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

ARIS TRI WAHYUDI

Abstract


Magnetospirillum magneticum AMB-1 synthesizes intracellular magnetic particles,
magnetite (Fe3
O4
), 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.
Key words:   Magnetospirillum magneticum AMB-1, Bacterial Magnetic Particle (BMP), ATP
Binding Cassette (ABC) Transporter, transposon mutagenesis.

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DOI: http://dx.doi.org/10.11598/btb.2009.16.1.62

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