Streptomyces coelicolor is a model organism for the GC-rich, Gram-positive actinobacteria. The strain is one of the best studied representatives of the genus Streptomyces. S. coelicolor has a very complex life cycle and, as a soil bacterium, must constantly adapt to changing environmental conditions. This is reflected, among other things, in the fact that about 1000 of the approximately 8000 annotated genes
have been attributed a regulatory function.
Among these are 64 sigma factors. These include the master regulator PhoP, which controls phosphate uptake, GlnR, which coordinates nitrogen metabolism, and DasR, which is considered as the central regulator of carbon metabolism and
combines the metabolism of the carbon source chitin, which occurs mainly in the natural environment, with various processes in the cell.
In contrast to transcriptional regulation, regulation at the post-transcriptional level in streptomyces has been studied little to date. The Rfam database lists a prediction with about 20 known cis-regulatory elements. In addition, as in many other organisms, more and more regulatory RNAs are being dentified
in streptomycetes, but their exact function often remains unknown.
These include small non-coding RNAs (sRNAs), which play an important role in posttranscriptional regulation. They are typically 50-200 nt long, structurally very diverse and show a large variance in their mode of action and target genes. As regulators of translation, they bind in many cases complementarily to the ribosome binding site of their target mRNAs, mask it and thus inhibit the initiation of translation.
The best studied sRNA in Streptomyces is scr5239 in S. coelicolor. It is encoded upstream of ORF5239, 159 nt long and highly conserved in about 2/3 of all known streptomycete genomes, both structurally and in large parts of its sequence.
At the beginning of this work two targets for scr5239 were already characterized: the genes for the agarase DagA and the methionine synthase MetE. Based on a proteome analysis, three additional targets for scr5239 were identified in this work. The genes coding for the phosphoenolpyruvate carboxykinase PEPCK, the malate oxidoreductase MOR and the acetyl-CoA acetyltransferase ThiL. Western blot and RT-qPCR analyses showed that the sRNA represses the expression of these targets at the post-transcriptional level. PEPCK, MOR and ThiL are closely associated with the central metabolism. PEPCK is a key enzyme of the primary metabolism that links glycolysis to the tricarboxylic acid cycle by catalyzing the conversion of
oxaloacetate to phosphoenolpyruvate (PEP). MOR is the enzyme that catalyzes the reaction from malate to pyruvate. MOR is essential in several parts of the metabolism, especially in the tricarboxylic acid cycle. ThiL catalyzes the acetylation of acetyl coenzyme A and its reverse reaction. These reactions
are necessary for the formation and degradation of ketone bodies and are therefore also closely linked to the tricarboxylic acid cycle.
Using proteinbiochemical methods it could be shown that scr5239 also influences intracellular PEP levels by regulating pepck. PEP plays a crucial role in glycolysis and glyconeogenesis and is therefore a key intermediate in carbon metabolism. The phosphotransferase system (PTS) of S. coelicolor also uses PEP as an energy source for the uptake and phosphorylation of N-acetylglucosamine, the monomer of
chitin. Since the expression of the scr5239-gene is dependent on DasR, the central regulator of the carbon metabolism, which in turn regulates the genes of PTS, a feedback regulation in the central metabolism can be described for scr5239 and DasR.
Taking the catalytic functions of the newly identified targets and the feeback regulation of DasR into account, this work showed that scr5239 plays a central role in the metabolism of S. coelicolor. | English |