Cloning and Overexpression of a Gene Encoding Protein Belong to Tetratricopeptide Involved in Magnetosome Synthesis in Magnetospirillum magneticum AMB-1

Abstract

A genomic DNA fragment involved in magnetosome synthesis was isolated from Magnetospirillum magneticum AMB-I through mini-Tn5 transposon mutagenesis. The DNA fragment flanking transposon from the genomic DNA of a non-magnetic mutant, designated as NMA42, was isolated by inverse polymerase chain reaction (inverse PCR), sequenced and aligned against the whole genome sequence of AMB-1. A 6020 bp genomic DNA sequence consisting of four open reading frames (ORFs) organized in an operon was determined. The deduced amino acid sequence of 1977 base pair from ORF4 directly interrupted by transposon homolog with tetratricopeptide repeat domain protein (TPR) from Chlamydophila pneumoniae (30% identity, 46% similarity). The presence of four repeats of a degenerate 34 amino acids consensus sequence indicate that the protein may interact with proteins in the cytoplasm to function during magnetosome synthesis. The ORF4 was subsequently isolated from AMB-l genome by polymerase chain reaction (PCR), cloned, and overcxpressed in Escherichia coli as a 70.8 kDa His-tagged polypeptide.