CHARACTERIZATION OF A NOVEL ACYL CARRIER PROTEIN, RKPF, ENCODED BY ANOPERON INVOLVED IN CAPSULAR POLYSACCHARIDE BIOSYNTHESIS IN SINORHIZOBIUM-MELILOTI

Rhizobial capsular polysaccharides (RKPs) play an important role in th e development of a nitrogen-fixing symbiosis with the plant host and i n Sinorhizobium meliloti AK631 functional rkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpre ssed and purified the derived protein product (RkpF) in Escherichia co li. Like acyl carrier protein (ACP), the RkpF protein ran be labeled i n vivo with radioactive beta-alanine added to the growth medium. If ho mogeneous RkpF protein is incubated with radiolabeled coenzyme A in th e presence of purified holo-ACP synthase from E. coli, an in vitro tra nsfer of 4'-phosphopantetheine to the RkpF protein can be observed, Th e conversion from apo-RkpF protein to holo-RkpF protein seems to go al ong with a major conformational change of the protein structure, becau se the holo-RkpF protein runs significantly faster on native polyacryl amide gel electrophoresis than the apo-RkpF protein, Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF prote in and a mass of 9,927 for the holo-RkpF protein. Our data show that R kpF is a novel ACP.