ACYL CARRIER PROTEIN OF AZOSPIRILLUM-BRASILENSE - PROPERTIES OF THE PURIFIED PROTEIN AND SEQUENCING OF THE CORRESPONDING GENE, ACPP

Acyl carrier protein (ACP) plays a crucial role in bacterial fatty aci d synthesis. Cloning genes encoding ACPs from Cram-negative bacteria i n Escherichia coil is difficult due to adverse effects of the cloned g ene on host cell viability, and we were unsuccessful in cloning the fu ll length ACP gene (acpP) from Azospirillum brasilense using conventio nal methods. Therefore, ACP from A. brasilense was purified to homogen eity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-te rminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs, The nucleotide sequence of the gene was obtained by cloning and sequencing inverse P CR products containing the acpP region generated by two oppositely ori ented internal primers designed from the partial acpP gene sequence us ing restriction-enzyme-digested, self-circularized chromosomal DNA fra gments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a hi ghly expressed protein (about 2.0-3.0 x 10(4) molecules per cell), (b) compared to E. coil ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potent ial mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.