Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M-1 resistance

Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newl y discovered pairs of a gamma-butyrolactone autoregulator and the correspon ding receptor protein of the Streptomyces species, and has been shown to re gulate the production of antibiotic virginiamycin (VM) in S. virginiae. A d ivergently transcribed barX gene is situated 259 bp upstream of the barA ge ne, and the BarX protein has been shown to be highly homologous (39.8% iden tity, 74.6% similarity) to S. griseus AfsA. Although AfsA is thought to be a biosynthetic enzyme for A-factor, another member of the family of gamma-b utyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison bet ween wild-type S. virginiae and a barX deletion mutant. With the same growt h rate as wild-type S. virginiae on both solid and liquid media, the barX m utant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. H owever, the barX mutant became more sensitive to virginiamycin M-1 than did the wild-type strain (minimum inhibitory concentration, 50 mu g ml(-1) com pared with > 200 mu g ml(-1)) and exhibited reduced VB and VM production. T he VM production was not restored by exogenous addition of VB, suggesting t hat BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regul atory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp down stream region of barX revealed the presence of five open reading frames (OR Fs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Strept omyces coelicolor RedD-like pathway specific regulator; varM, encoding a ho mologue of ATP-dependent transporters for macrolide antibiotics; orf4, enco ding a homologue of beta-ketoacyl ACP/CoA reductase; and orf5, encoding a h omologue of dNDP-glucose dehydratase. Reverse transcription polymerase chai n reaction (RT-PCR) analyses of the downstream five genes together with tho se of the three upstream genes (barA, barB, encoding a regulatory protein; and varS, encoding a virginiamycin S specific transporter) revealed that, i n the barX mutant, the transcriptions of barZ, orf2, varM and orf5 were com pletely repressed and those of barB and varS were derepressed. Because free BarA (BarA in the absence of VB) in wild-type S. virginiae represses the t ranscription of bicistronic barB-varS operon through binding to a specific DNA sequence (BarA-responsive element, BARE) overlapping the barB transcrip tional start site, the derepression of barB-varS transcription in the barX mutant suggested that the in vivo function of BarA was impaired by the lack of BarX protein. Gel-shift assays revealed that BarA easily lost its DNA-b inding activity in the absence of BarX but that the defect was restored by the presence of recombinant BarX as a fusion with maltose-binding protein ( MBP-BarX), whereas MBP-BarX itself showed no DNA-binding activity, indicati ng that BarX is likely to be a co-repressor of BarA, enforcing the DNA-bind ing activity of BarA through protein-protein interactions.