LIPOPROTEIN FROM THE OSMOREGULATED ABC TRANSPORT-SYSTEM OPUA OF BACILLUS-SUBTILIS - PURIFICATION OF THE GLYCINE BETAINE BINDING-PROTEIN ANDCHARACTERIZATION OF A FUNCTIONAL LIPIDLESS MUTANT

The OpuA transport system of Bacillus subtilis functions as a high-aff inity uptake system for the osmoprotectant glycine betaine. It is a me mber of the ABC transporter superfamily and consists of an ATPase (Opu AA), an integral membrane protein (OpuAB), and a hydrophilic polypepti de (OpuAC) that shows the signature sequence of lipoproteins (B. Kempf and E. Bremer, J. Biol. Chem. 270:16701-16713, 1995). The OpuAC prote in might thus serve as an extracellular substrate binding protein anch ored in the cytoplasmic membrane via a lipid modification at an amino- terminal cysteine residue. A malE-opuAC hybrid gene was constructed an d used to purify a lipidless OpuAC protein. The purified protein bound radiolabeled glycine betaine avidly and exhibited a K-D of 6 mu M for this ligand, demonstrating that OpuAC indeed functions as the substra te binding protein for the B. subtilis OpuA system. We have selectivel y expressed the opuAC gene under T7 phi 10 control in Escherichia call and have demonstrated through its metabolic labeling with [H-3]palmit ic acid that OpuAC is a lipoprotein. A mutant expressing an OpuAC prot ein in which the amino-terminal cysteine residue was changed to an ala nine (OpuAC-3),vas constructed by oligonucleotide site-directed mutage nesis. The OpuAC-3 protein aas not acylated by [H-3] palmitic acid, an d part of it was secreted into the periplasmic space of E. coli, where it could be released from the cells by cold osmotic shock The opuAC-3 mutation was recombined into an otherwise wild-type opuA operon in th e chromosome of B. subtilis. Unexpectedly, this mutant OpuAC system st ill functioned efficiently for glycine betaine acquisition in vivo und er high-osmolarity growth conditions. In addition, the mutant OpuA tra nsporter exhibited kinetic parameters similar to that of the wild-type system. Our data suggest that the lipidless OpuAC-3 protein is held i n the cytoplasmic membrane of B. subtilis via its uncleaved hydrophobi c signal peptide.