Actinobacterial metabolism: Exploring connections between the hybrid PDH-ODH complex, the signalling cascade GlnH-GlnX-PknG-OdhI and the glutamate exporter MscCG

• Applicants: Professor Dr. Michael Bott; Professor Dr. Marc Bramkamp
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545494849

Actinomycetes comprise a large and diverse group of bacteria including pathogens such as Mycobacterium tuberculosis, antibiotics producers such as streptomycetes, and industrial cell factories such as Corynebacterium glutamicum. Given their huge relevance in many economically and societally relevant sectors, a detailed molecular understanding of the physiology of Actinomycetes is important, both in the context of therapeutic applications and for biotechnological purposes. The GLUTACTINO proposal is based on the results of our previous DFG-ANR project METACTINO and focusses on three closely related exciting topics at the 2-oxoglutarate/glutamate hub of central metabolism. In our previous work, we identified a hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH) in C. glutamicum, with unusual structural and localisation properties. Fluorescence microscopic analyses showed that the PDH-ODH complex forms a cluster at each cell pole and, in larger cells, one or two additional clusters at the division site. In work package 1, the number and single molecule dynamics of the four subunits OdhA, AceE, AceF, and Lpd in these clusters will be quantitatively determined using high-resolution microscopy, as well as the dynamics of cluster formation. Furthermore, the molecular cause of cluster formation will be analysed. The second work package comprises the analysis of the signalling cascade identified by us, which regulates the ODH activity and thus the carbon flux between the citrate cycle and ammonium assimilation by glutamate dehydrogenase. The cascade comprises the periplasmic glutamate/aspartate binding protein GlnH, the membrane-integral transducer GlnX, the serine/threonine protein kinase PknG and the ODH inhibitor protein OdhI. The forkhead-associated (FHA) domain of OdhI binds with high affinity to the OdhA subunit of the PDH-ODH complex and thereby inhibits ODH activity. Phosphorylation of OdhI by PknG, that takes place when PknG is activated by GlnH and GlnX in the presence of glutamate or aspartate in the periplasm, hinders OdhI binding to OdhA. In work package 2, this signalling cascade will be quantified by single-molecule localisation microscopy techniques with regard to the number of protein copies and the stoichiometry of the components, and the subcellular localisation, the postulated colocalisation and the diffusion coefficients will be determined. Moreover, we will search for and explore additional OdhI interaction partners beyond OdhA. Work package 3 focusses on the glutamate exporter MscCG, as our preliminary data indicate that the MscCG function is linked to the GlnH-GlnX-PknG-OdhI signalling cascade. The role of MscCG in signal transduction and the necessity of both, OdhI and MscCG, for glutamate secretion will be analysed. Possible interaction partners as well as post-translational modifications of MscCG will be searched for and the cellular localisation of MscCG and its structure will be determined.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Privatdozent Marco Bellinzoni, Ph.D.