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We have demonstrated the portability of theophylline-dependent synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. The riboswitches mediate dose-dependent, up to 260-fold activation of reporter gene expression. Riboswitch regulation is a simple method requiring a sequence of only~85 nt to be inserted between a transcriptional start site and the start codon; no additional auxiliary factors are necessary. The promoters galP2, ermEp1 and SF14 worked well in concert with the riboswitches. They allowed theophyllinedependent expression of not only the heterologous b-glucuronidase reporter gene but also dagA, an endogenous agarase gene. The successful combination of all tested promoters with the riboswitches underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoters can be combined with the riboswitch.

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Transcriptional regulation of primary and secondary metabolism is well-studied in Streptomyces coelicolor, a model organism for antibiotic production and cell differentiation. In contrast, little is known about post-transcriptional regulation and the potential functions of small non-coding RNAs (sRNAs) in this Gram-positive, GC-rich soil bacterium. Here, we report the identification and characterization of scr5239, an sRNA highly conserved in the genus Streptomyces. The sRNA is 159 nt long, composed of five stem-loops, and encoded in the intergenic region between SCO5238 and SCO5239. scr5239 expression is constitutive under several stress and growth conditions but dependent on the nitrogen supply. scr5239 decreases the production of the antibiotic actinorhodin, and represses expression of the extracellular agarase dagA at the posttranscriptional level by direct base pairing to the coding region 33 nt downstream of the ribosome-binding site. INTRODUCTIONStreptomyces coelicolor is a model organism for Grampositive, filamentous, GC-rich soil-dwelling bacteria. It undergoes complex morphological differentiation, involving the development of sporophyte and mycelia colonies, and is characterized by a complex secondary metabolism. Streptomycetes produce two-thirds of all known antibiotics as well as a wide range of other bioactive compounds such as immunosuppressants, antivirals and herbicides, and are therefore of great industrial importance (Watve et al., 2001). Because of their ability to excrete correctly folded proteins, streptomycetes are also attractive for the production of recombinant human proteins (Binnie et al., 1997).Bacteria living in a highly variable environment, such as the soil, need the ability to adapt to sudden and drastic changes in environmental conditions (McAdams et al., 2004). This is reflected by the fact that a tenth of all proteins are predicted to have a regulatory function in S. coelicolor, controlling the metabolic and morphological changes that these bacteria undergo during their life cycle (Bentley et al., 2002). Transcriptional regulation of metabolic processes and the control of secondary metabolism have been studied intensely; however, little is known about post-transcriptional regulation in this organism (Chater, 2001;Claessen et al., 2006;Reuther & Wohlleben, 2007).Small non-coding RNAs (sRNAs), typically 50-200 nt in length, have recently emerged as an abundant class of posttranscriptional regulators in many and diverse bacteria (Sharma & Vogel, 2009). These sRNAs act by several mechanisms (Babitzke & Romeo, 2007;Gottesman, 2004;Vogel, 2009;Vogel et al., 2004), of which short and imperfect antisense pairing to trans-encoded target mRNAs seems to be the most common. Although several sRNAs are known to activate gene expression (Fröhlich & Vogel, 2009), the majority of sRNAs studied to date act to repress the translation of mRNAs by sequestering the RBS of target genes (Unoson & Wagner, 2008;Waters & Storz, 2009). The pairing often entails degradation of the interacting RNAs by end...

While transcriptional regulation of the primary and secondary metabolism of the model organism Streptomyces coelicolor is well studied, little is still known about the role small noncoding RNAs (sRNAs) play in regulating gene expression in this organism. Here, we report the identification of a second target of the sRNA scr5239, an sRNA highly conserved in streptomycetes. The 159 nt long sRNA binds its target, the mRNA of the cobalamin independent methionine synthase metE (SCO0985), at the 5’ end of its open reading frame thereby repressing translation. We show that a high methionine level induces expression of scr5239 itself. This leads, in a negative feedback loop, to the repression of methionine biosynthesis. In contrast to the first reported target of this sRNA, the agarase dagA, this interaction seems to be conserved in a wide number of streptomycetes.