000911589 001__ 911589 000911589 005__ 20240206204910.0 000911589 0247_ $$2doi$$a10.1128/spectrum.02677-22 000911589 0247_ $$2Handle$$a2128/33335 000911589 0247_ $$2pmid$$a36445153 000911589 0247_ $$2WOS$$aWOS:000891967400001 000911589 037__ $$aFZJ-2022-04848 000911589 041__ $$aEnglish 000911589 082__ $$a570 000911589 1001_ $$0P:(DE-Juel1)178698$$aSundermeyer, Lea$$b0$$eCorresponding author 000911589 245__ $$aCharacteristics of the GlnH and GlnX Signal Transduction Proteins Controlling PknG-Mediated Phosphorylation of OdhI and 2-Oxoglutarate Dehydrogenase Activity in Corynebacterium glutamicum 000911589 260__ $$aBirmingham, Ala.$$bASM$$c2022 000911589 3367_ $$2DRIVER$$aarticle 000911589 3367_ $$2DataCite$$aOutput Types/Journal article 000911589 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article$$bjournal$$mjournal$$s1707228312_14558 000911589 3367_ $$2BibTeX$$aARTICLE 000911589 3367_ $$2ORCID$$aJOURNAL_ARTICLE 000911589 3367_ $$00$$2EndNote$$aJournal Article 000911589 520__ $$aIn Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment.IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process. 000911589 536__ $$0G:(DE-HGF)POF4-5244$$a5244 - Information Processing in Neuronal Networks (POF4-524)$$cPOF4-524$$fPOF IV$$x0 000911589 536__ $$0G:(DE-HGF)POF4-2171$$a2171 - Biological and environmental resources for sustainable use (POF4-217)$$cPOF4-217$$fPOF IV$$x1 000911589 588__ $$aDataset connected to CrossRef, Journals: juser.fz-juelich.de 000911589 7001_ $$0P:(DE-HGF)0$$aBosco, Graziella$$b1 000911589 7001_ $$0P:(DE-Juel1)189001$$aGujar, Srushti$$b2 000911589 7001_ $$0P:(DE-Juel1)128946$$aBrocker, Melanie$$b3 000911589 7001_ $$0P:(DE-Juel1)145489$$aBaumgart, Meike$$b4 000911589 7001_ $$0P:(DE-Juel1)132029$$aWillbold, Dieter$$b5$$eCorresponding author 000911589 7001_ $$0P:(DE-Juel1)131988$$aWeiergräber, Oliver H.$$b6 000911589 7001_ $$00000-0002-8887-6917$$aBellinzoni, Marco$$b7 000911589 7001_ $$0P:(DE-Juel1)128943$$aBott, Michael$$b8 000911589 773__ $$0PERI:(DE-600)2807133-5$$a10.1128/spectrum.02677-22$$gp. e02677-22$$n6$$pe02677-22$$tMicrobiology spectrum$$v10$$x2165-0497$$y2022 000911589 8564_ $$uhttps://juser.fz-juelich.de/record/911589/files/Invoice_94317934%20.pdf 000911589 8564_ $$uhttps://juser.fz-juelich.de/record/911589/files/spectrum.02677-22-1.pdf$$yOpenAccess 000911589 8767_ $$894317934$$92022-11-21$$a1200187019$$d2022-12-13$$eAPC$$jZahlung erfolgt$$zUSD 2030,- 000911589 909CO $$ooai:juser.fz-juelich.de:911589$$pdriver$$pOpenAPC$$popen_access$$pdnbdelivery$$popenCost$$pVDB$$popenaire 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)178698$$aForschungszentrum Jülich$$b0$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-HGF)0$$aForschungszentrum Jülich$$b1$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)189001$$aForschungszentrum Jülich$$b2$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)128946$$aForschungszentrum Jülich$$b3$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)145489$$aForschungszentrum Jülich$$b4$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)132029$$aForschungszentrum Jülich$$b5$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)131988$$aForschungszentrum Jülich$$b6$$kFZJ 000911589 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)128943$$aForschungszentrum Jülich$$b8$$kFZJ 000911589 9131_ $$0G:(DE-HGF)POF4-524$$1G:(DE-HGF)POF4-520$$2G:(DE-HGF)POF4-500$$3G:(DE-HGF)POF4$$4G:(DE-HGF)POF$$9G:(DE-HGF)POF4-5244$$aDE-HGF$$bKey Technologies$$lNatural, Artificial and Cognitive Information Processing$$vMolecular and Cellular Information Processing$$x0 000911589 9131_ $$0G:(DE-HGF)POF4-217$$1G:(DE-HGF)POF4-210$$2G:(DE-HGF)POF4-200$$3G:(DE-HGF)POF4$$4G:(DE-HGF)POF$$9G:(DE-HGF)POF4-2171$$aDE-HGF$$bForschungsbereich Erde und Umwelt$$lErde im Wandel – Unsere Zukunft nachhaltig gestalten$$vFür eine nachhaltige Bio-Ökonomie – von Ressourcen zu Produkten$$x1 000911589 9141_ $$y2022 000911589 915pc $$0PC:(DE-HGF)0001$$2APC$$aLocal Funding 000911589 915__ $$0StatID:(DE-HGF)0200$$2StatID$$aDBCoverage$$bSCOPUS$$d2022-11-11 000911589 915__ $$0StatID:(DE-HGF)0300$$2StatID$$aDBCoverage$$bMedline$$d2022-11-11 000911589 915__ $$0StatID:(DE-HGF)1050$$2StatID$$aDBCoverage$$bBIOSIS Previews$$d2022-11-11 000911589 915__ $$0LIC:(DE-HGF)CCBY4$$2HGFVOC$$aCreative Commons Attribution CC BY 4.0 000911589 915__ $$0StatID:(DE-HGF)0100$$2StatID$$aJCR$$bMICROBIOL SPECTR : 2021$$d2022-11-11 000911589 915__ $$0StatID:(DE-HGF)0030$$2StatID$$aPeer Review$$bDOAJ : Peer review, Blind peer review$$d2022-02-02T17:55:23Z 000911589 915__ $$0StatID:(DE-HGF)0501$$2StatID$$aDBCoverage$$bDOAJ Seal$$d2022-02-02T17:55:23Z 000911589 915__ $$0StatID:(DE-HGF)1030$$2StatID$$aDBCoverage$$bCurrent Contents - 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