Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum

Abstract

The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.

Fig. 1

Immunoblot of the dinitrogenase reductase in UR2 (wild-type), UR2501 (wild-type NifA in wild-type background), UR2502 (NifA-M173I in wild-type background), UR2503 (NifA-M173V in wild-type background), UR2327 (wild-type NifA in draT mutant background), UR2328 (NifA-M173I in draT mutant background) and UR2329 (NifA-M173V in draT mutant background) when cells were grown in MN (NH₄⁺ -excess) medium. Proteins were extracted quickly with TCA, separated by SDS-PAGE, and immunoblotted with antibody against A. vinelandii dinitrogenase reductase. As described in Methods, active dinitrogenase reductase is unmodified and migrates as a single band (indicated by U), while inactive dinitrogenase reductase migrates as two bands with the upper band representing the modified subunit (indicated by M). A third band between the unmodified and the modified bands might be an alternative dinitrogenase reductase or another unmodified form of dinitrogenase reductase.