Axel Niebisch

Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein.

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 ΔpknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ΔodhI 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 Ki 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.

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.

Purification of a Cytochrome bc1 - aa3 Supercomplex with Quinol Oxidase Activity from Corynebacterium glutamicum: IDENTIFICATION OF A FOURTH SUBUNIT OF CYTOCHROME aa3 OXIDASE AND MUTATIONAL ANALYSIS OF DIHEME CYTOCHROME c1

The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves abc 1 complex with a diheme cytochromec 1 and a cytochrome aa 3oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochromeaa 3 always resulted in the copurification of the subunits of the bc 1 complex (QcrA, QcrB, QcrC) and the aa 3 complex (CtaD, CtaC, CtaE). The isolated bc 1 -aa 3supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c 1 and the CuA center of cytochrome aa 3. Besides the known bc 1 andaa 3 subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa 3. If either of the two CXXCH motifs for covalent heme attachment in cytochromec 1 was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectablec-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c 1 is not only required for the activity but also for the assembly and/or stability of the bc 1 complex.

Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG

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 ΔodhI 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 ΔpknG mutant showed significantly increased glutamate production, offering a new way to improve production strains.

Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases

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.

Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production

ABSTRACT Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1-aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by ∼45% and of the maximal biomass by ∼35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the l-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by ∼12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum.