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Notes: Many streptomycetes produce extracellular γ-butyrolactones. In several cases, these have been shown to act as signals for the onset of antibiotic production. Synthesis of these molecules appears to require a member of the AfsA family of proteins (AfsA is required for A-factor synthesis of the γ-butyrolactone A-factor and consequently for streptomycin production in Streptomyces griseus). An afsA homologue, scbA, was identified in Streptomyces coelicolor A3(2) and was found to lie adjacent to a divergently transcribed gene, scbR, which encodes a γ-butyrolactone binding protein. Gel retardation assays and DNase I footprinting studies revealed DNA binding sites for ScbR at − 4 to − 33 nt with respect to the scbA transcriptional start site, and at − 42 to − 68 nt with respect to the scbR transcriptional start site. Addition of the γ-butyrolactone SCB1 of S. coelicolor resulted in loss of the DNA-binding ability of ScbR. A scbA mutant produced no γ-butyrolactones, yet overproduced two antibiotics, actinorhodin (Act) and undecylprodigiosin (Red), whereas a deletion mutant of scbR also failed to make γ-butyrolactones and showed delayed Red production. These phenotypes differ markedly from those expected by analogy with the S. griseus A-factor system. Furthermore, transcription of scbR increased, and that of scbA was abolished, in an scbR mutant, indicating that ScbR represses its own expression while activating that of scbA. In the scbA mutant, expression of both genes was greatly reduced. Addition of SCB1 to the scbA mutant induced transcription of scbR, but did not restore scbA expression, indicating that the deficiency in scbA transcription in the scbA mutant is not solely due to the inability to produce SCB1, and that ScbA is a positive autoregulator in addition to being required for γ-butyrolactone production. Overall, these results indicate a complex mechanism for γ-butyrolactone-mediated regulation of antibiotic biosynthesis in S. coelicolor.

Notes: Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a γ-butyrolactone 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 γ-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 M1 than did the wild-type strain (minimum inhibitory concentration, 50 µg ml−1 compared with 〉 200 µg ml−1) 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 RedD-like pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of β-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-binding activity of BarA through protein–protein interactions.

Notes: Gamma-butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one γ-butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis (scbA) and binding (scbR) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the γ-butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway-specific regulatory gene, kasO, is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at −3 to −35 nt and the other at −222 to −244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO, indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a γ-butyrolactone-binding protein directly regulates a secondary metabolite pathway-specific regulatory gene in Streptomyces.

Notes: We report here that Streptomyces coelicolor A3(2) produces at least seven butyrolactone autoregulators: two of the IM-2 type, four virginiae butanolide type, and one A-factor type. The most abundant one corresponds to virginiae butanolide-C9 having a C2 side chain of nine carbons. Model butyrolactone compounds as well as extracts of S. coelicolor mycelia showed clear induction of morphological differentiation, implying that S. coelicolor A3(2) probably possessed butyrolactone-type autoregulator(s) controlling the morphological differentiation.

Notes: Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydroVB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydroVB-A reductase was found to require NADPH, not NADH, as a coenzyme. The Km values of the enzyme for NADPH and (±)-6-dehydroVB-A were determined to be 50±2 μM and 100±5 μM, respectively. Ultracentrifugation experiments revealed that 6-dehydroVB-A reductase was present almost exclusively in the 100 000×g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The Mr of the native 6-dehydroVB-A reductase was estimated to be 82 000±3000 by molecular sieve HPLC. The optimal pH was found to be 6.7±0.2.

Notes: Abstract IM-2, one of the butyrolactone autoregulators of Streptomyces, triggers blue pigment production in Streptomyces sp. FRI-5 at a concentration of 0.6 ng/ml. Two IM-2-induced metabolites were identified by comparison of the HPLC profiles of extracellular metabolites produced in the absence and presence of IM-2. The two compounds were purified to homogeneity from the culture broth, and assigned as tryptophol and guanosine, respectively. With respect to the IM-2-induced production of guanosine, the intracellular and extracellular nucleotide contents were investigated in the absence and presence of IM-2. No difference in nucleotide content, including that of ppGpp, was observed, indicating that ppGpp does not participate in the induction pathway of IM-2. It was therefore concluded that IM-2 is an independent regulator of Streptomyces metabolism, which not only induces blue pigment but also affects metabolism of tryptophan and guanosine.