AN ACCESSORY GENE, LIPB, REQUIRED FOR THE PRODUCTION OF ACTIVE PSEUDOMONAS-GLUMAE LIPASE

Pseudomonas glumae PG1 is able to secrete lipase into the extracellula r medium. The lipase is produced as a precursor protein, with an N-ter minal signal sequence. A second open reading frame (ORF) was found imm ediately downstream of the lipase structural gene, lipA, a situation f ound for the lipases of some other Pseudomonas species. Inactivation o f this ORF resulted in a lipase-negative phenotype, indicating its imp ortance in the production of active extracellular lipase. The ORF, lip B, potentially encodes a protein of 353-amino-acid residues, having a hydrophobic N-terminal (amino acids 1 to 90) and a hydrophilic C-termi nal part. As a first step in determining the role of LipB, its subcell ular location was determined. The protein was found to fractionate wit h the inner membranes. The expression of fusions of lipB fragments wit h phoA revealed an N(in)-C(out) topology for the LipB protein, which w as confirmed by protease accessibility studies on EDTA-permeabilized c ells and on inverted inner membrane vesicles. These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an N-terminal transmembrane hel ix, located between amino acids 19 and 40.