Molybdenum transport and Mo-dependent gene regulation in bacteria

• Applicant: Dr. Bernd Masepohl
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; General Genetics and Functional Genome Biology
• Term: from 2012 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 211163627

Biological nitrogen fixation, the reduction of chemically inert dinitrogen from air to bioavailable ammonia, is catalyzed by nitrogenases. Our model organism, the photosynthetic purple bacterium Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent and a Mo-free less efficient iron-only nitrogenase, and a high-affinity Mo transporter sustaining Mo-nitrogenase activity under Mo-limiting conditions. Rhodobacter possesses two structurally and functionally similar NifA copies, each of which is sufficient to activate Mo-nitrogenase genes, whereas AnfA activates transcription of Fe-nitrogenase genes. This project focusses on FdxD- and Mo-mediated regulatory mechanisms, which control the ratio of NifA and AnfA, and hence, balance synthesis of Mo- and Fe-nitrogenases.The first part of the proposal deals with the control of NifA and AnfA by FdxD, a ferredoxin, which protects Mo-nitrogenase against oxygen damage. FdxD stimulates accumulation of NifA1 and NifA2, and represses accumulation of AnfA. Hence, FdxD acts as a global regulator that controls the ratio of NifA and AnfA, and consequently, coordinates expression of Mo- and Fe-nitrogenases. We want to unravel FdxD-mediated regulation by dissecting transcription of nitrogen fixation (nif) genes activated by NifA, AnfA, or both. In addition, we plan to investigate the involvement of proposed oxygen-sensing domains in NifA (IDL) and AnfA (GAF) regarding FdxD control.The second part of the proposal deals with the Mo-responsive control of the ratio of NifA and AnfA. On the one hand, Mo represses anfA transcription. On the other hand, AnfA represses accumulation of NifA1 and NifA2. This indicates that the levels of NifA and AnfA are adapted to each other in response to the cellular Mo status. To dissect these Mo effects, we want to compare nif gene transcription in appropriate nifA and anfA deletion backgrounds at different Mo concentrations. In addition, we want to determine the role of GAF domains in NifA and AnfA regarding NifA repression by AnfA.The third part of the proposal deals with the mechanism underlying Mo control of IscN levels. IscN supports Fe-nitrogenase activity possibly by providing iron-sulfur clusters. IscN is the only Mo-repressed product of the iscN-nifU1-nifSVW operon, and its accumulation is likely controlled at the mRNA level. To verify this assumption, we want to identify cis-regulatory elements by exchange of the 5´ untranslated region and the introduction of neutral mutations into the coding region of the iscN gene.

• DFG Programme: Research Grants