In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae

Abstract

Ammonium suppresses nitrogenase activity in Anabaena flos-aquae (Lyng) Breb. at all pH values tested. L-Methionine-DL-sulfoximine at 1 millimolar totally inhibited glutamine synthetase, and 10 micromolar partially inhibited. Both concentrations protected nitrogenase activity from ammonium-induced suppression at pH 7.1 and 8.1. At pH 93 and 10.2, methionine sulfoximine did not alleviate the suppression of nitrogenase by ammonium. This pH-dependent protection of nitrogenase activity is a result of the noncompetitive inhibition of the ammonium transporter by methionine sulfoximine. At pH 7.1 and 8.2, ammonium is protonated and methionine sulfoximine inhibits its entry into the cell. At pH 93 and 10.2, unprotonated ammonia is abundant and may enter the cell independent of the transport system. The effects of ammonium are closely mimicked by the ammonium analog methylamine. These results suggest that ammoniumperse is an important in vivo regulator ofnitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re-evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter.In the absence ofcombined nitrogen, heterocystous blue-green algae fix atmospheric N2 and produce NH3 which is subsequently assimilated by the GS2/GOGAT pathway (2,8,20,21). Exogenous NH3 inhibits nitrogenase activity and heterocyst differentiation in nitrogen fixing algae (3). Stewart and Rowell (20) have shown that this effect can be alleviated by the presence ofthe GS inhibitor L-methionine-DL-sulfoximine (MSX). This result prompted Stewart and Rowell (20) to suggest that the inhibition of nitrogen fixation was not caused by ammonium directly but rather by GS, GOGAT, or a product ofammonium assimilation. Subsequent work (14) suggested that the size of the glutamine pool was an important factor in nitrogenase regulation. Recent Cultures were maintained at 30°C in 400-ml culture bottles bubbled with filter sterilized laboratory air which had been passed through an activated charcoal filter in series with two gas scrubbers containing 1 N H2SO4 and distilled H20, respectively. This treatment removed particulate matter and gaseous ammonia while at the same time saturating the air with water vapor. Continuous illumination was provided by two Gro-lux fluorescent tubes. The quantum flux density was attenuated to 40 gE m-2 s-' with neutral density screening. Experiments were carried out using 20-ml portions of algal cultures in mid-logarithmic phase of growth (1-2 ,ug Chl a ml-'). These portions were incubated in 40-ml air-bubbled test tubes. Culture pH was adjusted to pH 7.1, 8.1, and 10.2 with phosphate (20 mM), bicine (20 mM), and CHES (20 mM), respectively.Nitrogenase Activity. Nitrogenase activity was determined by acetylene reduction. Acetylene reduction was measured using 2-ml aliquots placed in 1 5-ml test tubes sealed with rubber septa. The tubes were preincubated for 10 min in a shaking water bath and illuminated with a quantum...

Transport and Assimilation of Nitrogen by Stichococcus bacillaris Grown in the Presence of Methionine Sulfoximine

“…This contrasts with the report of a direct inhibition of NH4+ transport system by MSX in Anabaenaflos-aquae (12). It has recently been pointed out (3) that methylammonium is metabolized to methylglutamine in cyanobacteria.…”

Nitrogen translocation in wheat inoculated with Azospirillum and fertilized with nitrogen

“…This increase in N content in the grain with inoculation without N was markedly higher than in the treatments that received additional N, probably due to higher N uptake after anthesis (Table 4). Such increment was above 66%, representing about 8.4 kg ha -1 of N. This effect was not observed in the treatments receiving N fertilizer, which could be attributed to the inhibitory effect of N compounds on the nitrogenase activity of the bacterium (Turpin et al, 1984). In this regard, Zimmer et al (1988) com-mented that the growth of the bacterium with nitrite or nitrate suppresses the biosynthesis of nitrogenase, since the cells synthetize nitrite reductase and nitrate reductase instead of using their limited energy in the nitrogenase reaction.…”

Methylamine uptake by zooxanthellae‐invertebrate symbioses: Insights into host ammonium environment and nutrition

“…The initial rapid phase may relate to the transport mechanism itself, while the second slower phase may relate to the metabolic transformation of MA to g-methylglutamine by glutamine synthetase (GS). In studies with cyanobacteria (Boussiba et al 1984;Turpin et al 1984;Kerby et al 1986), the second phase can be eliminated with the GS inhibitor methionine sulfoximine (MSX). In other studies that have demonstrated the biphasic nature of MA uptake in the dinoflagellate Gonyaulux (Balch 1986) and the diatom Phaeodactylum (Wright and Syrett 1983), the second phase was not related to the metabolism of MA.…”

“…Hackette et al 1970), free-living and symbiotic bacteria (Kleiner and Fitzke 198 1;Wiegel and Kleiner 1982), cyanobacteria (Turpin et al 1984), microalgae (Wheeler 1980), macroalgae (Wheeler 1979;MacFarlane and Smith 1982), lichens with algal endosymbionts (Tapper 1983), and angiosperms (Smith 1982). With few exceptions, [14C]MA has proved useful in understanding NH,+ transport, and generally the "affinity" of the transport system has been shown to be greater (i.e.…”