Purification and molecular cloning of a butyrolactone autoregulator receptor from Streptomyces virginiae.
In streptomyces, low molecular weight compounds termed "autoregulators" have been isolated as primary signal molecules for triggering secondary metabolism and/or cytodifferentiation. Streptomyces virginiae produces a set of autoregulators termed virginiae butanolide A-E which trigger virginiamycin production, and possesses a high-affinity virginiae butanolide receptor (Kim, H.S., Nihira, T., Tada, H., Yanagimoto, M., and Yamada, Y. (1989) J. Antibiot. (Tokyo) 42, 769-778). The virginiae butanolide receptor has now been purified to apparent homogeneity with 14,000-fold purification and an 8.6% activity yield. The purified receptor showed a Mr of 36,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a maximum ligand binding of 33.0 nmol/mg protein, indicating a 1:1 binding stoichiometry (1.18 mol of virginiae butanolide/36 kDa of protein) between virginiae butanolides and the receptor. Due to a blockage at the amino terminus, fragment peptides were generated by lysyl endopeptidase and five partial amino acid sequences were determined. The gene (vbrA) encoding the virginiae butanolide receptor was identified on a 5.0-kbp BamHI fragment by hybridization to synthetic oligonucleotide probes, cloned, and sequenced. Nucleotide-sequence analysis predicted a 319-amino acid open reading frame (vbrA) in which all the partial amino acid sequences of the receptor appeared, and 166 bp downstream from it another open reading frame for a 144-amino acid protein which was designated as a ribosomal protein L11 from its high homology (62-64%) to the amino acid sequences of ribosomal protein L11 of several origins, and thus denoted as rplK. The C-terminal half of VbrA showed 36% overall homology to the amino acid sequence of an essential protein (NusG) of Escherichia coli. Furthermore, the gene assembly of vbrA-rplk of S. virginiae closely resembled that of nusG-rplK of E. coli, suggesting that vbrA may constitute a part of an essential gene cluster encoding components of transcriptional and translational apparatuses.
Okamoto
S
S
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Nihira
T
T
Kataoka
H
H
Suzuki
A
A
Yamada
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C072187:virginiamycin butanolide receptor, Streptomyces