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Journal of Bacteriology, March 2008, p. 1588-1594, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01643-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Pier-Luc Tremblay and Patrick C. Hallenbeck*

Département de microbiologie et immunologie, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Received 10 October 2007/ Accepted 14 December 2007

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.

* Corresponding author. Mailing address: Département de microbiologie et immunologie, Université de Montréal, CP 6128, succursale Centre-ville, Montréal, Québec H3C 3J7, Canada. Phone: (514) 343-6278. Fax: (514) 343-5701. E-mail: patrick.hallenbeck{at}umontreal.ca

{triangledown} Published ahead of print on 21 December 2007.

Journal of Bacteriology, March 2008, p. 1588-1594, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01643-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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