A Novel RNA Aptamer as Synthetic Inducer of DasR Controlled Transcription.

Progress in the synthetic biology field is driven by the development of new tools for synthetic circuit engineering. Traditionally, the focus has relied on protein-based designs. In recent years, the use of RNA-based tools has tremendously increased, due to their versatile functionality and applicability. A promising class of molecules is RNA aptamers, small, single-stranded RNA molecules that bind to a target molecule with high affinity and specificity. When targeting bacterial repressors, RNA aptamers allow one to add a new layer to an established protein-based regulation. In the present study, we selected an RNA aptamer binding the bacterial repressor DasR, preventing its binding to its operator sequence and activating DasR-controlled transcription in vivo. This was made possible only by the combination of an in vitro selection and subsequent in vivo screening. Next-generation sequencing of the selection process proved the importance of the in vivo screening for the discovery of aptamers functioning in the cell. Mutational and biochemical studies led to the identification of the minimal necessary binding motif. Taken together, the resulting combination of bacterial repressor and RNA aptamer enlarges the synthetic biology toolbox by adding a new level of regulation.

sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism.

In contrast to transcriptional regulation, post-transcriptional regulation and the role of small non-coding RNAs (sRNAs) in streptomycetes are not well studied. Here, we focus on the highly conserved sRNA scr5239 in Streptomyces coelicolor. A proteomics approach revealed that the sRNA regulates several metabolic enzymes, among them phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the central carbon metabolism. The sRNA scr5239 represses pepck at the post-transcriptional level and thus modulates the intracellular level of phosphoenolpyruvate (PEP). The expression of scr5239 in turn is dependent on the global transcriptional regulator DasR, thus creating a feedback loop regulation of the central carbon metabolism. By post-transcriptional regulation of PEPCK and in all likelihood other targets, scr5239 adds an additional layer to the DasR regulatory network and provides a tool to control the metabolism dependent on the available carbon source.

Identification of metE as a second target of the sRNA scr5239 in Streptomyces coelicolor.

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.

Conditional control of gene expression by synthetic riboswitches in Streptomyces coelicolor.

Here we provide a step-by-step protocol for the application of synthetic theophylline-dependent riboswitches for conditional gene expression in Streptomyces coelicolor. Application of the method requires a sequence of only ~85 nt to be inserted between the transcriptional start site and the start codon of a gene of interest. No auxiliary factors are needed. All tested riboswitch variants worked well in concert with the promoters galP2, ermEp1, and SF14. Moreover, they allowed theophylline-dependent expression not only of the heterologous ß-glucuronidase reporter gene but also of dagA, an endogenous agarase gene. The right combination of the tested promoters with the riboswitch variants allows for the adjustment of the desired dynamic range of regulation in a highly specific and dose-dependent manner and underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoter can be combined with the presented riboswitches. Moreover, this system should easily be transferable to other Streptomyces species, and most likely to any other genetically manipulable bacteria.

Small non-coding RNAs in streptomycetes.

Streptomycetes are Gram-positive, GC-rich, soil dwelling bacteria, occurring ubiquitary throughout nature. They undergo extensive morphological changes from spores to filamentous mycelia and produce a plethora of secondary metabolites. Owing to their complex life cycle, streptomycetes require efficient regulatory machinery for the control of gene expression. Therefore, they possess a large diversity of regulators. Within this review we summarize the current knowledge about the importance of small non-coding RNA for the control of gene expression in these organisms.

Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor.

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 theophylline-dependent expression of not only the heterologous ß-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.

Streptomyces coelicolor sRNA scr5239 inhibits agarase expression by direct base pairing to the dagA coding region.

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 post-transcriptional level by direct base pairing to the coding region 33 nt downstream of the ribosome-binding site.

Deep sequencing-based identification of small non-coding RNAs in Streptomyces coelicolor.

Streptomyces coelicolor is considered the model organism among Gram positive, GC rich bacteria. Its genome has been sequenced but little is known about the occurrence and distribution of small non-coding RNAs in this biotechnologically relevant organism. Using deep sequencing we analyzed the transcriptome at the end of exponential growth, which corresponds to the onset of secondary metabolism. We mapped 193 transcriptional start sites of mRNA genes and identified putative new and alternative open reading frames. We identified 63 non-coding RNAs including 29 cis encoded antisense RNAs, and confirmed expression for 11, most of them being growth-phase dependent. A comparison between the sequencing results and bioinformatic sRNA predictions using Dynalign and RNAz revealed only a small overlap between the different approaches.