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.

SummaryGamma-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 g g g g -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 g g g g -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 g g g gbutyrolactone-binding protein directly regulates a secondary metabolite pathway-specific regulatory gene in Streptomyces .

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a ␥-butyrolactone of structure (2R,3R,1R)-2-(1-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide. Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.Small diffusible signaling molecules play regulatory roles in a wide variety of bacteria. The most intensively studied are the N-acyl homoserine lactones, which have diverse roles as signaling molecules in a wide range of Gram-negative bacteria (1). Among Gram-positive bacteria, structurally similar but chemically distinct ␥-butyrolactones play determining roles in antibiotic production and sporulation in Streptomyces species (2) (Fig. 1). Streptomycetes are mycelial soil bacteria with a developmental program that results in sporulation. They also produce a wide variety of antibiotics with important uses in medicine and in agriculture. These antibiotics are the products of complex biosynthetic pathways, activated typically in a growth phase-dependent manner (3). In liquid culture, antibiotic production generally occurs in stationary phase (4, 5), while in surface-grown cultures, it usually coincides with the onset of morphological differentiation, i.e. the formation of aerial hyphae.The most intensively studied ␥-butyrolactone is A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone), which is required for streptomycin production and sporulation in Streptomyces griseus (6). A-factor binds to a cytoplasmic protein that in its absence represses streptomycin production and morphological differentiation (7). Other ␥-butyrolactones have also been shown to induce antibiotic biosynthesis, e.g. the virginiae butanolides of Streptomyces virginiae (8, 9). Moreover, since at least 60% of Streptomyces species appear to produce ␥-butyrolactones (9), these compounds are likely to play important roles as extracellular signaling molecules in the biology of these organisms. Streptomyces coelicolor A3(2), the most genetically characterized streptomycete, produces at least four antibiotics, including the blue-pigmented polyketide actinorhodin (Act), 1 and the red-pigmented tri-pyrolle undecylprodigiosin (Red), both of which are produced in a growth phase-dependent manner (4, 5, 10). Earlier studies (11, 12) led to the isolation and partial structural determination of six ␥-butyrolactones made by S. coelicolor (Fig. 2), but there was no report of their biological activity in the producing strain.This paper identifies four low molecular weight compounds present in the supernatants of transition and stationary phase cultures of S. coelicolor...

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone–type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis —the producer of the important anthelmintic agent avermectin with annual world sales of $850 million—and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4 S ,10 R )-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae , Streptomyces ghanaensis , and Streptomyces griseoauranticus , suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.