Identification by Gene Deletion Analysis of a Regulator, VmsR, That Controls Virginiamycin Biosynthesis in Streptomyces virginiae

Abstract

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...

Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

“…In the left-hand extremity of the 77 kb virginiamycin biosynthetic gene cluster of S. virginiae, we have characterized in this study two pathway-specific regulatory genes, vmsS and vmsT, in addition to the previously identified vmsR gene (Kawachi et al, 2000b). VmsS is among the SARP-family proteins that are frequently present in biosynthetic gene clusters of Streptomyces origin and that regulate the production of corresponding secondary metabolites.…”

“…As the previously created vmsR-disrupted strain (DR1 strain) contained a deletion of a 145 bp 59-untranslated region (UTR) in addition to the deletion of a 698 bp region of vmsR (Kawachi et al, 2000b), we reconstructed an in-frame deletion mutant for vmsR (mutant IC104), as described in Methods, to completely avoid any indirect effects due to the deletion of the 59-UTR. The behaviour of mutant IC104 was similar to that of the DR1 strain: it did not produce any virginiamycin throughout the cultivation period.…”

“…These genes are clustered in a 10 kb region, and thus are considered as the virginiamycin regulatory island. This 10 kb regulatory island includes one pathway-specific regulatory gene, vmsR, which is essential for both VM and VS production and is transcriptionally controlled by higher-level regulators such as barA, thus indicating that VmsR is a lowerlevel regulator for virginiamycin production in the VBBarA regulatory system (Kawachi et al, 2000b). Recently, we characterized a 62 kb virginiamycin biosynthesis gene cluster in the flanking region of the virginiamycin regulatory island (Pulsawat et al, 2007).…”

Identification by Heterologous Expression and Gene Disruption of VisA as l -Lysine 2-Aminotransferase Essential for Virginiamycin S Biosynthesis in Streptomyces virginiae

“…The highly temporal transcrip- Fig. 6) (7,12,14). barA, VB receptor gene (8); barB, a kind of regulatory gene (unpublished data); varS, VS resistance gene (11); varR, varS repressor gene (12).…”

“…To date, its specific receptor protein, BarA, has been identified (14) and it was clarified that the binding of VB to BarA triggers the production of virginiamycins (8). Several genes relating to the regulation of virginiamycin biosynthesis have been found around the barA gene (7,12), but the structural genes for VS and VM 1 have not been identified in S. virginiae as yet.…”