetosomes. Such kind of strains was obtained, and they could inherit stably.
Magnetosomes have applying valve in medical treatment, environmental protecting etc. However, it is a long way to put magnetosomes to application. Because magnetotactic bacteria are difficult to culture, and costs of pure magnetosomes are high. During the investigating process of magnetosome assembling, magnetosomes were set free after 192 h culture.
引文
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