Modify of the bacterial magnetic particles by gene manipulation

磁旋藻。AMB-1是一种淡水磁性细菌，能合成细胞内的磁铁矿颗粒(Fe304)。通过引入转座子Tn5，获得了AMB-1的5个非磁性突变体。本研究从其中一个非磁性突变体Nm5中分离到了合成磁性颗粒所需的基因组DNA片段。我们已经确定了该片段的完整核苷酸序列。2975bp的区域包含两个推测的开放阅读框架。一个命名为MAGA的ORF编码一个多肽，它与来自Hirae肠球菌的阳离子外排蛋白、大肠杆菌钾离子转运蛋白KefC和可能的Na+/H+-逆向转运蛋白NapA同源。Northern杂交结果表明，MAGA基因转录本的大小为1.3kb，与MAGA基因的大小一致。在MAGA基因上游有一个功能性启动子，AMB-1的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，将多肽展示在细菌磁性颗粒的表面，便于利用MagA基因融合直接进行磁性纯化。编码要展示的蛋白质的基因在磁螺菌Maga基因下游融合，然后在磁性颗粒表面表达。为了证明这种方法的有效性，将萤火虫荧光素酶表达在磁性颗粒的表面，然后通过细胞裂解和磁性纯化来回收。从含有Maga-荧光素酶融合基因的AMB-1中分离的磁性颗粒上检测到荧光素酶活性。这种方法将大大增强蛋白质的筛选和纯化策略。

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.