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Analysis of genes concerned with bacterial magnetite production

与细菌磁铁矿生产相关的基因分析

基本信息

批准号：
03453128
负责人：
MATSUNAGA Tadashi
金额：
$ 1.22万
依托单位：
Tokyo University of Agriculture & Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03453128/
关键词：
Magnetic bacterium Transposon mutant Conjugation Magnetic particles Iron transport 16S rRNA MagA gene 磁性細菌粒子鉄還元

项目摘要

1. Development of a gene transfer system for magnetic bacteria.Broad-host-range plasmids have been transferred to the aerobic magnetic bacterium Magnetospirillum sp. AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high frequency transfer of the RK2 derivative pRK415 (4.5 x 10^<-3> transconjugants per recipient cell) and the RSF-1010 derivative pKT230 (3.0 x 10^<-3>). Optimum mating time was 6 hrs for both plasmids. These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E.coli, illustrating their potential as shuttle vectors.2. Genetic analysis of transposon mutant, NM5.The non-magnetic mutant NM5 is one of transposon mutagenized mutants. We cloned EcoRI genomic DNA fragment containing the Tn5 interrupted locus. Gene expression and nucleotide sequence of this cloned fragment were analyzed. Dot blot northern hybridization was performed using DNA fragments flanking the Tn5 insertion site, as a probe. Iron suff … More icient and iron limited media were prepared for cultivation of wild type AMB-1 cells from which total RNA was extracted. Strong hybridization was detected for samples from cells cultured in iron limited media. This result suggests that gene(s) whose expression was regulated by iron are present on the cloned EcoRI fragment from NM5. A 2640bp genomic DNA fragment from mutant NM5 was sequenced completely. Three open reading frames were found, one of which is interrupted by the Tn5 insertion. A putative promoter and a ribosome binding site exist upstream of this ORF. This ORF, named magA, was analyzed using the DNA / protein computer analysis software, DNASIS. The predicted amino acid sequence of the magA gene product has high homology with cation transport channel proteins, in particular the E.coli Kef C protein which functions as a potassium efflux protein for control of turgor. Furthermore the amino acid sequence of MagA shows high hydrophobicity. Therefore MagA is probably localized in the membrane fraction and may function as a cation transporter protein in the magnetic bacterium Magnetospirillum sp. AMB-1. Less

1. 磁性细菌基因转移系统的研制。广泛宿主范围的质粒已转移到好氧磁性细菌Magnetospirillum sp. AMB-1。与大肠杆菌S17-1的共轭结合允许RK2衍生物pRK415（每个受体细胞4.5 × 10^<-3>）和RSF-1010衍生物pKT230 （3.0 × 10^<-3>）的高频转移。两个质粒的最佳交配时间均为6 h。这些质粒在转共轭物中成功地形成了自主复制子，并且可以被分离并转化回大肠杆菌中，这说明了它们作为穿梭载体的潜力。转座子突变体NM5的遗传分析。非磁性突变体NM5是转座子诱变突变体之一。我们克隆了包含Tn5中断位点的EcoRI基因组DNA片段。分析了该克隆片段的基因表达和核苷酸序列。用Tn5插入位点两侧的DNA片段作为探针进行点印迹北杂交。为培养野生型AMB-1细胞制备了高效、限铁培养基，提取了总RNA。在限铁培养基中培养的细胞样品检测到强杂交。这一结果表明，受铁调控的基因存在于克隆的NM5的EcoRI片段上。对突变体NM5的2640bp基因组DNA片段进行了完整测序。发现三个开放的阅读框，其中一个被Tn5插入打断。一个假定的启动子和一个核糖体结合位点存在于这个ORF的上游。该ORF命名为magA，使用DNA /蛋白质计算机分析软件DNASIS进行分析。预测的magA基因产物的氨基酸序列与阳离子运输通道蛋白具有高度的同源性，特别是与大肠杆菌中控制腹胀的钾外排蛋白Kef C蛋白具有高度的同源性。此外，MagA的氨基酸序列具有较高的疏水性。因此，MagA可能定位于膜部分，并可能在磁性细菌Magnetospirillum sp. AMB-1中作为阳离子转运蛋白起作用。少