Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

Abstract

The PII regulatory protein family is unusually widely distributed, being found in all three domains of life. Three P II homologs called GlnB, GlnK, and GlnJ have been identified in the photosynthetic bacterium Rhodospirillum rubrum. These have roles in at least four distinct functions, one of which is activation of the nitrogen fixation-specific regulatory protein NifA. The activation of NifA requires only the covalently modified (uridylylated) form of GlnB. GlnK and GlnJ are not involved. However, the basis of specificity for different P II homologs in different processes is poorly understood. We examined this specificity by altering GlnJ to support NifA activation. A small number of amino acid substitutions in GlnJ were important for this ability. Two (affecting residues 45 and 54) are in a loop called the T-loop, which contains the site of uridylylation and is believed to be very important for contacts with other proteins, but other critical residues lie in the C terminus (residues 95-97 and 109 -112) and near the N terminus (residues 3-5 and 17). Because many of the residues important for P II-NifA interaction lie far from the T-loop in the known x-ray crystal structures of P II proteins, our results lead to the hypothesis that the T-loop of GlnB is flexible enough to come into proximity with both the C-and N-terminal regions of the protein to bind NifA. Finally, the results show that the level of P II accumulation is also an important factor for NifA activation.

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

“…First, GlnD might display a differential preference for some P II species over others for either modification or demodification. If a specific cellular process requires the interaction of a target protein with a specific P II homolog, as we have shown in R. rubrum for NifA activation (64), then the absolute level of only that P II -UMP species would be critical, although other physiological targets might have completely different requirements. Thus, a mutant with altered GlnD activity or accumulation might affect differential P II modification in a physiologically significant way.…”

“…That regulation probably occurs through the direct interaction between NifA and the uridylylated form of GlnB, which is required for the activation of NifA activity under NH 4 ϩ -limiting conditions (61,62,64). Neither GlnK nor GlnJ can replace GlnB to activate NifA (61,64).…”

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

“…In Rhodospirillum rubrum, GlnB-UMP, but not the other two P II homologues, interacts directly with NifA to activate it. Specific residues of GlnB that define this specific interaction have been analysed (Zhang et al, 2004).…”

Nitrogenase Switch-Off and Regulation of Ammonium Assimilation in Response to Light Deprivation in Rhodospirillum rubrum Are Influenced by the Nitrogen Source Used during Growth

“…The involvement of P II proteins in nitrogenase switch-off has been suggested to include binding to the ammonium transporter AmtB1 in R. rubrum, which in turn promotes sequestration of DRAG to the membrane (17,23). In addition, unmodified GlnB has been proposed to activate DRAT in A. brasilense (3) and R. rubrum (22). However, in N 2 -grown cells, there is a clear darkness switch-off (Fig.…”