Regulation der Phosphatmangelgene von $\textit{Corynebacterium glutamicum}$ durch das Zweikomponenten-System PhoRS

Schaaf, Sarah

Dissertation / PhD Thesis/Book

Regulation der Phosphatmangelgene von $\textit{Corynebacterium glutamicum}$ durch das Zweikomponenten-System PhoRS

Schaaf, S. (Corresponding author)FZJ*

2007
Forschungszentrum Jülich Gmbh Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-496-1

Jülich : Forschungszentrum Jülich Gmbh Zentralbibliothek, Verlag, Schriften des Forschungszentrums Jülich. Reihe Lebenswissenschaften / Life Sciences 36, VI, 114 S. (2007) = Universität Düsseldorf, Diss., 2007

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Please use a persistent id in citations: http://hdl.handle.net/2128/2626

Abstract: The Gram-positive soil bacterium $\textit{Corynebacterium glutamicum}$ possesses 13 two-component signal transduction systems. Among these the membrane-bound sensor kinase PhoS and the response regulator PhoR are involved in the phosphate (P$_{i}$) starvation response. To analyse the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible ($\textit{psi}$) genes, this response regulator and the kinase domain of its cognate sensor PhoS were overproduced and purified. The kinase domain showed constitutive autophosphorylation activity and a rapid phosphoryl group transfer from phosphorylated kinase domain to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR about 5-fold. The affinity of PhoR~P to different promoters varied and decreased in the order $\textit{pstSCAB > phoRS > phoC > ushA > porB > ugpA > pitA > nucH, phoH1 > glpQ1}$. The binding sites in front of $\textit{pstSCAB}$ and $\textit{phoRS}$ were localized at positions -194 to -176 and -61 to -43 upstream of the transcriptional start sites, respectively. Using mutational analysis these two 19-bp binding sites were analysed in detail and an alignment revealed a high identity in the 5’-terminal part, but not the 3’-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by three bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which represent putative PhoR binding sites. The $\textit{in vivo}$ relevance of the PhoR-binding site within the $\textit{phoRS}$ promoter was supported by reporter gene studies. $\textit{C. glutamicum}$ is used industrially for the production of more than two million tons of amino acids per year, mainly L-glutamate and L-lysine. In the second part of this work the influence of phosphate limitation and the overproduction of the PhoRS-system on the lysine production of DM1730 were analysed. Interestingly, cultivation under phosphate limitation resulted in an increased productivity of DM1730/pEKEx2 of about 12,5 % to 0,045 ± 0,001 g lysine·l-1·h-1 compared to cultivation under phosphate excess. Overproduction of PhoRS under phosphate limitation even led to an increased productivity of about 60 % in comparison to DM1730/pEKEx2 cultivated under phosphate excess.

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