趋磁细菌Magnetospirillum magneticumAMB-1反硝化代谢与DNA损伤修复体系对磁小体合成及其遗传稳定性的影响研究
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摘要
趋磁细菌是一类能在体内合成纳米级磁性矿物颗粒的兼性厌氧型原核生物,在自然界中分布广泛。趋磁细菌大部分分布在a,γ,δ变形菌门和硝化螺菌门,其生长环境为海水或者淡水的氧化还原交界面处,对生长环境的氧化还原状态要求较高。趋磁细菌由于能够通过生物矿化合成磁性颗粒——磁小体,所以可感受地磁场,在磁场的作用下进行定向游弋。磁小体在趋磁细菌体内排列成链状,而大大增大了菌体的总磁矩。磁小体结构均一,性能稳定,分散性好,在生物学、物理学、材料学上有非常广泛的应用。
磁小体作为趋磁细菌体内一类特殊的物质结构,其合成机制、作用机制引起了许多学者的研究兴趣。目前已获得的研究成果表明,磁小体合成是受一系列基因编码产物的控制而完成的复杂过程。大致包括铁源的吸收、运输、运输过程中的代谢以及价态变化,磁小体膜的起源和内陷,磁小体晶核的生物矿化,以及成熟磁小体的链状排列等等。这些过程互相影响、互相渗透,在不同种类的趋磁细菌中展示出多样性和种属特异性,涉及多种目前未曾全面了解的复杂生理活动和代谢途径,且对环境因素影响敏感。同时,遗传学研究发现,与磁小体合成的有关基因有明显的结构组成特征,以基因岛的形式存在于趋磁细菌基因组中,在基因岛内又可分为几个大的基因簇。岛内基因簇之间以及基因岛两端都存在正向重复序列,推测这些正向重复序列通过与某些相关重组蛋白的相互作用,会导致基因岛以一定的频率进行重排,最终引起基因岛序列部分或者全部的丢失,从而造成菌体磁小体合成能力的遗传不稳定性。
综上所述,磁小体合成过程可能会受到菌体多种代谢途径的影响。通过总结产磁过程中铁源的代谢研究成果,我们推测菌体内存在的反硝化蛋白组分可能与磁小体合成中铁价态变化过程相偶联。在趋磁细菌模式菌株MS-1(Magnetospirillum magnetotacticum MS-1)体内鉴定出了反硝化组分——硝酸盐还原酶和亚硝酸盐还原酶。通过对两种酶体外活性的研究发现,硝酸盐还原酶可能在微好氧条件下以及磁小体合成过程中的能量供应方面起作用。而亚硝酸盐还原酶具有一种与趋磁细菌相关的特异活性：二价铁：亚硝酸盐氧化还原酶活性。推测该酶在反硝化途径中参与了磁小体合成过程中铁源的氧化还原。基于上述两种酶的初步研究,我们推测趋磁细菌在以硝酸盐作为唯一氮源的培养条件下合成菌体所需蛋白质进行生长的同时,可偶联铁的氧化还原从而参与或影响磁小体合成过程。但是由于MS-1在厌氧条件下不能以硝酸盐作为唯一氮源进行生长,所以目前还不能确定MS-1体内有独立的反硝化系统存在。趋磁细菌AMB-1是趋磁细菌研究的另一模式菌株,与MS-1不同的是,其能够在厌氧条件下以硝酸盐为唯一氮源进行生长和产磁。
作为兼性厌氧微生物,趋磁细菌在氧气存在条件下进行生长,但是氧在体内代谢产生的活性氧也同时会对菌体产生毒害作用,这种作用主要表现在对蛋白、核酸等生物大分子的损伤。我们曾在趋磁细菌模式菌株,淡水趋磁螺菌AMB-1(Magnetospirillum magneticum AMB-1)中报道了三个过氧化还原酶,研究发现它们在活性氧自由基的清除与磁小体合成能力遗传稳定性的维持方面发挥重要作用。尤为重要的是这些酶组分的缺失会明显提高菌体在传代过程中基因组磁小体岛的丢失频率。因此,趋磁细菌的氧化抗逆性途径可能在好氧条件下维持磁小体基因岛遗传稳定性方面起作用,但是具体作用机制并不清楚。在趋磁螺菌MSR-1(Magnetospirillum gryphiswaldense)中,重组酶RecA可以通过重组活性参与磁小体基因岛遗传过程中大片段的丢失,是磁小体岛遗传不稳定性的直接原因之一。
本文以淡水趋磁螺菌AMB-1为实验材料,对两个方面进行了研究。第一,AMB-1反硝化代谢途径的分析鉴定以及功能验证。基于对趋磁细菌体内铁源代谢以及价态变化的推测,对AMB-1中反硝化途径在细胞能量供应和对磁小体合成过程的影响进行了探讨。第二,针对AMB-1的磁小体合成过程及可能的影响因素,通过转座诱变对AMB-1进行突变筛选,探索了磁小体岛外与其生理代谢功能相关的基因在磁小体合成过程中的作用。通过对一个DNA损伤修复基因功能失活突变菌株的研究,探讨了DNA损伤修复系统在高氧化压力的环境下对AMB-1磁小体基因岛以及菌体产磁功能的影响。具体工作内容和创新性研究结果如下：
1.对AMB-1亚硝酸盐还原酶的编码基因进行预测和功能分析,探讨了该酶在AMB-1反硝化代谢以及磁小体合成过程中的作用。
通过AMB-1基因组的生物信息学分析,获得了三个可能的亚硝酸盐还原酶编码基因：amb1395、amb1408、amb4165。在建立体外酶活测定方法的基础上,对该酶在菌体内的活性分布和产酶适宜条件进行摸索。结果表明,亚硝酸盐还原酶酶活在菌体静置培养状态下的周质空间组分中分布较高。对静置培养的AMB-1周质空间组分进行分离纯化,得到电泳纯的活性纯蛋白组分。质谱鉴定结果表明,该蛋白的编码基因为AMB-1基因组中的amb1395。
通过荧光实时定量PCR进行的转录分析表明,amb1395在静置培养条件下转录水平明显高于振荡培养条件,并且在静置培养条件下,amb1395的转录水平在磁小体合成阶段明显升高,说明该基因的表达产物与磁小体的合成密切相关。但采用单交换插入策略对amb1395进行了功能失活后,功能失活突变株的生理表型却显示,菌体在厌氧条件下的生长和磁小体合成与野生型相比均无明显区别。这些结果表明,虽然该酶在趋磁细菌AMB-1的反硝化代谢及磁小体合成过程中不发挥主要作用,但可能参与了上述过程。另外,由于该酶的催化底物亚硝酸盐对菌体有毒性,推测菌体内应该存在其他途径组分能够分解亚硝酸盐,导致菌体在缺失amb1395之后没有明显的表型变化。
2.通过对AMB-1在不同氮源条件下的生长以及对硝酸盐还原酶基因的预测分析,确定了反硝化途径对AMB-1在厌氧条件下的生长及产磁过程的重要性。
AMB-1能够以硝酸盐作为唯一氮源进行生长。硝酸盐在体内可以通过铵化作用供应菌体大分子物质的合成。以铵盐作为唯一氮源的生长实验表明,菌体在好氧振荡条件下能够正常生长,但是在静置培养或者厌氧条件下生长则受到明显抑制,这说明铵盐在静置或厌氧条件下不能完全代替硝酸盐。结合MS-1中的研究,可以推测在AMB-1体内可能存在依赖于硝酸盐的厌氧产能途径。通过生物信息学分析,在AMB-1基因组中存在一个硝酸盐还原酶编码基因,ammb2690。在建立其体外酶活测定方法的基础上,对amb2690进行了插入失活。发现硝酸盐还原酶功能失活突变株在以硝酸盐作为唯一氮源的有氧培养条件下可正常生长,但不能在厌氧条件下生长和产磁。在以0.3L/min的流速通入空气的恒定通氧发酵条件下,菌体能够在获得氧气的基础上进行生长,此时菌体的铁吸收水平和磁小体合成水平不受影响。这些结果说明反硝化途径可能仅在厌氧条件下作为趋磁细菌AMB-1生长和磁小体合成过程的能量来源,与磁小体合成过程并没有特异性的联系。当有氧气存在时,反硝化途径的功能丧失对菌体生长和产磁过程没有影响。
3.DNA损伤修复体系UvrABC在好氧条件下具有维持AMB-1磁小体岛遗传稳定性的重要作用。
利用转座子对AMB-1进行随机插入突变,获得了一株产磁异常突变株。该突变株的生长与野生型AMB-1没有明显差别,但是磁小体合成能力明显低于野生型。通过分子生物学鉴定,发现该突变株的DNA损伤修复蛋白复合体UvrABC的UvrA亚基编码基因uvrA被转座子插入导致其功能失活。进一步对uvrA的定点插入失活研究表明,该组分的功能缺失使得菌体在恒定通氧发酵条件下的产磁能力受到不可逆的损伤。突变株经过好氧培养后,保留产磁能力的菌体只占群体的38%左右(野生型AMB-1的产磁菌体达94%)。同时,产磁菌体的平均磁小体合成数目也明显降低,突变株平均为1-4个,远低于野生型的8-20个。通过荧光定量PCR分析发现,突变株在好氧条件传代过程中,磁小体岛基因拷贝数与基因组其他区段基因拷贝数的比值明显降低,突变株子代的磁小体岛拷贝数在经20次左右的传代后仅为突变株原始菌株的20-30%,而同样条件下的野生型AMB-1子代能保持初始菌株的80%。这表明突变株的磁小体岛遗传稳定性在UvrA缺失的情况下明显下降。进一步转录分析表明,突变株中的重组酶基因recA转录水平明显高于野生型AMB-1,达到AMB-1的11.9倍。recA的插入失活可以降低因UvrA功能失活而引发的磁小体岛区域的不稳定性。因此,uvrA缺失菌中recA转录活性的上升可能是导致磁小体岛遗传稳定性下降的原因。通过以上实验,我们认为UvrABC体系的功能丧失会造成趋磁细菌在好氧条件下DNA随机损伤的积累,这种损伤积累到一定水平能够导致基于RecA蛋白的同源重组修复途径的激活而引发基因组磁小体岛遗传稳定性的降低。该研究结果进一步补充和完善了有关前期研究所证明的趋磁细菌AMB-1氧化抗逆能力的存在是维持磁小体岛遗传稳定性的重要因素的观点。
Magnetotactic bacteria (MTBs) are kinds of facultative anaerobic prokaryote which are able to synthesis membrane-bounded, nano-sized magnetic particles, magnetosomes, which make them capable to response to magnetic field. MTBs can be identified in the phylum of α-, γ-,γ-Proteobacteia and Nitrospira. They can grow rigorously in oxic-anoxic transition zone of seawater or fresh water. Magnetosomes are composed by crystal nucleus, which are derived from crystallization of Fe3O4or Fe3S4under complicated genetic controlling, and membrane structure, which originated from cell inner membrane. Magnetosomes can be arranged in chains in parallel to long axis of the cell in order to maximize the cell magnetic dipolemoment. The uniformity, stable property and dispersibility of magnetosomes make them applicable in Biology, Physics and Material science.
As kinds of species specific structure, the function and synthesis procedures of magnetosome having been studied for decades. So far, the results proved that the magnetosome production was an intricate process which regulated by dozens of genes and proteins. The step-wise procedures are composed of iron assimilation, transportation, oxidoreduction, magnetosome membrane origin, nucleus crystallization and magnetosome chain formation, etc,al.. Genomic analysis showed that the genes involved in magnetosome formation were arranged in a large genomic island, which called magnetosome island (MAI), and the horizontal gene transfer of the MAI between species may provide evidence for the original of MTBs.
The procedures of magnetosome formation can be influenced by several metabolic pathways. Considering the iron metabolism involved in magnetsome formation, including iron transportation and oxidation state, it may coupled with denitrification pathway. Nitrate can be used in biomass synthesis by reducing to ammonium, which called assimilation. MTBs can grow with nitrate as sole nitrogen source. Two components of denitrification pathway were identified in Magnetospirillum magnetotacticum MS-1. It showed that nitrate reductase (Nar) may have function in energy supplement when cells were cultured in microaerobic condition or in magnetsome formation process. Besides, a novel nitrite reductase (Nir) with Fe(II):nitrite oxidoreductase was identified in MS-1. It was considered that the Fe(II) oxidase activity of Nir may contribute the iron metabolism which involved in magnetosome formation. Enzymology research of the two components revealed that they may participate in the process of magnetosome formation, but with non-critical function. Strains with inactivated enzymes can grow normally, but with a tiny weak in magnetosome formation. MS-1cannot grow with nitrate as sole nitrogen source without oxygen, so it is hard to confirm that there is independent denitrification pathway worked as energy supplement process under micro-or anaerobic condition.
As kinds of facultative anaerobic prokaryote, MTBs can grow aerobically, with reactive oxygen species (ROS) causing damage to protein or nucleic acid. It was reported that three peroxiredoxin components were identified in Magnetospirillum magneticum AMB-1, which play a role in ROS elimination and genetic stability maintenance. The deficient of peroxiredoxin components may cause instability of the magnetosome synthesis capacity during subculture. Antioxidant mechanism of MTBs can take effect in maintaining the genetic stability of MAI. In Magnetospirillum giyphiswaldense, a recombinase, which mediate the frequent loss of MAI, RecA was identified. Strains lacking RecA showed a high stability of MAI during subculture.
Different to MS-1, AMB-1can grow with nitrate as sole nitrogen source without oxygen. In this article, we intend to do two piece of work using AMB-1as material.1, we attempt to characterize the members involved in denitrification pathway, and to study the function of the pathway to the field of energy supplement and magnetosome formation process.2, we make effort in genetic screening of AMB-1to obtain some mutant with abnormal capacity in magnetosome synthesis. We obtained a mutant with defective in the function of DNA damage repair system, and learned more information of the relationship between DNA repair system and magnetosome synthesis process. The major results are follows:
1. Function of Nir in denitrification pathway and magnetosome synthesis process
Three Nir coding genes (nir) were predeterminated by analyzing in bioinformatics. The main Nir activity component was purified on the basis of enzyme activity assay, and then characterized by MOLDI-TOF and sequence blast. The Nir activity measurement results showed that the activity was higher in periplasmic space than cytoplasm, higher in static condition than aerobic condition.
Transcript analysis of nir showed that the transcript level of nir in micro-or anaerobic condition is higher than aerobic condition, and it would increased in magnetosome synthesis phase in the time-course of life-cycle. We studied the function of nir by disrupting the coding sequence. Growth and magnetosome synthesis of the nir deficient mutant were not obviously influenced by the inactivated Nir. The results showed that Nir may have a hand but is not significant in energy supplement or magnetosome synthesis process.
2. AMB-1can grow anaerobically by virtue of nitrate-dependent energy supplement pathway
AMB-1can grow with nitrate as sole nitrogen source. Ammonium used for biomass synthesis can be generated by nitrate reduction. AMB-1can grow with ammonium as sole nitrogen source under aerobic condition, but ammonium cannot support AMB-1's growth under anaerobic condition, which means the bacteria need an energy supplement pathway depending on nitrate. The Nar coding genes (nar) were predeterminated by analyzing in bioinformatics. The function of nar was studied by disrupting the coding sequence. Growth and magnetosome synthesis of nar deficient mutant in anaerobic condition were obviously inhibited by the inactivated Nar. The phenotype of the mutant was the same as wildtype AMB-1when oxygen was existed. The results suggested that energy supplement pathway depending on nitrate, which was denitrification pathway also, may existed in AMB-1, and may have function in anaerobic condition, but it may not involved in magnetosome synthesis process.
3. DNA damage repair system UvrABC complex had function in the maintenance of MAI genetic stability.
To learn more information about the genetic factors of magnetosome synthesis process, a genetic screen was carried out by transposon mutagenesis to obtain nonmagnetic mutants of AMB-1. A mutant with defect in UvrABC system was obtained. Growth, magnetosome synthesis capacity and MAI stability were further found to be influenced by inactivited UvrA. Site-specific insertion of uvrA led to weak magnetosome formation under aerobic condition. Only38%of the mutant cells can synthesis magnetosome, while the ratio in wildtype AMB-1is94%. The magnetosome number which produced by mutant cells is1-4, while8-20in wildtype AMB-1cells. Quantitative PCR analysis showed that the deficiency in UvrA also leads to lower MAI stability under aerobic conditions. These results indicated that the DNA repair system guarded against the instability of the MAI. Studies on the transcript level of a recombinase RecA showed that, the transcript level of recA in the mutant is11.9times higher than that in wildtype AMB-1, indicating the recombinase may involved in the loss of MAI. The insertion mutagenesis of recA in uvrA mutant rescues the phenotype of the MAI instability. These results suggested that the accumulation of unrepaired mutations caused by the absence of UvrA may account for the elevated recombination activity, which then led to the high instability level of MAI.
磁小体作为趋磁细菌体内一类特殊的物质结构,其合成机制、作用机制引起了许多学者的研究兴趣。目前已获得的研究成果表明,磁小体合成是受一系列基因编码产物的控制而完成的复杂过程。大致包括铁源的吸收、运输、运输过程中的代谢以及价态变化,磁小体膜的起源和内陷,磁小体晶核的生物矿化,以及成熟磁小体的链状排列等等。这些过程互相影响、互相渗透,在不同种类的趋磁细菌中展示出多样性和种属特异性,涉及多种目前未曾全面了解的复杂生理活动和代谢途径,且对环境因素影响敏感。同时,遗传学研究发现,与磁小体合成的有关基因有明显的结构组成特征,以基因岛的形式存在于趋磁细菌基因组中,在基因岛内又可分为几个大的基因簇。岛内基因簇之间以及基因岛两端都存在正向重复序列,推测这些正向重复序列通过与某些相关重组蛋白的相互作用,会导致基因岛以一定的频率进行重排,最终引起基因岛序列部分或者全部的丢失,从而造成菌体磁小体合成能力的遗传不稳定性。
综上所述,磁小体合成过程可能会受到菌体多种代谢途径的影响。通过总结产磁过程中铁源的代谢研究成果,我们推测菌体内存在的反硝化蛋白组分可能与磁小体合成中铁价态变化过程相偶联。在趋磁细菌模式菌株MS-1(Magnetospirillum magnetotacticum MS-1)体内鉴定出了反硝化组分——硝酸盐还原酶和亚硝酸盐还原酶。通过对两种酶体外活性的研究发现,硝酸盐还原酶可能在微好氧条件下以及磁小体合成过程中的能量供应方面起作用。而亚硝酸盐还原酶具有一种与趋磁细菌相关的特异活性：二价铁：亚硝酸盐氧化还原酶活性。推测该酶在反硝化途径中参与了磁小体合成过程中铁源的氧化还原。基于上述两种酶的初步研究,我们推测趋磁细菌在以硝酸盐作为唯一氮源的培养条件下合成菌体所需蛋白质进行生长的同时,可偶联铁的氧化还原从而参与或影响磁小体合成过程。但是由于MS-1在厌氧条件下不能以硝酸盐作为唯一氮源进行生长,所以目前还不能确定MS-1体内有独立的反硝化系统存在。趋磁细菌AMB-1是趋磁细菌研究的另一模式菌株,与MS-1不同的是,其能够在厌氧条件下以硝酸盐为唯一氮源进行生长和产磁。
作为兼性厌氧微生物,趋磁细菌在氧气存在条件下进行生长,但是氧在体内代谢产生的活性氧也同时会对菌体产生毒害作用,这种作用主要表现在对蛋白、核酸等生物大分子的损伤。我们曾在趋磁细菌模式菌株,淡水趋磁螺菌AMB-1(Magnetospirillum magneticum AMB-1)中报道了三个过氧化还原酶,研究发现它们在活性氧自由基的清除与磁小体合成能力遗传稳定性的维持方面发挥重要作用。尤为重要的是这些酶组分的缺失会明显提高菌体在传代过程中基因组磁小体岛的丢失频率。因此,趋磁细菌的氧化抗逆性途径可能在好氧条件下维持磁小体基因岛遗传稳定性方面起作用,但是具体作用机制并不清楚。在趋磁螺菌MSR-1(Magnetospirillum gryphiswaldense)中,重组酶RecA可以通过重组活性参与磁小体基因岛遗传过程中大片段的丢失,是磁小体岛遗传不稳定性的直接原因之一。
本文以淡水趋磁螺菌AMB-1为实验材料,对两个方面进行了研究。第一,AMB-1反硝化代谢途径的分析鉴定以及功能验证。基于对趋磁细菌体内铁源代谢以及价态变化的推测,对AMB-1中反硝化途径在细胞能量供应和对磁小体合成过程的影响进行了探讨。第二,针对AMB-1的磁小体合成过程及可能的影响因素,通过转座诱变对AMB-1进行突变筛选,探索了磁小体岛外与其生理代谢功能相关的基因在磁小体合成过程中的作用。通过对一个DNA损伤修复基因功能失活突变菌株的研究,探讨了DNA损伤修复系统在高氧化压力的环境下对AMB-1磁小体基因岛以及菌体产磁功能的影响。具体工作内容和创新性研究结果如下：
1.对AMB-1亚硝酸盐还原酶的编码基因进行预测和功能分析,探讨了该酶在AMB-1反硝化代谢以及磁小体合成过程中的作用。
通过AMB-1基因组的生物信息学分析,获得了三个可能的亚硝酸盐还原酶编码基因：amb1395、amb1408、amb4165。在建立体外酶活测定方法的基础上,对该酶在菌体内的活性分布和产酶适宜条件进行摸索。结果表明,亚硝酸盐还原酶酶活在菌体静置培养状态下的周质空间组分中分布较高。对静置培养的AMB-1周质空间组分进行分离纯化,得到电泳纯的活性纯蛋白组分。质谱鉴定结果表明,该蛋白的编码基因为AMB-1基因组中的amb1395。
通过荧光实时定量PCR进行的转录分析表明,amb1395在静置培养条件下转录水平明显高于振荡培养条件,并且在静置培养条件下,amb1395的转录水平在磁小体合成阶段明显升高,说明该基因的表达产物与磁小体的合成密切相关。但采用单交换插入策略对amb1395进行了功能失活后,功能失活突变株的生理表型却显示,菌体在厌氧条件下的生长和磁小体合成与野生型相比均无明显区别。这些结果表明,虽然该酶在趋磁细菌AMB-1的反硝化代谢及磁小体合成过程中不发挥主要作用,但可能参与了上述过程。另外,由于该酶的催化底物亚硝酸盐对菌体有毒性,推测菌体内应该存在其他途径组分能够分解亚硝酸盐,导致菌体在缺失amb1395之后没有明显的表型变化。
2.通过对AMB-1在不同氮源条件下的生长以及对硝酸盐还原酶基因的预测分析,确定了反硝化途径对AMB-1在厌氧条件下的生长及产磁过程的重要性。
AMB-1能够以硝酸盐作为唯一氮源进行生长。硝酸盐在体内可以通过铵化作用供应菌体大分子物质的合成。以铵盐作为唯一氮源的生长实验表明,菌体在好氧振荡条件下能够正常生长,但是在静置培养或者厌氧条件下生长则受到明显抑制,这说明铵盐在静置或厌氧条件下不能完全代替硝酸盐。结合MS-1中的研究,可以推测在AMB-1体内可能存在依赖于硝酸盐的厌氧产能途径。通过生物信息学分析,在AMB-1基因组中存在一个硝酸盐还原酶编码基因,ammb2690。在建立其体外酶活测定方法的基础上,对amb2690进行了插入失活。发现硝酸盐还原酶功能失活突变株在以硝酸盐作为唯一氮源的有氧培养条件下可正常生长,但不能在厌氧条件下生长和产磁。在以0.3L/min的流速通入空气的恒定通氧发酵条件下,菌体能够在获得氧气的基础上进行生长,此时菌体的铁吸收水平和磁小体合成水平不受影响。这些结果说明反硝化途径可能仅在厌氧条件下作为趋磁细菌AMB-1生长和磁小体合成过程的能量来源,与磁小体合成过程并没有特异性的联系。当有氧气存在时,反硝化途径的功能丧失对菌体生长和产磁过程没有影响。
3.DNA损伤修复体系UvrABC在好氧条件下具有维持AMB-1磁小体岛遗传稳定性的重要作用。
利用转座子对AMB-1进行随机插入突变,获得了一株产磁异常突变株。该突变株的生长与野生型AMB-1没有明显差别,但是磁小体合成能力明显低于野生型。通过分子生物学鉴定,发现该突变株的DNA损伤修复蛋白复合体UvrABC的UvrA亚基编码基因uvrA被转座子插入导致其功能失活。进一步对uvrA的定点插入失活研究表明,该组分的功能缺失使得菌体在恒定通氧发酵条件下的产磁能力受到不可逆的损伤。突变株经过好氧培养后,保留产磁能力的菌体只占群体的38%左右(野生型AMB-1的产磁菌体达94%)。同时,产磁菌体的平均磁小体合成数目也明显降低,突变株平均为1-4个,远低于野生型的8-20个。通过荧光定量PCR分析发现,突变株在好氧条件传代过程中,磁小体岛基因拷贝数与基因组其他区段基因拷贝数的比值明显降低,突变株子代的磁小体岛拷贝数在经20次左右的传代后仅为突变株原始菌株的20-30%,而同样条件下的野生型AMB-1子代能保持初始菌株的80%。这表明突变株的磁小体岛遗传稳定性在UvrA缺失的情况下明显下降。进一步转录分析表明,突变株中的重组酶基因recA转录水平明显高于野生型AMB-1,达到AMB-1的11.9倍。recA的插入失活可以降低因UvrA功能失活而引发的磁小体岛区域的不稳定性。因此,uvrA缺失菌中recA转录活性的上升可能是导致磁小体岛遗传稳定性下降的原因。通过以上实验,我们认为UvrABC体系的功能丧失会造成趋磁细菌在好氧条件下DNA随机损伤的积累,这种损伤积累到一定水平能够导致基于RecA蛋白的同源重组修复途径的激活而引发基因组磁小体岛遗传稳定性的降低。该研究结果进一步补充和完善了有关前期研究所证明的趋磁细菌AMB-1氧化抗逆能力的存在是维持磁小体岛遗传稳定性的重要因素的观点。
Magnetotactic bacteria (MTBs) are kinds of facultative anaerobic prokaryote which are able to synthesis membrane-bounded, nano-sized magnetic particles, magnetosomes, which make them capable to response to magnetic field. MTBs can be identified in the phylum of α-, γ-,γ-Proteobacteia and Nitrospira. They can grow rigorously in oxic-anoxic transition zone of seawater or fresh water. Magnetosomes are composed by crystal nucleus, which are derived from crystallization of Fe3O4or Fe3S4under complicated genetic controlling, and membrane structure, which originated from cell inner membrane. Magnetosomes can be arranged in chains in parallel to long axis of the cell in order to maximize the cell magnetic dipolemoment. The uniformity, stable property and dispersibility of magnetosomes make them applicable in Biology, Physics and Material science.
As kinds of species specific structure, the function and synthesis procedures of magnetosome having been studied for decades. So far, the results proved that the magnetosome production was an intricate process which regulated by dozens of genes and proteins. The step-wise procedures are composed of iron assimilation, transportation, oxidoreduction, magnetosome membrane origin, nucleus crystallization and magnetosome chain formation, etc,al.. Genomic analysis showed that the genes involved in magnetosome formation were arranged in a large genomic island, which called magnetosome island (MAI), and the horizontal gene transfer of the MAI between species may provide evidence for the original of MTBs.
The procedures of magnetosome formation can be influenced by several metabolic pathways. Considering the iron metabolism involved in magnetsome formation, including iron transportation and oxidation state, it may coupled with denitrification pathway. Nitrate can be used in biomass synthesis by reducing to ammonium, which called assimilation. MTBs can grow with nitrate as sole nitrogen source. Two components of denitrification pathway were identified in Magnetospirillum magnetotacticum MS-1. It showed that nitrate reductase (Nar) may have function in energy supplement when cells were cultured in microaerobic condition or in magnetsome formation process. Besides, a novel nitrite reductase (Nir) with Fe(II):nitrite oxidoreductase was identified in MS-1. It was considered that the Fe(II) oxidase activity of Nir may contribute the iron metabolism which involved in magnetosome formation. Enzymology research of the two components revealed that they may participate in the process of magnetosome formation, but with non-critical function. Strains with inactivated enzymes can grow normally, but with a tiny weak in magnetosome formation. MS-1cannot grow with nitrate as sole nitrogen source without oxygen, so it is hard to confirm that there is independent denitrification pathway worked as energy supplement process under micro-or anaerobic condition.
As kinds of facultative anaerobic prokaryote, MTBs can grow aerobically, with reactive oxygen species (ROS) causing damage to protein or nucleic acid. It was reported that three peroxiredoxin components were identified in Magnetospirillum magneticum AMB-1, which play a role in ROS elimination and genetic stability maintenance. The deficient of peroxiredoxin components may cause instability of the magnetosome synthesis capacity during subculture. Antioxidant mechanism of MTBs can take effect in maintaining the genetic stability of MAI. In Magnetospirillum giyphiswaldense, a recombinase, which mediate the frequent loss of MAI, RecA was identified. Strains lacking RecA showed a high stability of MAI during subculture.
Different to MS-1, AMB-1can grow with nitrate as sole nitrogen source without oxygen. In this article, we intend to do two piece of work using AMB-1as material.1, we attempt to characterize the members involved in denitrification pathway, and to study the function of the pathway to the field of energy supplement and magnetosome formation process.2, we make effort in genetic screening of AMB-1to obtain some mutant with abnormal capacity in magnetosome synthesis. We obtained a mutant with defective in the function of DNA damage repair system, and learned more information of the relationship between DNA repair system and magnetosome synthesis process. The major results are follows:
1. Function of Nir in denitrification pathway and magnetosome synthesis process
Three Nir coding genes (nir) were predeterminated by analyzing in bioinformatics. The main Nir activity component was purified on the basis of enzyme activity assay, and then characterized by MOLDI-TOF and sequence blast. The Nir activity measurement results showed that the activity was higher in periplasmic space than cytoplasm, higher in static condition than aerobic condition.
Transcript analysis of nir showed that the transcript level of nir in micro-or anaerobic condition is higher than aerobic condition, and it would increased in magnetosome synthesis phase in the time-course of life-cycle. We studied the function of nir by disrupting the coding sequence. Growth and magnetosome synthesis of the nir deficient mutant were not obviously influenced by the inactivated Nir. The results showed that Nir may have a hand but is not significant in energy supplement or magnetosome synthesis process.
2. AMB-1can grow anaerobically by virtue of nitrate-dependent energy supplement pathway
AMB-1can grow with nitrate as sole nitrogen source. Ammonium used for biomass synthesis can be generated by nitrate reduction. AMB-1can grow with ammonium as sole nitrogen source under aerobic condition, but ammonium cannot support AMB-1's growth under anaerobic condition, which means the bacteria need an energy supplement pathway depending on nitrate. The Nar coding genes (nar) were predeterminated by analyzing in bioinformatics. The function of nar was studied by disrupting the coding sequence. Growth and magnetosome synthesis of nar deficient mutant in anaerobic condition were obviously inhibited by the inactivated Nar. The phenotype of the mutant was the same as wildtype AMB-1when oxygen was existed. The results suggested that energy supplement pathway depending on nitrate, which was denitrification pathway also, may existed in AMB-1, and may have function in anaerobic condition, but it may not involved in magnetosome synthesis process.
3. DNA damage repair system UvrABC complex had function in the maintenance of MAI genetic stability.
To learn more information about the genetic factors of magnetosome synthesis process, a genetic screen was carried out by transposon mutagenesis to obtain nonmagnetic mutants of AMB-1. A mutant with defect in UvrABC system was obtained. Growth, magnetosome synthesis capacity and MAI stability were further found to be influenced by inactivited UvrA. Site-specific insertion of uvrA led to weak magnetosome formation under aerobic condition. Only38%of the mutant cells can synthesis magnetosome, while the ratio in wildtype AMB-1is94%. The magnetosome number which produced by mutant cells is1-4, while8-20in wildtype AMB-1cells. Quantitative PCR analysis showed that the deficiency in UvrA also leads to lower MAI stability under aerobic conditions. These results indicated that the DNA repair system guarded against the instability of the MAI. Studies on the transcript level of a recombinase RecA showed that, the transcript level of recA in the mutant is11.9times higher than that in wildtype AMB-1, indicating the recombinase may involved in the loss of MAI. The insertion mutagenesis of recA in uvrA mutant rescues the phenotype of the MAI instability. These results suggested that the accumulation of unrepaired mutations caused by the absence of UvrA may account for the elevated recombination activity, which then led to the high instability level of MAI.
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