台勾霉素生产菌指孢囊菌NRRL 18085遗传操作体系的建立

【目的】建立新型大环内酯类抗生素台勾霉素的生产菌指孢囊菌Dactylosporangium aurantiacum NRRL18085的遗传操作体系,实现台勾霉素相关生物合成基因的敲除突变。【方法】以整合型质粒pSET152为载体,建立了外源DNA通过接合转移进入指孢囊菌NRRL18085的操作方法和培养条件,利用PCR-targeting系统在体外构建了一个台勾霉素卤化酶基因敲除的cosmid质粒,通过接合转移转入到指孢囊菌NRRL18085野生菌中。【结果】获得了台勾霉素卤化酶基因敲除的指孢囊菌NRRL18085的双交换突变株,该突变株失去了产生台勾霉素的能力。【结论】成功建立和优化了指孢囊菌NRRL18085菌株的遗传操作体系,使得在体内分析和鉴定台勾霉素生物合成基因的功能成为可能,同时也为建立其他类似放线菌的遗传操作体系提供了参考。     

趋磁螺菌中磁小体链装配相关基因及其功能

趋磁细菌(MTB)依赖于体内磁小体结构在磁场中取向,多个磁小体以一定的组 织形式排列是形成菌体内生物磁罗盘的重要环节．多数趋磁细菌中磁小体成链排列,有效增加了细胞磁偶极矩,从而使菌体表现出在环境磁场中定向的能力．趋磁螺菌M. magneticum AMB－1和M. gryphiswaldense MSR－1中磁小体均沿细胞长轴形成一条磁 小体链．通过对相关基因突变体表型的研究,结合对磁小体链形成过程的实时动态观 察,人们已初步了解MamJ、MamK和MamA等基因在磁小体链装配和维护过程中的功能．本文介绍了近年来趋磁螺菌磁小体链装配过程中重要功能性基因的研究进展,并重点分析了AMB-1和MSR-1中磁小体链装配的差异．     

子囊霉素产生菌吸水链霉菌FIM260840基因转移系统的建立

目的:建立子囊霉素产生菌吸水链霉菌FIM260840的接合基因转移体系,以便基因敲除和外源基因表达等遗传操作。方法:以整合型质粒p SET152为出发质粒,通过接合转移构建并子囊霉素产生菌FIM260840的基因转移系统。结果:12.5μg/m L安普霉素可有效筛选接合子。经PCR验证,质粒成功整合到菌株FIM260840基因组DNA中,所获接合子的安普霉素抗性高达400μg/m L以上。接合子经多次传代后,导入的质粒p SET152仍稳定整合于接合子基因组DNA上。结论:建立了高效、简便的吸水链霉菌FIM260840的基因转移系统,为该菌的生物合成基因改造奠定了基础。     

安丝菌素产生菌拟诺卡氏菌EGI80425接合转移体系的建立与应用

安丝菌素是一种有强抗肿瘤作用的天然产物,拟诺卡氏菌Nocardiopsis ansamitocini EGI80425作为安丝菌素的产生菌具有重要的研究价值,因此需要建立其遗传操作体系,并实施对该菌株的遗传改造。首先,通过优化菌丝体接合转移相关的培养时间、供受体比例和培养基中Mg2+浓度等,建立了效率为6.6×10^-3的整合型载体p IB139的接合转移方法;然后在此基础上进一步调整相关条件,建立了效率为6.2×10^-4的游离型质粒p JTU1278的接合转移方法;最后,利用游离型质粒对潜在的PKS竞争基因簇ClusterⅠ和ClusterⅦ进行了缺失。结果显示,ClusterⅠ缺失突变株CXY01中安丝菌素的产量为12.6 mg/L,相比野生型提高了95.3%;ClusterⅦ缺失突变株CXY02中安丝菌素产量为7.4 mg/L,相比野生型提高了15.2%。本研究建立了N. ansamitocini EGI80425的遗传操作体系,并在此基础上对该菌进行了基因组片段敲除,有效提升了安丝菌素产量,为利用N. ansamitocini大量产生安丝菌素奠定了基础。     

矿物作用下A.ferrooxidans中磁小体相关基因的差异表达

目的:研究氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans,A.f)中与磁小体形成相关的mpsA、magA、thy和mamB基因分别在黄铁矿、黄铜矿、磁黄铁矿和闪锌矿的作用下的表达差异,寻找有利于磁小体形成的最佳培养矿物能源。方法:测量以不同硫化矿为能源时的菌体生长特性,用实时定量PCR方法研究与磁小体形成相关基因的表达差异。结果:在以磁黄铁矿为能源时,菌的生长量及多数基因的表达量优于其它三种矿,四个基因相对表达量分别为1.15、2.35、1.32、2.68。结论:磁黄铁矿是A.f中磁小体形成的最佳矿物能源。     

Magnetospirillum gryphiswaldense MSR-1磁小体缺失突变株NM4 Tn5侧翼序列的克隆及功能分析

利用mini-Tn5 lacZ2对格瑞菲斯瓦尔德磁螺菌(Magnetospirillum gryphiswaldense)MSR-1进行转座插入突变, 获得磁小体缺失突变株NM4. 通过锚定PCR(anchored PCR)从NM4中克隆出Tn5插入位点的侧翼序列, 获得长5045 bp的DNA 片段, 其中含有6个ORFs, Tn5插入在ORF4中. 功能互补实验证明该片段与磁小体的合成有关. 对ORF4编码的蛋白进行同源比较和功能分析, 发现ORF4编码的蛋白与Caulobacter crescentus CB15的长为200 AA的趋化蛋白CheYIII的同源性为25% (30/116), 且ORF4编码的蛋白也具有与CheYIII相同的接收磷酸基团的REC结构域, 可进行信号传递, 因此推测ORF4编码的蛋白可能参与磁小体合成过程中的某种(低氧分压或铁离子浓度)信号的转导.     

磁小体的生物合成及用于肿瘤靶向治疗的研究进展

趋磁细菌产生的磁小体是生物膜包被的磁性纳米颗粒,具有优良的纳米磁特性;相比化学合成的磁性纳米材料,其生物来源赋予磁小体更好的生物相容性和遗传可操作性.在生物医学领域,除了用于磁热疗进行肿瘤治疗外,最近几年其作为靶向药物载体、可能参与肿瘤微环境调控的性质得到研究者的广泛关注;同时DNA重组技术的发展解决了磁小体的产率低而趋磁细菌难培养的问题.本文综述了磁小体的生物合成及其相关研究进展,并对其应用前景进行了展望.     

Acidthiobacillus ferrooxidans中磁小体的提取

At.f和趋磁细菌在生理特性和生长环境有一定的相似性,而且镜检发现At.f具有趋磁性,所以本文采用了趋磁细菌中磁小体的提取方法尝试提取At.f中的磁小体,用超声波破碎At.f后,以磁铁吸取其体内的磁性颗粒,经过检测,发现其体内确实存在含铁元素的磁性颗粒。提取粗样品经过电镜分析,证实其体内存在着少量由脂质包裹的磁小体。磁小体悬浮液经过蔗糖密度梯度离心纯化后,对其作透射电镜,可以清晰的看到磁小体。实验结果表明,At.f体内存在少量的磁小体,正是由于磁小体的存在,才使得At.f在外加磁场作用下发生磁生物效应。这是首次发现从酸性矿坑水分离的At.f具有趋磁性,并从中提取到了磁小体,可以利用At.f的趋磁性将其按照不同磁性进行分离,从而获得活性高的、对不同磁性矿物有特异性的高效浸矿菌种。     

趋磁细菌的磁小体

趋磁细菌是一类对磁场有趋向性反应的细菌,其菌体能吸收外界环境中铁元素并在体内合成包裹有膜的纳米磁性颗粒Fe₃O₄或Fe₃O_{3S4晶体即磁小体。综述了趋磁细菌的磁小体生物矿化的条件,以及趋磁细菌的铁离子吸收、磁小体囊泡的形成、铁离子的转运到磁小体囊泡及囊泡中受控的Fe3O4生物矿化的分子生物学和生物化学等方面的研究进展,重点介绍了趋磁细菌磁小体合成机制的研究进展及未来研究磁小体的发展方向。}     

趋磁细菌纳米磁小体的培养与分离及其在医学上的应用进展和前景

趋磁细菌是一种对磁场有趋向性反应的细菌,其原因是它们体内能合成一种特殊的细胞器-磁小体;由于磁小体有着大小合适,磁性强,表面易修饰等诸多优点,在诸多领域,尤其是医学领域有广泛的应用和广阔的前景.本文主要就从环境中区分和分离趋磁细菌;对其不同培养条件的优化与选择;从细菌体内提取磁小体并加以纯化;将不同药物偶联于磁小体之上的方法及其在医学上如,制造磁性细胞,磁分离技术,生物传感与检测技术,并将其作为靶向药物的载体,肿瘤治疗,基因治疗等方面的应用现状和前景作简要论述.     

Interruption of the denitrification pathway influences cell growth and magnetosome formation in Magnetospirillum magneticum AMB-1

Aims: Intracellular magnetosome synthesis in magnetotactic bacteria has been proposed to be a process involving functions of a variety of proteins. To learn more about the genetic control that is involved in magnetosome formation, nonmagnetic mutants are screened and characterized. Methods and Results: Conjugation‐mediated transposon mutagenesis was applied to screen for nonmagnetic mutants of Magnetospirillum magneticum AMB‐1 that were unable to respond to the magnetic field. A mutant strain with disruption of a gene locus encoding nitric oxide reductase was obtained. Growth and magnetosome formation under different conditions were further characterized. Conclusions: Interruption of denitrification by inactivating nitric oxide reductase was responsible for the compromised growth and magnetosome formation in the mutant with shorter intracellular chains of magnetite crystals than those of wild‐type cells under anaerobic conditions. Nevertheless, the mutant displayed apparently normal growth in aerobic culture. Significance and Impact of the Study: Efficient denitrification in the absence of oxygen is not only necessary for maintaining cell growth but may also be required to derive sufficient energy to mediate the formation of magnetosome vesicles necessary for the initiation or activation of magnetite formation.     

Compromised DNA Damage Repair Promotes Genetic Instability of the Genomic Magnetosome Island in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) are capable of synthesizing nano-sized, intracellular membrane-bound magnetosomes. To learn more about the genetic factors involved in magnetosome formation, transposon mutagenesis was carried out by conjugation using a hyperactive mariner transposon to obtain nonmagnetic mutants of Magnetospirillum magneticum AMB-1. A mutant with defect in uvrA gene encoding the DNA binding subunit of the UvrABC complex responsible for the process of nucleotide excision repair, was obtained. Growth, magnetosome formation and maintenance of magnetosome island (MAI) were further analyzed in the absence of UvrA. Interruption of uvrA led to decreased capacity to form magnetosome when cultured in the presence of oxygen. The deficiency in UvrA also resulted in an accelerated loss of the MAI under aerobic conditions indicating that the nucleotide excision repair system guards against the instability of the MAI. The incapacity of MTB to efficiently initiate recombination mediated by RecA rescued the instability of MAI observed in uvrA mutant. Elevated recombination activity resulting from the accumulation of unrepaired mutations may thus account for the instability of MAI in the absence of UvrA.     

A hypervariable 130-kilobase genomic region of Magnetospirillum gryphiswaldense comprises a magnetosome island which undergoes frequent rearrangements during stationary growth

Genes involved in magnetite biomineralization are clustered in the genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense. We analyzed a 482-kb genomic fragment, in which we identified an approximately 130-kb region representing a putative genomic "magnetosome island" (MAI). In addition to all known magnetosome genes, the MAI contains genes putatively involved in magnetosome biomineralization and numerous genes with unknown functions, as well as pseudogenes, and it is particularly rich in insertion elements. Substantial sequence polymorphism of clones from different subcultures indicated that this region undergoes frequent rearrangements during serial subcultivation in the laboratory. Spontaneous mutants affected in magnetosome formation arise at a frequency of up to 10(-2) after prolonged storage of cells at 4 degrees C or exposure to oxidative stress. All nonmagnetic mutants exhibited extended and multiple deletions in the MAI and had lost either parts of or the entire mms and mam gene clusters encoding magnetosome proteins. The mutations were polymorphic with respect to the sites and extents of deletions, but all mutations were found to be associated with the loss of various copies of insertion elements, as revealed by Southern hybridization and PCR analysis. Insertions and deletions in the MAI were also found in different magnetosome-producing clones, indicating that parts of this region are not essential for the magnetic phenotype. Our data suggest that the genomic MAI undergoes frequent transposition events, which lead to subsequent deletion by homologous recombination under physiological stress conditions. This can be interpreted in terms of adaptation to physiological stress and might contribute to the genetic plasticity and mobilization of the magnetosome island.     

The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.     

Development of a genetic system for <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis>

Genetic analysis of bacterial magnetosome biomineralization has been hindered by the lack of an appropriate methodology for cultivation and genetic manipulation of most magnetotactic bacteria. In this report, a genetic system for Magnetospirillum gryphiswaldense is described. The system includes a plating technique that allows the screening of magnetic vs non-magnetic colonies, and a protocol for the transfer of foreign DNA by electroporation and high-frequency conjugation. Various broad-host-range vectors of the IncQ, IncP, and pBBR1 groups were found to be capable of replication in M. gryphiswaldense. Several antibiotic resistance markers that can be expressed in M. gryphiswaldense were identified. Tn 5 transposons delivered on a suicide plasmid showed transpositional insertion into random chromosomal sites.     

A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.     

A Tailored galK Counterselection System for Efficient Markerless Gene Deletion and Chromosomal Tagging in Magnetospirillum gryphiswaldense

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.     

Analysis of Magnetosome Chains in Magnetotactic Bacteria by Magnetic Measurements and Automated Image Analysis of Electron Micrographs

Magnetotactic bacteria (MTB) align along the Earth''s magnetic field by the activity of intracellular magnetosomes, which are membrane-enveloped magnetite or greigite particles that are assembled into well-ordered chains. Formation of magnetosome chains was found to be controlled by a set of specific proteins in Magnetospirillum gryphiswaldense and other MTB. However, the contribution of abiotic factors on magnetosome chain assembly has not been fully explored. Here, we first analyzed the effect of growth conditions on magnetosome chain formation in M. gryphiswaldense by electron microscopy. Whereas higher temperatures (30 to 35°C) and high oxygen concentrations caused increasingly disordered chains and smaller magnetite crystals, growth at 20°C and anoxic conditions resulted in long chains with mature cuboctahedron-shaped crystals. In order to analyze the magnetosome chain in electron microscopy data sets in a more quantitative and unbiased manner, we developed a computerized image analysis algorithm. The collected data comprised the cell dimensions and particle size and number as well as the intracellular position and extension of the magnetosome chain. The chain analysis program (CHAP) was used to evaluate the effects of the genetic and growth conditions on magnetosome chain formation. This was compared and correlated to data obtained from bulk magnetic measurements of wild-type (WT) and mutant cells displaying different chain configurations. These techniques were used to differentiate mutants due to magnetosome chain defects on a bulk scale.     

A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.