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...

Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a butyrolactone autoregulator and a corresponding receptor protein of Streptomyces species and regulates the production of the antibiotic virginiamycin (VM) in S. virginiae. The gene vmsR was found to be situated 4.7 kbp upstream of the barA gene, which encodes the VB-specific receptor. The vmsR product was predicted to be a regulator of VM biosynthesis based on its high homology to some Streptomyces pathway-specific transcriptional regulators for the biosynthetic gene clusters of polyketide antibiotics, such as Streptomyces peucetius DnrI (47.5% identity, 84.3% similarity), which controls daunorubicin biosynthesis. A vmsR deletion mutant was created by homologous recombination. Neither virginiamycin M 1 nor virginiamycin S was produced in the vmsR mutant, while amounts of VB and BarA similar to those produced in the wild-type strain were detected. Reverse transcription-PCR analyses confirmed that the vmsR deletion had no deleterious effects on the transcription of the vmsR-surrounding genes, indicating that VmsR is a positive regulator of VM biosynthesis in S. virginiae.Streptomycetes are gram-positive filamentous bacteria that are well-known for producing a vast array of bioactive compounds, including more than 70% of commercially important antibiotics. The production of antibiotics by these organisms is regulated by a variety of physiological and nutritional conditions and is coordinated with processes of morphological differentiation, such as the formation of aerial mycelia and spores. Despite many years of research on antibiotics driven by their commercial importance, the overall regulatory pathway governing antibiotic production is still poorly understood. A detailed knowledge of the signal cascade and the genetic components involved in antibiotic production should permit construction of strains that can overproduce these commercially important compounds.Antibiotic production and/or morphological differentiation is controlled in several Streptomyces species by low-molecularweight compounds called butyrolactone autoregulators (32). To date, 11 butyrolactone autoregulators have been chemically identified and classified into three types based on minor structural differences in their C-2 side chains: (i) the virginiae butanolide (VB) type, containing a 6-␣-hydroxy group (13, 31); (ii) the IM-2 type, containing a 6-␤-hydroxy group (25, 27); and (iii) the A-factor type, containing a 6-keto group (16). Their effectiveness at nanomolar concentrations, as well as the presence in these species of specific receptor proteins (BarA as a VB-specific receptor in Streptomyces virginiae [9,10,21], FarA as an IM-2-specific receptor in Streptomyces lavendulae 28], and ArpA as an A-factor-specific receptor in Streptomyces griseus [22]) as mediators of autoregulator signaling, implies that they should be regarded as Streptomyces hormones.VB-BarA of S. virginiae has been among the most frequently studied pairs and is known to regula...