Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M₁ resistance

Abstract

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...

γ-Butyrolactone autoregulator-receptor system involved in lankacidin and lankamycin production and morphological differentiation in Streptomyces rochei

“…The orf85 gene (named srrX, Streptomyces rochei regulatory gene X) encodes a neutral protein (pI 6.85) with 331 amino acids and a molecular mass of 36.8 kDa. The SrrX protein is most similar to BarX (46 % identity), which is involved in the biosynthesis of virginiae butanolides in S. virginiae, leading to virginiamycin production (Kawachi et al, 2000).…”

“…Search for additional AdpAbinding DNA sequences identified an ECF sigma factor gene (Yamazaki et al, 2000), a metalloendopeptidase gene (Kato et al, 2002) and many additional genes, which make an AdpA regulon functioning for secondary metabolism and morphological development (Ohnishi et al, 2005). Following the afsA-arpA system, similar c-butyrolactone receptor systems have been found in streptomycetes, for example, the barX-barA system in Streptomyces virginiae (Kawachi et al, 2000), farX-farA in Streptomyces lavendulae (Kitani et al, 2001) and scbA-scbR in S. coelicolor A3(2) (Takano et al, 2001). However, their effects on antibiotic production and morphological differentiation are different from species to species.…”

“…The orf85 gene (named srrX, Streptomyces rochei regulatory gene X) encodes a neutral protein (pI 6.85) with 331 amino acids and a molecular mass of 36.8 kDa. The SrrX protein is most similar to BarX (46 % identity), which is involved in the biosynthesis of virginiae butanolides in S. virginiae, leading to virginiamycin production (Kawachi et al, 2000).To reveal the function of srrX in S. rochei 7434AN4, its disruptant was constructed from strain 51252, which carries only the largest linear plasmid pSLA2-L. The srrX disruptant KY85 did not produce lankacidin or lankamycin (Fig.…”

Biosynthesis of γ-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces

“…However, the AfsA function has been controversial (12,13). Concerning the biosynthesis pathway, Sakuda et al (1,14,15) proposed a route on the basis of feeding experiments in VB-producing Streptomyces antibioticus (see pathway A in Fig.…”

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

“…The flanking regions of barA contain other important genes that are involved in VB biosynthesis (barX, barS1 and barS2) and mechanisms of resistance to VM and VS (varM, varR and varS) (Kawachi et al, 2000a;Namwat et al, 2001) (Fig. 1).…”