RESUMEN
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 Δhmp 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hemoproteínas/metabolismo , Nitrato-Reductasa/metabolismo , Nitritos/metabolismo , Estrés Fisiológico/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Corynebacterium glutamicum/enzimología , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/fisiología , Hemoproteínas/química , Hemoproteínas/genética , Mutación , Nitrato-Reductasa/química , Nitrato-Reductasa/genética , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Fenotipo , Estrés Fisiológico/genéticaRESUMEN
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 delta pknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a delta 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 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.