Identification and characterization of sRNAs in Streptomyces coelicolor

Streptomyces coelicolor ist der Modellorganismus der GC reichen, Gram+ Actinomyceten, die mehr als zwei Drittel aller bekannten Antibiotika produzieren. Phänotypisch zeichnet er sich durch die Bildung eines Substrat- und eines Luftmyzels aus, welches im Laufe der weiteren Differenzierung Sporen bildet. Streptomyceten produzieren neben Antibiotika noch eine Vielzahl biotechnologisch interessanter Metaboliten. Der komplexe Lebenszyklus und Stoffwechsel erfordern eine genaue Regulation der Genexpression. Die letzten Jahre haben gezeigt, dass neben Proteinen auch die RNA eine regulatorische Funktion hat. Verschiedene regulatorisch aktive RNA Elemente wie Riboswitche, RNA-Thermometer und kleine nicht kodierende RNAs (small noncoding RNAs – sRNAs) wurden identifiziert. sRNAs wirken meist als antisense Riboregulatoren, indem sie ihre Ziel-mRNA binden und dadurch die Translation hemmen oder fördern. In dieser Arbeit wurden verschiedene bioinformatische Methoden verwendet, um sRNAs im Genom von S. coelicolor vorherzusagen. Es wurden Terminatorstrukturen und konservierte Sekundärstrukturen in den intergenen Regionen vorhergesagt, die keinem Gen zuzuordnen waren. In einem weiteren Ansatz wurden Bindestellen des Regulatorproteins DasR vorhergesagt, um DasR kontrollierte sRNAs zu identifizieren. Zusätzlich wurde mittels 454 Sequenzierung erstmalig das Transkriptom von S. coeliocolor analysiert. Auf diese Weise konnten etwa 500 sRNAs vorhergesagt werden. Eine der beiden charakterisierten sRNAs, sc32, ist 139 nt lang. Ihr Promoter liegt im kodierenden Bereich des Gens bldC und sie wird spezifisch durch Kälteschock induziert. Die zweite charakterisierte sRNA, sc1, ist 159 nt lang und in allen sequenzierten Streptomyceten konserviert. Ihre Expression wird nur bei Stickstoffmangel in der Stationärphase reprimiert. Durch molekularbiologische Analysen konnte ein Zielgen von sc1 identifiziert werden, die extrazelluläre Agarase DagA. Es konnte gezeigt werden, dass sc1 an die dagA-mRNA bindet und dadurch die Translation inhibiert. Als zweites mögliches Ziel von sc1 konnte die Histidinkinase SCO5239 identifiziert werden. Hier wurde gezeigt, dass Koexpression von sc1 die Expression einer SCO5239 Reportergenfusion um den Faktor acht steigert. Durch Analyse des Proteoms von sc1 Mutanten, konnte die differenzierte Expression von elf weiteren Proteinen gezeigt werden. Sc1 scheint als Regulator zu agieren, indem es auf die Stickstoffversorgung der Zelle reagiert und den Sekundärmetabolismus deaktiviert.Streptomyces coelicolor is the model organism for the class of GC rich, gram+ actinomycetes that produce more than two thirds of all known antibiotics. It grows in a characteristic substrate and areal mycelium that during further development diffentiaties into spores. Next to antibiotics, Streptomycetes produce a variety of biotechnological interesting metabolites. This complex life cycle and metabolism demands a strict regulation of gene expression. The last years have shown, that next to proteins also RNA can have a regulatory function. Numerous regulatory active RNA elements like riboswitches, RNA-thermometers and small non coding RNAs (sRNAs) have been identified. In most cases sRNAs act as antisense riboregulators by binding to their target-mRNA and thereby blocking or activating its translation. In this work, several different bioinformatic methods were used to predict sRNAs in the genome of S. coelicolor. With these methods terminators and other conserved secondary structures in the intergenic regions of the genome that could not be assigned to an annotated gene were predicted. In another approach binding sites for the global regulator protein DasR were predicted to identify DasR-controlled sRNAs. Additionally for the first time the transcriptome of S. coelicolor was analyzed by 454 deep sequencing. Using this methods ~ 500 sRNAs could be predicted in the genome of S. coelicolor. One of the two characterized sRNAs – sc32 – is 139nt long. Its promoter resides in the conding region of the gene bldC and its expression is specifically induced by cold-shock conditions. The second characterized sRNA – sc1 – is 159nt long and conserved among all Streptomyces species sequenced today. Its expression is repressed by nitrogen starvation in stationary phase. By biochemical analyses a target mRNA of sc1 could be identified – the extracellular agarase dagA. It could be shown that sc1 binds to the dagA-mRNA and thereby hinders translation. As a second putative target of sc1 the histidine kinase SCO5239 could be identified. It was shown that coexpression of sc1 increases the expression of an SCO5239 reporter gene fusion eight times. Analysis of the proteome of sc1 mutants showed a differential expression of further eleven proteins. Sc1 seems to act as a regulator by sensing the nitrogen supply of the cell and in response deactivating the secondary metabolism

Radiochemical determination of 129I and 36Cl inMEGAPIE, a proton irradiated lead-bismuth eutectic spallation target

The concentrations of the long-lived nuclear reaction products 129I and 36Cl have been measured in samples from the MEGAPIE liquid metal spallation target. Samples from the bulk target material (lead-bismuth eutectic, LBE), from the interface of the metal free surface with the cover gas, from LBE/steel interfaces and from noble metal absorber foils installed in the cover gas system were analysed using Accelerator Mass Spectrometry at the Laboratory of Ion beam Physics at ETH Zürich. The major part of 129I and 36Cl was found accumulated on the interfaces, particularly at the interface of LBE and the steel walls of the target container, while bulk LBE samples contain only a minor fraction of these nuclides. Both nuclides were also detected on the absorber foils to a certain extent (≪ 1% of the total amount). The latter number is negligible concerning the radio-hazard of the irradiated target material; however it indicates a certain affinity of the absorber foils for halogens, thus proving the principle of using noble metal foils for catching these volatile radionuclides. The total amounts of 129I and 36Cl in the target were estimated from the analytical data by averaging within the different groups of samples and summing up these averages over the total target. This estimation could account for about half of the amount of 129I and 36Cl predicted to be produced using nuclear physics modelling codes for both nuclides. The significance of the results and the associated uncertainties are discussed

Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli

An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur

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

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. The riboswitch regulation offers a simple method since only a sequence of ~85 nucleotides has to be inserted between a transcriptional start site and the start codon and no further auxiliary factors are necessary. The promoters galP2, ermEp1 and SF14 worked well in concert with the riboswitches. They allowed theophylline-dependent expression of the heterologous β-glucuronidase reporter gene but also of an endogenous gene for the agarase dagA. The successful combination of all tested promoters with the riboswitches underlines the orthogonality of riboswitch regulation. We anticipate that any further natural or synthetic promoter can be combined with the riboswitch

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

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

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

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 ~85nt 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 β-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

Identification of <i>metE</i> as a Second Target of the sRNA scr5239 in <i>Streptomyces coelicolor</i>

<div><p>While transcriptional regulation of the primary and secondary metabolism of the model organism <i>Streptomyces coelicolor</i> 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 <i>metE</i> (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 <i>dagA</i>, this interaction seems to be conserved in a wide number of streptomycetes.</p></div