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CLONING OF A GENE ENCODING PROTEIN BELONGING TO ABC TRANSPORTER INVOLVED IN BACTERIAL MAGNETIC PARTICLE SYNTHESIS IN MAGNETOSPIRILLUM MAGNETICUM AMB-1

Keywords: Magnetospirillum magneticum AMB-1 , Bacterial Magnetic Particle (BMP) , ATP Binding Cassette (ABC) Transporter , transposon mutagenesis.

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Abstract:

Magnetospirillum magneticum AMB-1 synthesizes intracellular magnetic particles, magnetite (Fe3O4), enveloped by membrane called magnetosome under micro-aerobic conditions. Initial study of random transposon-based mutagenesis generated 62 nonmagnetic mutants of AMB-1 in a mini-Tn5 library. In order to identify a gene involved in bacterial magnetic particle (BMP) synthesis in the magnetic bacterium M. magneticum AMB-1, a nonmagnetic mutant from the library designated as NMA38-4, was analyzed. h e amino acid sequence deduced from the gene directly interrupted by transposon, ORF4 (1482 bp), showed homology to ATP binding cassette (ABC) transporter of Mesorhizobium loti with 62 % identity and 74 % similarity. It was strongly indicated by the occurrence of putative consensus sequence of ATP-binding motifs (ATP-binding protein). h e ORF4 was subsequently cloned in pET-15b and the recombinant ORF4-Histag fusion protein was heterologously expressed in Escherichia coli BL21 (DE3) pLysS. A 55 kDa protein corresponding to the ORF4-Histag fusion protein was obtained after purifi cation using Ni-NTA column. h is is the fi rst report describing a gene cluster containing gene encoding protein belonging to ABC transporter organized in an operon which is involved in BMP synthesis.

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