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

Abstract

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.

Keywords: DasR; PEPCK; Streptomyces; carbon metabolism; phosphoenolpyruvate; sRNA.

FIGURE 1

Quantitative proteomics analysis of three different S. coelicolor strains. (A) LS-MS/MS workflow. S. coelicolor Δscr5239, M145, and scr5239+ were grown on rich media and harvested in the transition phase. The samples of the three strains were labeled with different TMT labels. The peptides of the three strains where pooled and the fragments where analyzed in tandem MS. As a result, a fragment of the TMT label is cleaved of each peptide. The mass of the cleaved part of the label differs with each label type. In consequence, we were able to quantify the relative amount of each peptide fragment in the input strains. The signal strength of the cleaved-off part of the labels was then used for input normalization. Thus, this method allows relative quantification of proteins. (B) Results of the quantitative proteomics and subsequent functional analysis. Bars indicate the x-fold change in protein expression in the Δscr5239 and scr5239+ strain compared to the wild type. Only hits with a minimum change of 50% in expression are shown (Δ vs. wt or wt vs. +). Colored boxes show categorization of proteins depending on their functional characteristics. Numbers on the x-axis refer to the SCO numbers of the expressed genes. The SCO number of PEPCK is highlighted in red.

FIGURE 2

Expression analysis of PEPCK in different strains. The strains were grown on rich media and harvested in the transition phase. (A) Western blot analysis using FE01, FE02, and FE03 to detect PEPCK-F3 under control of its endogenous promoter. (B) Western blot analysis using FE04, FE05, and FE06 to detect PEPCK-F3 under control of the synthetic promoter SP4. (C) RT-qPCR results. mRNA levels of pepck were detected in S. coelicolor M145, Δscr5239, and scr5239+ and normalized to the levels in M145. Measurements were normalized to the respective strain expressing wild type scr5239; error bars represent the standard deviation calculated from three independent experiments.

FIGURE 3

PEP levels in M145, Δscr5239, and scr5239+. (A) PEP levels were determined in S. coelicolor M145, Δscr5239, and scr5239+ and normalized to the levels in M145; error bars represent the standard deviation calculated from three independent experiments. (B) Schematic overview of the connection between scr5239, PEPCK, and PEP levels in S. coelicolor.

FIGURE 4

Analysis of the interaction of scr5239 and PEPCK mRNA. (A) Predicted interaction site. The sequence of the sRNA is shown completely, that of the pepck mRNA only partially. The RBS (red) and the start codon (blue) of pepck are highlighted. The interaction site of dagA and metE is highlighted in red on the sRNA. (B) PEPCK was mutated at the predicted interaction site (PEPCmut-F3) and integrated in the genome of the M145, Δscr5239, and scr5239+. Mutated bases in the sequence are underlined. Western blot analysis using FE07, FE08, and FE09 and thus detecting PEPCmut-F3. (C) scr5239 was mutated at the predicted interaction site. Mutated bases in the sequence are underlined. The mutated and the wild type scr5239 in combination with PEPC-F3 were integrated into Δscr5239. Western blot analysis using FE10 and FE11 and thus detecting with PEPC-F3. Measurements were normalized to the respective strain expressing wild type scr5239; errors represent the standard deviation calculated from three independent experiments.

FIGURE 5

Regulation of scr5239 by DasR. (A) Northern blot of scr5239 with RNA harvested from strains with different DasR expression levels. (B) Reporter gene measurements of different variants of the scr5239 promoter fused to a gusA gene. (A) Scheme of the used constructs. The dre site of scr5239 is indicated. The sequences of the wild type and the mutated dre site are shown. The mutated bases are highlighted in red. (B) Results of the reporter gene measurements for FE13, FE14, and FE15. dre: DasR responsive element, wt: scr5239 Promoter with wild type dre site, M1: scr5239 Promoter with mutated dre site, M2: First 50 bp of scr5239 Promoter without dre site. Measurements were normalized to FE13; error bars represent the standard deviation calculated from three independent experiments.

FIGURE 6

Model of the scr5239 feedback loop. scr5239 is regulated by the global transcriptional regulator DasR that also regulates the antibiotic production and the carbon catabolite repression. scr5239 inhibits the expression of PEPCK and thus influences the PEP levels. Higher PEP levels lead to an enhanced GlcNAc uptake, which as a result inhibits DasR.