Ammonia-Induced Formation of an AmtB-GlnK Complex Is Not Sufficient for Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

ABSTRACT A series of Rhodobacter capsulatus AmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.

Comparing Short-Term Effects of Ammonia and Methylamine on Nitrogenase Activity in Anabaena variabilis (ATCC 29413)

Cyanobacterium . Anabaena variabilis, Nitrogenase Regulation, Ammonia, Methylamine Using the heterocystous cyanobacterium A nabaena variabilis (ATCC 29413) in an alkaline environment its nitrogenase activity is rapidly inhibited by ammonia and methylamine. Nitro­genase inhibition by ammonia is probably caused by a mechanism comparable to the switch-off effect, which has been described for several species of the Rhodospirillaceae, whereas methyl-amine-induced inhibition is caused by an uncoupling effect only. Evidence for these different effects is obtained by comparing nitrogenase activity in cell-free extracts of filaments pretreated by ammonia or methylamine. In addition, ammonia-dependent nitrogenase inhibition is shown to be dependent on protein synthesis and on light intensity.