Introduction

The Azotobacter vinelandii NifL-NifA two-component regulatory system integrates metabolic signals for redox, carbon and nitrogen status to fine tune regulation of nitrogenase synthesis (Dixon 1998). The NifL protein utilizes discrete mechanisms to perceive these signals leading to the formation of a protein-protein complex which inhibits NifA activity. The binding of adenosine nucleotides to NifL plays a key role in transducing environmental signals to form the inhibitory protein complex (Eydmann et al. 1995; Money et al. 1999). We have recently demonstrated that an additional ligand, 2-oxoglutarate, allosterically modulates the activity of the complex to antagonize the influence of adenosine nucleotides on NifL activity (Little et al. 2000). Redox signaling is mediated by the N-terminal FAD-containing PAS domain in NifL (Soderback et al. 1998) and the nitrogen status is sensed via interaction with the non-modified form of the PH-like signal transduction protein (Av GlnK) (Little et al. 2000) encoded by A. vinelandii glnK gene (Meletzus et al. 1998).

The mechanism of nitrogen sensing by the A. vinelandii system (Figure 3) is clearly different from that in K. pneumoniae since in the latter system, GlnK is required to prevent NifL from inhibiting NifA under nitrogen-limiting conditions (Jack et al. 1999; He et al. 1998), whereas in the former system both in vitro and in vivo experiments suggest that Av GlnK increases the inhibitory activity of NifL under conditions of nitrogen excess (Little et al. 2000; Reyes-Ramirez et al. 2001). Moreover, whereas the uridylylation status of GlnK has no apparent influence on nitrogen regulation of the K. pneumoniae NifL-NifA system, glnD mutants of A. vinelandii are unable to fix nitrogen (Contreras et al. 1991; Colnaghi et al. 2001) and uridylylation of Av GlnK abrogates its ability to enhance the inhibitory activity of NifL (Little et al. 2000). In order to further dissect the molecular mechanisms of signal transduction in this system, we have analyzed the interaction of the PII protein and 2-ketoglutarate with the individual component proteins and have isolated mutant forms of NifL and NifA which discriminate between redox and fixed nitrogen sensing, suggesting that these signals generate different inhibitory conformers.

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