Charakterisierung von Proteinen einer neuartigen Signaltransduktionskaskade in $\textit{Corynebacterium glutamicum}$

A reduced activity of the 2-oxoglutarate dehydrogenase complex (ODHc) is required for L-glutamate production with $\textit{Corynebacterium glutamicum}$. The unphosphorylated protein OdhI inhibits ODHc activity by binding to the OdhA subunit. Binding of OdhI to OdhA is abolished by phosphorylation of OdhI by the serine/threonine protein kinase PknG and other kinases. In corynebacteria and mycobacteria the $\textit{pknG}$ gene is located in a putative operon together with the genes $\textit{glnX}$ and $\textit{glnH}$, which were annotated to encode an integral membrane protein and a glutamine-binding lipoprotein, respectively. The object of the present study was the analysis of the functional association of the proteins GlnX, GlnH and PknG in $\textit{C. glutamicum}$. Genetic characterisation of the putative $\textit{glnX-glnH-pknG}$ operon revealed that the $\textit{glnX}$ gene is transcribed as a leaderless transcript. A common transcript could be detected only for $\textit{glnH}$ and $\textit{pknG}$ under the tested conditions. Biochemical investigations confirmed (i) GlnX to be an integral membrane protein with four transmembrane helices and the N- and C- terminus located in the cytoplasm, and (ii) GlnH to be a membrane-associated lipoprotein which is modified at cysteine-27 of its lipobox. Co-purification experiments of these proteins indicated a direct interaction of GlnX and GlnH. Although PknG is a soluble serine/threonine protein kinase, the protein could be detected in the soluble and membrane fraction of $\textit{C. glutamicum}$. The $\Delta \textit{glnX}$, $\Delta \textit{glnH}$ and $\Delta \textit{pknG}$ mutants showed a 2-fold reduced amount of phosphorylated OdhI protein in comparison to $\textit{C. glutamicum}$ wild type. In line with these findings, glutamate production by these mutants was considerably increased compared to the wild type, presumably as a consequence of stronger ODHc inhibition by unphosphorylated OdhI. The $\Delta \textit{glnX}$, $\Delta \textit{glnH}$ as well as $\Delta \textit{pknG}$ mutants were not able to use L-glutamine as sole carbon source. The analysis of $\Delta \textit{glnX}$ suppressor mutants arising after prolonged incubation with L-glutamine showed a preference for mutations in OdhI which negatively influence the stability or activity of this protein. The most prominent mutation OdhI-Arg87Pro still allowed interaction of the mutated OdhI protein with OdhA, but ODHc activity was inhibited much weaker than by wild type OdhI. These results and the fact that a deletion of the $\textit{odhI}$ gene in a $\Delta \textit{glnX}$ mutant abolished the observed phenotype on L-glutamine indicate a functional association of the proteins GlnX, GlnH and PknG, which form a novel type of signal transduction cascade in $\textit{C. glutamicum.}