Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness

Abstract

Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+-or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool rqmained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4' azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4L substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivatioi. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4' switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4' switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determnining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4' (glutamine) switch-off.The nonsulfur photosynthetic bacteria are among a group of N2-fixing bacteria with nitrogenase activity that is subject to regulation by NH4'. Carithers et al. (4) and Nordlund and Eriksspn (17) showed that the inhibitory effect of NH4' on nitrogenase activity (switch-off) is related to the covalent modification and concurrent inactivation of the nitrogenase Fe protein extracted from glutamate-and N2-grown cells (13,14,18). Similar to the inactive Fe protein isolated from glutamate-grown cells, Fe protein extracted from NH4+-inhibited cells (4, 17) was inactive and could be reactivated by a membrane-bound activating enzyme (13, 18). Switch-off of nitrogenase activity by NH4' catalyzes the covalent modification of one of the two Fe protein subunits (6, 23) with one molecule of ADP-ribose (22).The role of NH4' as an in vivo signal to activate an Fe protein-inactivating enzyme has been the subject of numerous investigations. Glutamine mimics NH4' (16) and methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), prevents NH4' switch-off in Rhodospirillum rubrum (5,25,30) and all other N2 fixers that are capable of switch-off regulation (3, 9). 6-Diazo-5-oxo-L-norleucine (30) and azaserine (7) are inhibitors of glutamate synthase (GOGAT), and both initiate switch-off in the absence of NH4', presumably by blocking the normal flux of nitrogen through the GS-GOGAT pathway. This results in a glutamine buildup sufficient to initiate switch-off.The confirmed and extended to show that R. rubrum could rapidly reverse this process in the light (10). It is not readily apparent what glutamine and darkness have in common that leads to the activation of the Fe...

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.…”

“…Previous reports showed that MSX completely blocked the effect of NH z 4 on nitrogenase activity, while the effect of MSX on the darkness response has been controversial in R. rubrum (Kanemoto & Ludden, 1984;Li et al, 1987;Sweet & Burris, 1981;Yoch & Gotto, 1982). We again used a draG mutant (UR832) to obtain more interpretable results.…”

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).…”

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

“…Although there have been several studies of possible metabolic signals for these stimuli, including the effect of energy (11,20), pools of amino acids and pyridine nucleotides (11,14,18,24), and metal ions (19,27), the nature of the signals for the DRAT/DRAG system is still unknown. In A. brasilense, NH 4 ϩ and anaerobic stimuli may have at least partially divergent signal transduction pathways, as the mutational loss of ntrBC causes a dramatically altered NH 4 ϩ switch-off but has little effect on anaerobic switch-off (29).…”