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| Home > Publications database > Analyse des Mechanismus der Phosphatregulation in $\textit{Corynebacterium glutamicum}$ |
| Home > Publications database > Analyse des Mechanismus der Phosphatregulation in $\textit{Corynebacterium glutamicum}$ |
| Dissertation / PhD Thesis/Book | PreJuSER-55617 |
2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 3-89336-466-8
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Please use a persistent id in citations: http://hdl.handle.net/2128/2470
Abstract: In this work the regulation mechanism of the phosphor metabolism in $\textit{C. glutamicum}$ was characterized. It was based on the knowledge that the two-component regulatory system PhoRS is involved in this regulation, but it is not essential for the induction of the $\textit{pstSCAB}$ operon, which encodes a high-affinity uptake system for phosphate, upon phosphate starvation. Consequently, the goal was to characterize the PhoRS independent regulation of the $\textit{pstSCAB}$ operon. Initially, it was determined whether the phosphate starvation inducible genes are controlled by mRNA degradation. For the first time mRNA stability experiments were performed in $\textit{C. glutamicum}$. In terms of mRNA stability, the genes of the phosphate starvation stimulon did not differ from all other genes of the C. glutamicum genome and showed an average mRNA half life of 5 min. Thus, the regulation of the phosphate starvation inducible genes primarily occurs on the transcriptional level and not by mRNA degradation. A detailed analysis of the long-term effects of PhoRS on the regulation of phosphor metabolism genes revealed that PhoRS is neither essential for growth under phosphate starvation conditions nor for growth with 5’adenosine monophosphate, L-α-glycerinphosphate or UDP-glucose as alternative sources of phosphorus. However, the delayed induction of the genes of the phosphate starvation stimulon observed in the absence of PhoRS showed its importance for the fast adaption to phosphate starvation. The binding region of PhoR, which is located in a 35 bp long region, -217 to -182 bp upstream of the transcriptional start, could be determined employing deletion analysis of the $\textit{pstS}$ promoter. By DNA affinity chromatography and MALDI-MS analysis the transcriptional regulators of acetate metabolism RamB and GlxR as well as of the uncharacterized response regulator CgtR4 could be identified as proteins binding to the $\textit{pstS}$ promotor. RamB and CgtR4 interacted with the pstS promoter after cultivation on glucose medium under phosphate excess as well as on acetate medium under phosphate limiting conditions. However, binding of GlxR could only be detected after cultivation on acetate medium upon phosphate starvation. In electro mobility shift assays purified GlxR bound to the $\textit{pstS}$ promoter $\textit{in vitro}$ with high affinity. Binding of GlxR to the pstS promoter was only detected in the presence of cAMP. The binding region of GlxR is located in a region of the $\textit{pstS}$ promoter ranging from bp –129 to bp +126 related to the transcriptional start site. Based on the known RamB binding motif, 5’-AA/GAACTTTGCAAA-3’, two incompletely conserved RamB binding motifs could be identified in the $\textit{pstS}$ promoter. In electro mobility shift assays with different fragments of the $\textit{pstS}$ promoter and with fragments carrying mutated binding motifs it could be shown that both binding motifs are necessary for the interaction of RamB with the $\textit{pstS}$ promoter. Transcriptional fusion analyses revealed that the regulator RamB activates the $\textit{pstS}$ promoter after a shift from phosphate-containing to phosphate-free glucose medium $\textit{in vivo}$. However, RamB represses the $\textit{pstS}$ promoter after a shift from phosphate-containing to phosphate-free acetate medium. In addition, transcriptional fusion analyses and transcriptome analyses revealed that the induction level of the $\textit{pstSCAB}$ operon upon phosphate starvation depends on the carbon source. The induction of the $\textit{pstSCAB}$ operon after a shift from phosphate-containing to phosphate-free medium was higher with glucose as carbon source than with acetate as carbon source. These results and the observations that the genes of the two component system PhoRS showed a higher expression on phosphate-free glucose medium than on phosphate-free acetate medium and that a potential RamA binding site could be identified upstream of the $\textit{phoRS}$ operon by bioinformatical analysis revel link between phosphate- and carbon-dependent regulation of genes of the phosphorus metabolism. Thus, this work for the first time shows that the regulation of carbon and phosphor metabolism is connected by the carbon regulators RamA, RamB and GlxR.
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