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The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δndh) and/or one of the alternative NADH-oxidizing enzymes, L-lactate dehydrogenase LdhA (ΔldhA) and malate dehydrogenase Mdh (Δmdh). Together with the menaquinone-dependent L-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δndh mutant, but not the ΔldhA and Δmdh strains, showed reduced growth and a lowered NAD+/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants ΔndhΔmdh and ΔndhΔldhA, but not of strain ΔmdhΔldhA, in glucose medium was stronger impaired than that of the Δndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L-lactate minimal medium the Δndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L-lactate dehydrogenase LldD. The ΔndhΔmdh mutant failed to grow in L-lactate medium and acetate medium. Growth with L-lactate could be restored by additional deletion of sugR, suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L-lactate medium. Attempts to construct a ΔndhΔmdhΔldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum.

The supply and usage of energetic cofactors in metabolism is a central concern for systems metabolic engineering, particularly in case of energy intensive products. One of the most important parameters for systems wide balancing of energetic cofactors is the ATP requirement for biomass formation YATP/Biomass. Despite its fundamental importance, YATP/Biomass values for non-fermentative organisms are still rough estimates deduced from theoretical considerations. For the first time, we present an approach for the experimental determination of YATP/Biomass using comparative 13C metabolic flux analysis (13C MFA) of a wild type strain and an ATP synthase knockout mutant. We show that the energetic profile of a cell can then be deduced from a genome wide stoichiometric model and experimental maintenance data. Particularly, the contributions of substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP) to ATP generation become available which enables the overall energetic efficiency of a cell to be characterized. As a model organism, the industrial platform organism Corynebacterium glutamicum is used. C. glutamicum uses a respiratory type of energy metabolism, implying that ATP can be synthesized either by SLP or by ETP with the membrane-bound F1FO-ATP synthase using the proton motive force (pmf) as driving force. The presence of two terminal oxidases, which differ in their proton translocation efficiency by a factor of three, further complicates energy balancing for this organism. By integration of experimental data and network models, we show that in the wild type SLP and ETP contribute equally to ATP generation. Thus, the role of ETP in respiring bacteria may have been overrated in the past. Remarkably, in the genome wide setting 65% of the pmf is actually not used for ATP synthesis. However, it turns out that, compared to other organisms C. glutamicum still uses its energy budget rather efficiently.

In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ΔcyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ΔcyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1FO-ATP synthase and numerous transport processes. The impaired membrane potential of the ΔcyaB mutant could be due to a decreased expression of the cytochrome bc1-aa3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ΔcyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ΔcyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ΔcyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.

Corynebacterium glutamicum , a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l -lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD 600 ) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO 2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.

The influence of nitrate and nitrite on growth of Corynebacterium glutamicum under aerobic conditions in shake flasks was analysed. When dissolved oxygen became limiting at higher cell densities, nitrate was reduced almost stoichiometrically to nitrite by nitrate reductase (NarGHJI). The nitrite concentration also declined slowly, presumably as a result of several reactions including reduction to nitric oxide by a side-activity of nitrate reductase. The flavohaemoglobin gene hmp was most strongly upregulated (19-fold) in the presence of nitrite. Hmp is known to catalyse the oxygen-dependent oxidation of nitric oxide to nitrate and, in the absence of oxygen, with a much lower rate the reduction of nitric oxide to nitrous oxide. A Δ hmp mutant showed strong growth defects under aerobic conditions in the presence of nitrate, nitrite and the NO-donating reagent sodium nitroprusside, but also under anaerobic nitrate-respiring conditions. Therefore, Hmp is likely to be responsible for nitric oxide conversion to either nitrate or nitrous oxide in C. glutamicum . The results suggest that a cyclic nitrate–nitrite conversion takes place in C. glutamicum under microaerobic conditions.

In this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc1–aa3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1–aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc1–aa3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain–heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons.