SummaryVirginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a gbutyrolactone autoregulator and the corresponding receptor protein of the Streptomyces species, and has been shown to regulate the production of antibiotic virginiamycin (VM) in S. virginiae. A divergently transcribed barX gene is situated 259 bp upstream of the barA gene, and the BarX protein has been shown to be highly homologous (39.8% identity, 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 g-butyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison between wild-type S. virginiae and a barX deletion mutant. With the same growth rate as wild-type S. virginiae on both solid and liquid media, the barX mutant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. However, the barX mutant became more sensitive to virginiamycin M 1 than did the wild-type strain (minimum inhibitory concentration, 50 mg ml 21 compared with . 200 mg ml 21 ) and exhibited reduced VB and VM production. The VM production was not restored by exogenous addition of VB, suggesting that BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regulatory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp downstream region of barX revealed the presence of five open reading frames (ORFs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Streptomyces coelicolor RedDlike pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of b-ketoacyl ACP/CoA reductase; and orf5, encoding a homologue of dNDP-glucose dehydratase. Reverse transcription polymerase chain reaction (RT-PCR) analyses of the downstream five genes together with those of the three upstream genes (barA, barB, encoding a regulatory protein; and varS, encoding a virginiamycin S specific transporter) revealed that, in the barX mutant, the transcriptions of barZ, orf2, varM and orf5 were completely 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 transcription of bicistronic barB±varS operon through binding to a specific DNA sequence (BarA-responsive element, BARE) overlapping the barB transcriptional 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-binding 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, indicating that BarX is likely to be a co-repressor of BarA, enforcing the DNA-bindin...

BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barBtermination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was namedvarS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in virginiamycin S-specific resistance, suggesting that varS encoded a virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of virginiamycin M1 and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic varS transcript.