Analysis of genes concerned with bacterial magnetite production

1.磁性细菌基因转移系统的建立将广宿主范围的质粒转移到好氧磁性细菌磁细菌AMB-1中。与大肠杆菌S17-1的接合交配允许RK 2衍生物pRK 415（每个受体细胞4.5 × 104个<-3>转移接合物）和RSF-1010衍生物pKT 230（3.0 × 104个<-3>）的高频转移。两种质粒的最佳交配时间均为6小时。这些质粒成功地在接合子中形成了自主复制子，并且可以被分离并转化回大肠杆菌中，说明了它们作为穿梭载体的潜力.转座子突变体NM 5的遗传分析非磁性突变体NM 5是转座子诱变突变体之一。我们克隆了含有Tn 5中断位点的EcoRI基因组DNA片段。对该克隆片段进行基因表达和核苷酸序列分析。使用Tn 5插入位点侧翼的DNA片段作为探针进行斑点印迹北方杂交。铁SUFF 关于我们 制备用于培养提取总RNA的野生型AMB-1细胞的充足和铁限制培养基。在铁限制培养基中培养的细胞样品中检测到强杂交。该结果表明，在从NM 5克隆的EcoRI片段上存在表达受铁调节的基因。对突变株NM 5的基因组DNA进行了全序列测定。发现三个开放的阅读框，其中一个被Tn 5插入中断。一个假定的启动子和核糖体结合位点存在于该ORF的上游。利用DNA /蛋白质计算机分析软件DNASIS对该ORF进行分析，命名为magA。预测的magA基因产物的氨基酸序列与阳离子转运通道蛋白，特别是大肠杆菌Kef C蛋白具有高度同源性，其作为控制膨压的钾流出蛋白发挥作用。此外，MagA的氨基酸序列显示出高疏水性。因此，MagA可能是本地化的膜部分，并可能作为一个阳离子转运蛋白在磁性细菌磁细菌。AMB-1。少

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