Analysis of genes concerned with bacterial magnetite production

1.磁性细菌基因转移系统的建立：将大宿主范围的质粒转移到好氧磁性细菌Magnetspirillum sp.AMB-1。与大肠杆菌S17-1的配对允许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。两种载体的最佳交配时间均为6h。这些质粒成功地在转接子中形成了自主复制，并可以被分离并转化回大肠杆菌，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。我们克隆了含有Tn5突变位点的EcoRI基因组DNA片段。对该克隆片段的基因表达和核苷酸序列进行了分析。以Tn5插入位点两侧的DNA片段为探针，进行斑点杂交。铁绒…为培养野生型AMB-1细胞，制备了较为适宜的、铁受限的培养液，提取了总RNA。在铁限制培养液中培养的细胞样本检测到强杂交。这一结果表明，在克隆的NM5EcoRI片段上存在受铁调控的基因(S)。对突变体NM5的2640bp基因组DNA片段进行了全序列测定。发现了三个开放阅读框，其中一个被Tn5插入中断。该ORF的上游存在一个可能的启动子和一个核糖体结合部位。用DNA/蛋白质计算机分析软件DNASIS对这个名为MAGA的ORF进行了分析。预测的MAGA基因产物的氨基酸序列与阳离子转运通道蛋白有很高的同源性，特别是E.ColiKef C蛋白，它作为一种钾外排蛋白来控制肿胀。此外，MAGA的氨基酸序列具有很强的疏水性。因此，MagA可能定位于膜组分，并可能作为一种阳离子转运蛋白在磁性细菌Magnetspirillum sp.中发挥作用。AMB-1。较少

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