Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium

Abstract

In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase activity is regulated by ADPribosylation of component H in response to the addition of ammonium to cultures or to the removal of light. The ammonium stimulus results in a fast and almost complete inhibition of the in vivo acetylene reduction activity, termed switch-off, which is reversed after the ammonium is exhausted. In the present study of tion of nitrogenase activity. This modification is performed by the enzyme dinitrogenase reductase ADP-ribosyl transferase (DRAT) (39). When the stimulus is exhausted or removed, the ADP-ribose moiety is removed from component II, restoring nitrogenase activity. This ADP-ribose removal is catalyzed by dinitrogenase reductase-activating glycohydrolase (DRAG) (59). For R. rubrum and Azospirillum brasilense, which is a microaerobic nitrogen-fixing bacterium, the draT and draG genes, coding for DRAT and DRAG, respectively have been cloned, sequenced, and expressed in different backgrounds (10,12,71). Mutations of these genes have been analyzed, and posttranslational regulation of nitrogenase in these mutants has been found to be strictly dependent on the presence of draTG (37,71).Posttranslational regulation of nitrogenase in the photosynthetic bacterium Rhodobacter capsulatus also involves ADP-ribosylation of component II (19,29,47). Removal of a culture from the light causes ADP-ribosylation (50), and addition of ammonium to cultures results in an immediate inhibition of nitrogenase activity (20,23,28,68). However, none of these reports established a good correlation between ADP-ribosylation of component II and loss of activity resulting from the ammonium stimulus, either because the relative kinetics of the two effects were not determined or because the ADP-ribosylation was not quantified. Moreover, cells were often subjected to the other stimulus, darkness, during centrifugation steps.We previously described mutants whose component II proteins are no longer substrates for . The observation that their in vivo nitrogenase activity continued to respond to ammonium addition resulted in the detection of a second regulatory response that regulates nitrogenase activity in response to this stimulus.

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

“…In contrast, both the modification of dinitrogenase reductase and the loss of nitrogenase activity are rather slow in R. rubrum following NH 4 ϩ addition or a shift to darkness (10,15). R. capsulatus shows rapid loss of nitrogenase activity but a slow rate of modification of dinitrogenase reductase during NH 4 ϩ switch-off (22). In this organism, the difference between the rate of loss of nitrogenase activity and the rate of modification of dinitrogenase reductase reflects the presence of another uncharacterized regulatory system that responds to NH 4 ϩ (22).…”

“…R. capsulatus shows rapid loss of nitrogenase activity but a slow rate of modification of dinitrogenase reductase during NH 4 ϩ switch-off (22). In this organism, the difference between the rate of loss of nitrogenase activity and the rate of modification of dinitrogenase reductase reflects the presence of another uncharacterized regulatory system that responds to NH 4 ϩ (22). The response to darkness of R. capsulatus nitrogenase activity and modification is also slow, similar to that in R. rubrum (10,15,22).…”

“…First, the DRAT/DRAG system responds to different negative stimuli in these bacteria. In the photosynthetic bacteria R. rubrum and R. capsulatus, dinitrogenase reductase is modified in response to addition of fixed nitrogen, such as NH 4 ϩ , or a shift of the culture to darkness, which deprives the cells of energy (10,15,17,22). A. brasilense is a nonphotosynthetic, microaerobic nitrogen-fixing bacterium, and its DRAT/DRAG system negatively regulates nitrogenase activity in response to exogenous NH 4 ϩ or anaerobic conditions, because the latter treatment removes the preferred energy source (8,9,28).…”

Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

“…The characterization of R. capsulatus NifA1 mutants which are able to activate nif gene transcription in the presence of ammonium revealed that the N-terminal domain of NifA is involved in this post-translational control mechanism (Paschen et al, 2001). Finally, at a third level of control, the activity of both nitrogenases is regulated in response to ammonium and darkness via reversible ADP-ribosylation of the dinitrogenase reductases NifH and AnfH mediated by DraT (dinitrogenase reductase ADP-ribosyltransferase) and DraG (dinitrogenase-reductase-activating glycohydrolase) and by a DraT/DraG-independent switch-off mechanism Pierrard et al, 1993;Yakunin & Hallenbeck, 1998). Recently it has been shown that the (methyl-) ammonium transporter AmtB, but not its homologue AmtY, may act as an ammonium sensor which is involved in controlling nitrogenase activity via DraT/DraG and also via the DraT/DraG-independent mechanism .…”

Photofermentative Hydrogen Production in Outdoor Conditions

“…This process is catalyzed by two non-nif-specific enzymes: dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). Another post-translational regulation that does not involve ADP-ribosylation was also proposed in R. capsulatus [22,23]. Due to the modulation of nitrogenase activity according to cellular nitrogen status, hydrogen production studies on PNSB are carried out on media with limited nitrogen source.…”