Isolation and Analysis of Surface Protein Gene from Bacterial Magnetic Particles

Abstract

Proteins located within the lipid bilayer, surrounding the intracellular bacterial magnetic particles from Magnetospirillum sp. AMB-1 were purified and characterized using two dimensional polyacrylamide gel-electrophoresis. Three major proteins of approximate molecular weight 55.9, 36.4 and 28.4kDa were identified. The N-terminal amino acid sequence of one of these proteins, designated MpsA, was determined and used to design a pair of PCR primers which amplified a 105bp DNA fragment from AMB-1 genomic DNA. Gene-walking, using anchored PCR, was used to determine the complete nucleotide sequence (951bp) of the mpsA gene. mpsA encodes a 317 amino acid protein which does not have an N-terminal cytoplasmic transport signal sequence. Intracellular localization studies were carried out using a mpsA-luc gene fusion expressed in AMB-1 following gene transfer by conjugation. The gene fusion was constructed by cloning a 1.6kb mpsA fragment upstream of luc in the conjugal plasmid pKLC. The MpsA-Luc fusion protein was preferentially located on the magnetic particle membrane. Although the function of MpsA remains unknown, homologies indicate a similarity with biotin dependent carboxylase/decarboxylases.