Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr→Phe mutations on NifA and its interaction with GlnB

Abstract

Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.

Interaction between GlnB and the N-terminal domain of NifA in Azospirillum brasilense

“…This is consistent with the previous results determined in A. brasilense that the double mutant protein NifA-Y18/53F displayed a higher activity than the wild type protein [12] . Surprisingly, the most strengthened GlnB signal is in the elution fraction from cells expressing His6-NifA-N-Y18F.…”

“…Replacement of the Tyr residues at positions 18, 43 and 53 in A. brasilense NifA N-terminal domain resulted in NifA proteins that retained high nitrogenase activity in both nifA and glnB mutant backgrounds [12] . However, whether GlnB regulates NifA activity by directly interaction with N-terminal of NifA (abbreviated to NifA-N) or by covalent modification of NifA is still unknown.…”

Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501

“…It has been successfully used to explore binding between several components of the ntr and nif regulatory systems (Lei et al, 1999;Martínez-Argudo et al, 2002;Rudnick et al, 2002;Pawlowski et al, 2003;Chen et al, 2005). Thus, a direct protein-protein binding was detected between NifA and NifL in K. pneumoniae, Azotobacter vinelandii and Enterobacter cloacae (Lei et al, 1999;Martínez-Argudo et al, 2002;Liao et al, 2002).…”

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

“…In some organisms, for example, A. brasilense, Herbaspirillum seropedicae, and Rhodospirillum rubrum, the uridylylated form of the P II protein is apparently required to prevent intramolecular repression of NifA activity by the GAF domain under nitrogen-limiting conditions (71)(72)(73). In contrast, in Rhodobacter capsulatus and Azorhizobium caulinodans, P II proteins are required to inactivate NifA under conditions of nitrogen excess (74,75).…”