VIRGINIAE BUTANOLIDE BINDING-PROTEIN FROM STREPTOMYCES-VIRGINIAE - EVIDENCE THAT VBRA IS NOT THE VIRGINIAE BUTANOLIDE BINDING-PROTEIN AND REIDENTIFICATION OF THE TRUE BINDING-PROTEIN

Virginiae butanolides (VBs) A-E are butyrolactone autoregulators that control virginiamycin production in Streptomyces virginiae. We have pr eviously reported the purification and molecular cloning of VbrA, a pu tative VB binding protein (Okamoto, S., Nihira, T., Kataoka, H., Suzuk i, A., and Yamada, Y. (1992) J. Biol. Chem. 267, 1093-1098). However, VbrA protein overexpressed in Escherichia coli did not show any detect able VB binding activity nor did the immunoprecipitation of native Vbr A from a cell-free extract of S. virginiae cause any decrease in such activity, indicating that VbrA is not the true VB binding protein. Thi s finding prompted us to seek the true VB binding protein by repurific ation. After successive purification by anion exchange, gel filtration , heparin, and hydrophobic interaction chromatography, a 26-kDa protei n (p26k) was identified as the true VB binding protein. Partial amino acid sequences of p26k were determined, and the gene (barA) that encod es this protein was isolated and cloned using degenerate oligonucleoti de probes. When the barA gene was expressed in Streptomyces lividans a nd E. coli, strong VB binding activity appeared, demonstrating unambig uously that the S. virginiae p26k protein is the true VB binding prote in.