Stefan Nordlund

Research Project
Nitrogen fixation and ammonium asssimilation in photosynthetic bacteria; regulation and electron transport.

Address
e-mail: stefan@dbb.su.se
phone: (+46)-8-16 2932
fax: (+46)-8-15 7794

Research group & Co-workers
Tomas Edgren, Anders Jonsson, Jeanette Lindh, Agneta Norén, He Wang.

Project description
Biological nitrogen fixation, a process occurring in bacteria and cyano-bacteria, is the reduction of molecular nitrogen to ammonia. The reaction is catalyzed by nitrogenase, an enzyme complex consisting of two proteins, the Fe-protein and the MoFe-protein. Nitrogenase is a highly conserved enzyme and all nitrogenases are irreversibly inhibited by oxygen. In the reaction catalyzed by nitrogenase at least 16 ATP are hydrolyzed per N2 reduced.

In all nitrogen fixing organisms nitrogen fixation is regulated transcriptionally in response to "fixed" nitrogen. This regulation involves a number of proteins which act in concert to turn on transcription of the genes encoding the proteins required for nitrogen fixation. In addition to the genetic regulation, nitrogen fixation in phototrophic bacteria is controlled by a sensitive metabolic regulatory system, involving reversible covalent modification. Upon the addition of ammonia the Fe-protein is inactivated by ADP-ribosylation in a reaction catalyzed by DRAT. When the ammonia added is metabolized, DRAG catalyzes the reactivation by removal of the ADP-ribose moiety.

This metabolic regulation was first demonstrated in Rhodospirillum rubrum and one of our projects aims at identifying the metabolic signals involved. We suggest that in R. rubrum, changes in the red-ox status are involved in controlling DRAT/DRAG. This model, which includes membrane association as a regulatory feature, offers a unifying concept for the action of all conditions leading to regulatory inhibition of nitrogen fixation in this bacterium.Another area of major interest concerns the identity of the organic electron donor(s) and the electron transfer to nitrogenase in R.rubrum. We have recently established that this pathway involves the Fix proteins that reduce ferredoxin, possibly in a reaction driven by the membrane potential. We have shown that ferredoxin is the direct electron donor to the Fe-protein.

In most nitrogen fixing organisms the ammonia produced during nitrogen fixation is assimilated by reactions catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The activity of GS in R.rubrum is also regulated and it has been shown to be ADP-ribosylated as well as adenylylated, i. e. modified with AMP. Our interest concerns the enzymes involved in the regulation of GS in R.rubrum, the role of the modifications and to understand in what way the regulation of GS is coupled to that of nitrogenase. We are focussing on the PII proteins, which have a central role in the regulation of GS as well as a crucial function in the transcriptional regulation of nitrogen fixation, but also as recently discovered in the metabolic regulation of nitrogenase.

Our overall goal is to understand the molecular details of the regulation of nitrogen fixation and ammonia assimilation in R.rubrum as well as the interaction between these and other metabolic processes, for example photosynthesis.

References
* Rees, D.C. and Howard, J.B.. (2000) Curr. Opin. Chem. Biol. 4, 559-566.
* Cheng, J., Johansson, M. and Nordlund, S. (1999) J. Bacteriol. 181, 6530- 6534.
* Norén, A. and Nordlund, S. (1997) J. Bacteriol. 179, 7872 – 7874.
* Edgren, T. and Nordlund, S. (2004) J. Bacteriol. 186, 2052-2060.
* Nordlund, S. and Ludden, P.W. (2004) in “Genetics and regulation of nitrogen fixing bacteria”, (Klipp, W., Masephol, B., Gallon, J.R. and Newton, W.E. eds.) Kluwer Academic Press, the Netherlands, pp 175-196.

Acknowledgement
Supported by grants from the Swedish Research Council, Wenner-Gren Foundation, FORMAS, Swedish Institute and Carl Tryggers Stiftelse för Vetenskaplig Forskning.