Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity

Abstract

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions. Nitrogenase activity in the phototrophic bacterium Rhodospirillum rubrum is regulated at the level of expression and by posttranslational modification (8,10,12,28,33, and references therein). Oxygen and fixed sources of N such as glutamine and ammonia lead to the repression of nitrogenase; when glutamate is provided as the N source, nitrogenase is expressed.The inhibition of nitrogenase activity in vivo by ammonia was first noted by Gest et al. (8). The molecular basis for the inhibition is the ADP-ribosylation of dinitrogenase reductase (the iron protein) at a specific arginyl residue (26). Enzymes that attach (14) and remove (15,23,29) the ADP-ribosyl residue have been isolated from R. rubrum and have been found under a variety of culturing conditions (14,33).Factors that lead to the loss of activity in vivo include ammonia (10, 22, 31), glutamine (22), darkness (12, 40), and phenazine methosulfate (PMS) (12,25). Cells grown on N2 or glutamate as the N source are competent to switch-off nitrogenase activity (switch-off is the term given by Zumft and Castillo [44] to the reversible, in vivo loss of nitrogenase activity), whereas cells grown on limiting-ammonia (i.e., N-starved) medium are incapable of switch-off (38). Nitrogen assimilation in R. rubrum under nif-derepressed conditions is via glutamine synthetase (GS) and glutamate synthase (GOGAT) (3,21,32,36), and ...

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

“…These results indicate that glutamine is not the signal for the darkness response. Consistent with this, a rapid and drastic increase in the glutamine pool has been seen after addition of NH z 4 , but only a very small change was seen after the darkness shift in R. rubrum (Kanemoto & Ludden, 1987;Li et al, 1987). These data suggest that the modification of GS and the regulation of nitrogenase activity are two independent events, and both are regulated by P II in response to NH z 4 and darkness stimuli.…”

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum

“…NH 4 ϩ itself is not the direct signal for the DRAT-DRAG system, as L-methionine-D-sulfoximine, an inhibitor of GS, can significantly block the NH 4 ϩ effect on nitrogenase activity in both A. brasilense (12) and R. rubrum (14). Consistent with this, the intracellular glutamine concentration increases rapidly after NH 4 ϩ treatments in both R. rubrum and A. brasilense (11,15,18). GS activity is reduced in the A. brasilense ntrBC mutants (40), and the products of ntrBC regulate GS synthesis in other nitrogen-fixing bacteria, such as K. pneumoniae (6), Rhizobium meliloti (4,34), and B. japonicum (22).…”

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

“…The in vitro studies performed with GlnD from R. rubrum (6) and Escherichia coli (4,5) indicate that glutamine is the main signal inducing GlnD deuridylylating activity. In fact, it has been shown that in glutamate-grown cells, there is a small increase in the glutamine concentration upon transfer to darkness (8). This observation might explain the observed P II protein deuridylylation upon exposure to darkness in glutamate-grown cells.…”