Ammonia assimilation and nitrogen fixation in phototrophic bacteria : studies on glutamine synthease and the metabolic regulation of nitrogenase in Rhodospirillum rubrum

Sammanfattning: The phototrophic bacteria and a few other species, are able to control nitrogen fixation on the metabolic level. This is a fast and reversible mechanism, which “switches off and on” the nitrogenase activity in response to the accessibility of combined nitrogen, the energy supply and the level of oxygen in the medium. By the modification of dinitrogenase reductase the switch-off mechanism leads to the inhibition of the catalytic activity of the whole complex. On the physiological level the details of this mechanism are inadequately understood. The present investigation focus on two aspects of regulation; i) glutamine synthetase; a central enzyme in both ammonia assimilation and metabolic regulation of; nitrogenase activity, and ii) the identity of the signal that initiates the switch-off mechanism.Glutamine synthetase was purified to electrophoretic, homogeneity from Rsp.rubrum before and after switch-off by affinity chromatography as major step: The enzyme purified was in either high or low-activity form; due to using cultures growing, om N2. either before- or after switch-off respectively. The molecular mass of the enzyme as well as its kinetic properties are similar to other bacterial glutamine synthetases. Except the difference- in: activity between the high and: the tow active forms of Rsp. rubrum glutamine synthetase, the enzyme has- several properties which are different from glutamine synthetase of E. coli as well a» glutamine synthetase from other phototrophic bacteria: i) the different forms of the enzyme do not show an isoactivity pH in the transferase reaction, ii)in the presence of Mn^+, the transferase activity of both, forms is- inhibited by Mg2+, iii) snake venom phosphodiesterase has no effect on the low activity form;, iiw)) there are no differences in the sensitivity of the two forms of towards feedback inhibitors and' Vf the activity of glutamine synthetase from Rsp. rubrum shows switch-off behaviour similar to dinitrogenase reductase from the same organism. The present study could not reveal which type of modification regulates glutamine synthetase activity in Rsp. rubrum growing on N2.The metabolic regulation of nitrogen fixation in Rsp. rubrum, as presented in this thesis, involves both nitrogen and energy metabolism, therefore such a complex phenomenon can only, so far, be studied in vivo. The present studies demonstrated the following: i) The elevation of the cellular level of NAD+ could be the common signal molecule which triggers ADP-ribosylation of dinitrogenase reductase. Using exogenous NAD+, we found that it was taken up by the cells leading to switch-off, iijunlike glutamine as switch-off effector, exogenous NAD+ exerts its effect without participation of glutamate synthase. This result supports our assumption that, upon the addition of NFLf1-, extensive ammonia assimilation in the glutamine synthetase/glutamate synthase pathway leads to oxidation of NADPH. In the transhydrogenase reaction, the regeneration of NADPH would cause a sudden elevation of the cellular level NAD+, iii) switch-off by NAD+ was more pronounced under low light intensity and this observation made possible the use of this compound as a switch-off effector in Nstarved cells. The influence of light intensity on switch-off by exogenous NAD+, is also in line with the assumption that switch-off by darkness is due to direct elevation of the cellular level of this compound and v) the addition of reduced carbon compounds accelerate switch-on, whereas blocking the reduction of NAD+ favours switch-off. Furthermore, it was found that oxaloacetate and 2-oxoglutarate were able to trigger switch-off. Taken together with the results from other investigators, the present results are the basis for the formulation of a scheme which suggest that, the elevation of the cellular level of NAD+ could be the common signal between switch-off effectors and the regulatory cycle of nitrogenase activity in Rsp. rubrum.