Nitrogenase Switch-Off and Regulation of Ammonium Assimilation in Response to Light Deprivation in Rhodospirillum rubrum Are Influenced by the Nitrogen Source Used during Growth

Abstract

Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P II modification is dependent on the nitrogen source used for growth, N 2 or glutamate, although both lead to nitrogenase derepression.The reduction of molecular nitrogen to ammonia is carried out only by some prokaryotes, diazotrophs. This process is catalyzed by nitrogenase, a metalloenzyme complex composed of two components, the Fe protein and the MoFe protein (14). In the photosynthetic purple bacterium Rhodospirillum rubrum, but also in, e.g., Rhodobacter capsulatus and Azospirillum brasilense, the activity of nitrogenase is posttranslationally regulated by reversible mono-ADP-ribosylation of one of the subunits of the Fe protein, the so-called "switch-off" effect (reviewed in reference 12). The result is loss of nitrogenase activity, for instance, in response to addition of ammonium ions or shift to darkness, i.e., changing the nitrogen or energy status. The enzymes involved are dinitrogenase reductase ADP-ribosyl transferase (DRAT), catalyzing the modification, and dinitrogenase reductase-activating glycohydrolase (DRAG), catalyzing hydrolysis of the ADP-ribose moiety when light is turned on or the ammonium added has been metabolized, leading to recovery of nitrogenase activity.Ammonium (ammonia and/or ammonium ions) is further assimilated into glutamate/glutamine primarily through the glutamine synthetase (GS)/glutamate synthase pathway (2, 9, 15). In R. rubrum, GS is posttranslationally regulated through reversible adenylylation, a reaction catalyzed by the bifunctional enzyme adenylyltransferase (GlnE). R. rubrum GlnE requires the presence of an unmodified P II protein in vitro to catalyze adenylylation of GS, leading to a decrease in GS activity (7). P II signal transduction proteins have a central role in controlling nitrogen metabolism by integrating signals such as energy and carbon status by binding ATP/ADP (4) and 2-oxoglutarate (10). In most proteobacteria, P II proteins are also regulated through reversible uridylylation catalyzed by another bifunctional enzyme, uridylyltransferase or GlnD. In R. rubrum, 2-oxoglutarate is the signal for uridylylation while glutamine stimulates deuridylylation; GlnD is therefore a link between cellular nitrogen status and P II modification (6). In R. rubrum, three P II paralogs, GlnB, GlnK, and GlnJ, have been identified (20). These are highly similar but nevertheless show both unique and overlapping functions in the cell, including the posttranslational regulation of nitrogenase and GS activities (7, 16-18, 20, 23).In a number of studies, the molecular events in the regulation of nitrogenase and GS activities in R. rubrum have been investigated, but different nitrogen sources, glutamate and N 2 , have been used during growth, often in an inconsistent way. For instance, in a recent investigation of the role of the ammonium transport protein AmtB1 in nitrogenas...

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

“…2b). This in fact underlines the differences in metabolic responses depending on the nitrogen source used for growth, as we have reported previously (Teixeira et al, 2010).…”

Enhanced hydrogen production performance of Rubrivivax gelatinosus M002 using mixed carbon sources

“…The results showed no hydrogen production when 0.5 g/L of NH 4 Cl was employed as nitrogen source and regular hydrogen yield when 0.5 g/L of glutamic acid was used as nitrogen source (data not shown). To our best knowledge, the activity of nitrogenase could be deactivated by certain amount of ammonium, and the activity of hydrogenase is not sensitive to ammonium [36,37]. This result indicates that nitrogenase is responsible for hydrogen production in this study.…”

PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity

“…However, this hypothesis is challenged by several sets of experimental data. In R. rubrum, neither GlnB nor GlnJ are de-uridylylated under darkness switch-off when cells are cultivated using N 2 as nitrogen source instead of glutamate (Teixeira et al, 2010). Hence, as fully uridylylated P II cannot interact with AmtB (Rodrigues et al, 2011), AmtB-P II complex formation seems dispensable for the darkness response in R. rubrum (Teixeira et al, 2010).…”

“…Western blot analysis revealed that in response to darkness, GlnJ and GlnB are de-uridylylated and GlnJ associates with the membrane in an AmtBdependent manner Teixeira et al, 2010). The signal triggering P II protein de-uridylylation in response to darkness is not known, though a slight increase in glutamine levels has been reported (Kanemoto & Ludden, 1987).…”

“…In R. rubrum, neither GlnB nor GlnJ are de-uridylylated under darkness switch-off when cells are cultivated using N 2 as nitrogen source instead of glutamate (Teixeira et al, 2010). Hence, as fully uridylylated P II cannot interact with AmtB (Rodrigues et al, 2011), AmtB-P II complex formation seems dispensable for the darkness response in R. rubrum (Teixeira et al, 2010). Furthermore, AmtB is not required for darkness-induced NifH ADPribosylation in R. capsulatus (Yakunin & Hallenbeck, 2002) or for anaerobically induced NifH ADP-ribosylation in A. brasilense (L. F. Huergo, unpublished data) and Azoarcus sp.…”