A novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the ⌬pknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ⌬odhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a K i value of 2.4 nM. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle.Increasing numbers of eukaryotic-like serine/threonine protein kinases found in bacteria implicate that they play important roles in cell signaling, but their targets and specific functions are largely unknown (1). The genome of the important human pathogen Mycobacterium tuberculosis encodes 11 members of this protein family (2). Among these, protein kinase G (PknG) 2 gained particular interest because it was reported to inhibit phagosome-lysosome fusion, thus allowing for intracellular survival of mycobacteria. Deletion of the pknG gene in Mycobacterium bovis BCG resulted in lysosomal localization and mycobacterial cell death in infected macrophages. PknG was detected in the cytosol of infected macrophages and was therefore suggested to interfere with host cell signaling pathways (3). A pknG deletion mutant of M. tuberculosis displayed decreased viability upon infection of immunocompetent mice but also reduced growth in vitro (4), implying that PknG function is not restricted to the pathogenic life style. This is supported by the fact that genes encoding PknG homologs are not only present in pathogenic mycobacteria but also in all other members of the suborder Corynebacterineae with known genome sequence, i.e. species of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus, as well as in Streptomyces species. To determine the function of PknG, we chose Coryneb...

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.

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We recently showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC) in Corynebacterium glutamicum is controlled by a novel regulatory mechanism that involves a 15-kDa protein called OdhI and serine/threonine protein kinase G (PknG). In its unphosphorylated state, OdhI binds to the E1 subunit (OdhA) of ODHC and, thereby, inhibits its activity. Inhibition is relieved by phosphorylation of OdhI at threonine-14 by PknG under conditions requiring high ODHC activity. In this work, evidence is provided that the dephosphorylation of phosphorylated OdhI is catalyzed by a phospho-Ser/Thr protein phosphatase encoded by the gene cg0062, designated ppp. As a decreased ODHC activity is important for glutamate synthesis, we investigated the role of OdhI and PknG for glutamate production under biotin limitation and after addition of Tween-40, penicillin, or ethambutol. A DeltaodhI mutant formed only 1-13% of the glutamate synthesized by the wild type. Thus, OdhI is essential for efficient glutamate production. The effect of a pknG deletion on glutamate synthesis was dependent on the induction conditions. Under strong biotin limitation and in the presence of ethambutol, the DeltapknG mutant showed significantly increased glutamate production, offering a new way to improve production strains.

We previously showed that the 2-oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (ΔpknA, ΔpknB, ΔpknL), five double (ΔpknAG, ΔpknAL, ΔpknBG, ΔpknBL, ΔpknLG) and two triple deletion mutants (ΔpknALG, ΔpknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C. glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho-Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division.