Short‐Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum

Abstract

1. Evidence is presented that the direct depressing effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii is due to inhibition of the electron transport system to nitrogenase. Furthermore, we were able to confirm the observation [Houwaard, F. (1979) Appl. Environ. Microbiol. in the press] that ammonium chloride has no short-term effect on nitrogen fixation by isolated bacteroids of Rhizobium leguminosarum.2. By means of the flow dialysis technique it could be demonstrated that in A . vinelandii ammonium is taken up as a cation in response to the A $ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the A $ across the cytoplasmic membrane. In A . vinelandii, like in bacteroids, the generation of reducing equivalents at a potential low enough to reduce nitrogenase was found to be extremely sensitive towards changes in A$. At A$ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a d $ value of 110 mV nitrogenase is supplied optimally with reducing equivalents. The nature of the ammonium transport system in A. vinelandii and its significance as a regulator for the rapid 'switch off/switch on' of nitrogenase activity is discussed.3. Bacteroids of R. leguminosarum did not accumulate ammonium and no effect of ammonium on A $ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the A pH.It has been firmly established that the process of nitrogen fixation by free-living bacteria [l-41 as well as by symbiotic associations [5,6] is inhibited in the presence of fixed nitrogen, especially ammonium. In the free-living nitrogen-fixer Azotobacter two effects of ammonium can be distinguished. Firstly, the energetically expensive nitrogenase reaction is rapidly switched off (short-term effect) [l]. Secondly, the synthesis of nitrogenase is repressed, resulting in a decline of total activity (long-term effect) [2,7]. On the other hand, fixed nitrogen does not affect the nitrogenase activity of isolated bacteroids of Rhizobium leguminosarum [8]. However, when these bacteroids are in symbiosis with the plant host cell, fixed nitrogen affects the nitrogenase activity [5,6].Little is known about the short-term effect of am- enough energy in the form of ATP and reducing equivalents. Since cell-free nitrogenases are insensitive to ammonium concentrations that completely inhibit the nitrogenase activity in whole cells [9,10], it has been suggested that ammonium in Azotobacter depresses the flow of reducing equivalents and/or the supply of ATP to nitrogenase [l, 1 I].In the present paper we investigated the shortterm effect of ammonium chloride on nitrogen fixation by the free-living organism Azotobacter vinelandii and the symbiotic nitrogen-fixer R. leguminosarum. Evidence is presented that electron transfer to nitrogenase in A. vinelandii, like in R. leguminosarum bacteroids [12], is regulated exclusively by the electrical component (A$) of the protonmotive for...

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii

“…vinelandii is known to at least partially switch off nitrogenase activity when exposed to a sudden increase in the concentration of ammonium (14). In contrast to the oxygen stress and in agreement with a previous report (23) a slight increase in the cellular ATP pool could be observed under the conditions of the present investigation.…”

Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH3 in Soybean

“…(a) NH3 could have a direct effect on nitrogenase, on the ATP supply for nitrogenase, or on the electron transfer system to nitrogenase (8). However, a direct effect of NH3 on nitrogenase activity seems unlikely.…”

Key Role of Bacterial NH 4 + Metabolism in Rhizobium-Plant Symbiosis

“…In Azotobacter vinelandii, it was observed that the addition of NH 4 ϩ to N 2 -fixing cells rapidly inhibits nitrogenase activity by decreasing the flow of reducing equivalents, whereas at high concentrations it also reduces the intracellular ATP/ADP ratio (92). In contrast, addition of NH 4 ϩ does not affect nitrogenase activity of isolated SBs of R. leguminosarum, which are instead unable to take up NH 4 ϩ actively, and therefore it was concluded that NH 4 ϩ could affect the energized state of the membrane only when it is taken up (92). The amtB-mediated negative effect on BT growth supports the notion that, unlike free-living diazotrophs, BTs are programmed not only to fix N 2 but also to release most of the N fixed and that the N 2 fixation process in BTs is regulated to satisfy the plant requirement for N.…”

“…Addition of NH 4 ϩ (and NO 3 Ϫ ) also inhibits nitrogenase activity, possibly not directly but after conversion to glutamine, or through the regulation of a plant function (173), e.g., the reduction of the O 2 or C supply by the plant (15). In fact, it has been shown that isolated SBs do not take up NH 4 ϩ and that SBs in the nodule do not express the amtB gene, which is required for high-affinity NH 4 ϩ uptake activity (92,179). Moreover, externally added NH 4 ϩ may never reach the SBs, since the NH 4 ϩ -assimilating enzymes are highly expressed in the cytoplasm of ICs.…”

“…This operon is expressed from a promoter located upstream of glnK, whereas no promoter activity was detected in the intergenic region (179). The glnK promoter becomes inactive very early during symbiosis, and in correlation isolated BTs do not show high-affinity NH 4 ϩ uptake activity (92,134,179,180). Downregulation of amtB expression is essential for the normal function of BTs (see below).…”