ADP‐ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB‐dependent membrane sequestration of DraG

Abstract

SummaryNitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADPribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P II proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADPribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P II family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

“…The model is supported by copurification experiments and gel filtration with purified components that demonstrate the protein-protein interactions and in vitro nitrogenase assays with purified components. The mechanism is relatively simple and contrasts with the more complex regulation found in some Proteobacteria involving ADP-ribosylation of nitrogenase reductase by DraT and DraG (3), binding and membrane sequestration of DraG to the ammonia transporter AmtB by PII proteins, and covalent modification of the PII proteins by the glutamine sensor GlnD (12). The wide phylogenetic distribution of nifI 1 and nifI 2 genes, and their almost exclusive linkage to nifHDK genes, suggests that this mode of regulation may be conserved in a variety of diazotrophic Archaea and Bacteria (30).…”

“…8 and 9), including ADP-ribosylation of NifH (10)(11)(12). The best studied examples, GlnB and GlnK of Escherichia coli and other Proteobacteria, are homotrimers (13).…”

The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel

“…We therefore have suggested that AmtB activity might be regulated by GlnK (16). Indeed, in vivo, GlnK binds rapidly and reversibly to AmtB in response to an extracellular ammonium shock (Ն50 M extracellular ammonium) (17), and this interaction has also been shown in a number of other eubacterial species (16,(18)(19)(20). Methylammonium uptake by AmtB is also inhibited by a GlnK variant that forms an irreversible complex, supporting the concept that GlnK regulates ammonium flux through AmtB (17).…”

“…The structure emphasizes that the primary function of GlnK is almost certainly regulation of ammonium flux into the cell, and the almost ubiquitous linkage of the glnK and amtB genes suggests that regulation of the ammonia channel may be the ancestral role of P II proteins (38). However, in some systems, complex formation might also serve other functions such as sequestration of cytoplasmic proteins involved in regulation of nitrogen metabolism as suggested by recent studies in Azospirillum brasilense (19) and Bacillus subtilis (39).…”

“…The occurrence of the AmtB-GlnK pair is highly conserved in both eubacteria and archaea, and the interaction of these proteins has already been demonstrated in a number of cases (16,(18)(19)(20). Hence, we might expect that the mode of binding is also conserved.…”