Localization Of The Protein Encoded By A Gene That Is Closely Related To The Synthesis Of Magnetosome In Magnetospirillum Gryphiswaldense MSR-1

Magnetospirillum gryphiswaldense MSR-1, the modal strain of genus Magnetospirillum, is capable of synthesizing specific intracellular structures, magnet- osomes, which are chain-linked and membrane-enclosed crystals of magnetic iron oxide. At the beginning of our experiments, a magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment contained three putative ORFs. Analysis of the protein encoded by ORF1 suggested that it probably located on the membrane of magnetosomes, take part in the biosyn- thesis of magnetosomes as Fe2+ transporter. In confirmation of the hypothesis, the subcellular localization of a protein encoded by ORF1 from 3073-bp fragment in Magnetospirillum gryphiswaldense MSR-1 was studied.Analysis of some bio-software predicted that ORF1 encoded a putative protein of 320 amino acids. There were no signal peptide in protein ORF1 which contained 5 hydrophobic regions, 4 transmembrane domains, 18 protein phosphorylation sites, 8 -helixes and 2β-sheets, most probably located on plasma membrane or extracellular. These informations will help us to study the ORF1 protein.The ORF1 was PCR amplified with genomic DNA of MSR-1 as a template, the EGFP was amplified from plasmid pEGFPN-1 respectively; The ORF1'was PCR am- plified with genomic DNA of MSR-1 as a template, the EGFP'was amplified from plasmid pEGFPN-1 respectively. Both of them were subcloned into the broad- host-range vector pBBR1MCS-2, then transformed into E. coli S17-1 to generate fusion expression vector pBBR1MCS-ORF1-EGFP and pBBR1MCS- EGFP'-ORF1'. They were transferred into MSR-1 by conjugation from E. coli S17-1. Localization of ORF1 protein was examined by fluorescence microscopy after the positive clone were induced for protein expression with a result of on the membrane of magnetosomes.The ORF1'was PCR amplified with genomic DNA of M. gryphiswaldense as a template then cloned into the pET-29a vector. The recombinant plasmid pET-29a- ORF1'was transformed into E. coli BL 21 and induced for expression. SDS-PAGE profile showed there was no clear increased protein band.Research preliminarily demonstreted that the protein encoded by ORF1 precisely located on the membrane of magnetosomes. The result laid a foundation for the research of its function, as well as the further research of the role of the 3073-bp and its flanking fragment in the biosynthesis of magnetosome in MSR-1.