Modify of the bacterial magnetic particles by gene manipulation

Magnetospirillum sp. AMB-1是一种淡水磁性细菌，可合成细胞内磁铁矿（Fe_30_4）颗粒。先前通过引入转座子Tn5产生了5个AMB-1的非磁性突变体。在这项研究中，从一个非磁性突变体NM5中分离出合成磁性颗粒所需的基因组DNA片段。我们已经确定了这个片段的完整核苷酸序列。2975 bp区域包含两个假定的开放阅读帧。其中一个ORF，被命名为magA，编码一种多肽，该多肽与阳离子外排蛋白、大肠杆菌钾离子转运蛋白KefC和来自hirae肠球菌的Na^+/H^+-反转运蛋白NapA同源。Northern杂交表明，magA mRNA转录物的大小为1.3 kb，与magA基因的大小相对应。在AMB-1中，magA基因上游有一个功能性启动子，其转录受环境铁浓度的调控。我们开发了一种简单而通用的方法，该方法可以将多肽显示在细菌磁性颗粒表面，便于使用magA基因融合对其进行直接磁性纯化。编码要显示的蛋白质的基因在磁螺旋藻magA基因的下游融合，然后在磁颗粒表面表达。为了证明该方法的有效性，荧光素酶在磁性颗粒表面表达，然后通过细胞裂解和磁性纯化回收。利用从AMB-1中分离的磁性颗粒检测了荧光素酶的活性，该磁性颗粒含有磁性荧光素酶融合基因。该方法将显著提高蛋白质筛选和纯化策略。

Magnetospirillum sp. AMB-1 is a freshwater magnetic bacterium which synthesizes intracellular particles of magnetite (Fe_30_4). Five nonmagnetic mutants of AMB-1 were previously generated by introduction of transposon Tn5.In this study, a genomic DNA fragment required for synthesis of magnetic particles was isolated from one of the nonmagnetic mutants, NM5. We have determined the complete nucleotide sequence of this fragment. The 2975 bp region contains two putative open reading frames. One ORF,designated magA,encodes a polypeptide which is homologous to the cation efflux proteins, the Escherichia coli potassium ion translocating protein, KefC,and the putative Na^+/H^+-antiporter, NapA,from Enterococcus hirae. Northern hybridization demonstrated that the magA mRNA transcript is 1.3 kb in size, corresponding to the size of the magA gene. A functional promoter was located upstream from the magA gene, and the transcription was regulated by environmental iron concentration in AMB-1.We devel ped a simple and general method in which peptides can be displayd on the surface of bacterial magnetic particles facilitating their direct magnetic purification using the magA gene fusion. Genes encoding the proteins to be displayd are fused downstream from the Magnetospirillum magA gene which are then expressed on the magnetic particle surface. To demonstrate the effectiveness of this method firefly luciferase was expressed on the surface of the magnetic particles which could then be recovered by cell lysis and magnetic purification. Luciferase activity was detected on the magnetic particles which were isolated from AMB-1 harboring the magA-luciferase fusion gene. This approach will significantly enhance protein screening and purification strategies.