Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein

Abstract

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

“…Recall that nifL-R306C is competent to inhibit NifA under all conditions in the absence of the PAS domain in vivo (Table 1). In the presence of ADP, NifL (147-519) inhibits NifA, but this inhibition is relieved by the binding of 2-oxoglutarate to the GAF domain of NifA (19,29,30). As anticipated, N his6 NifL (147-519) and N his6 NifL-R306C (147-519) were both effective in inhibiting open-promoter complex formation by NifA in the presence of ADP (Fig.…”

“…The GlnK-NifL-NifA ternary complex is formed under nitrogen-excess conditions when GlnK is primarily in the nonuridylylated form. Uridylylation of GlnK under nitrogen-limiting conditions prevents this interaction (17)(18)(19). We infer that the R306C substitution locks NifL in a conformation that is analogous to that shown in C and D, so that it is competent to inhibit NifA irrespective of other signals.…”

“…Thus, although signal transmission by NifL does not involve phosphoryl transfer reactions, the interdomain movements that are necessary to modulate the inhibitory function of NifL may be similar to those required to control the catalytic function of the HPKs. In the absence of 2-oxoglutarate, both the reduced and oxidized forms of NifL inhibit the activity of NifA, provided that adenosine nucleotide is bound to NifL (8,15,19). (B) Binding of 2-oxoglutarate to the GAF domain of NifA (indicated by stars) induces a conformational change that releases inhibition by the reduced form of NifL, enabling NifA to activate transcription (29,30).…”

“…Unlike the HPKs, the GHKL domain of NifL does not exhibit ATP hydrolysis or transphosphorylation activity, but the binding of ADP to this domain strongly stimulates the inhibitory activity of NifL and the stability of the NifL-NifA complex (8,15,16). Also, the GHKL domain is involved in sensing the nitrogen status because it is the site of interaction with the signal transduction protein GlnK (17)(18)(19), the PII-like protein of A. vinelandii (20)(21)(22)(23).…”

Direct Interaction of the NifL Regulatory Protein with the GlnK Signal Transducer Enables the Azotobacter vinelandiiNifL-NifA Regulatory System to Respond to Conditions Replete for Nitrogen

“…NifL is a flavoprotein that senses redox status via an N-terminal FAD-containing PAS domain (6 -8). The mechanism whereby the NifL-NifA system perceives the nitrogen status is less well understood although recent evidence implicates direct interaction with PII-like signal transduction proteins (9).…”