Methylammonium transport

Abstract: Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to gamma-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH4+.

Abstract: The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH3, we recently speculated that Rh proteins are biological gas channels for CO2. Like NH3, CO2 differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii. Expression of RH1 was high for cells grown in air supplemented with 3% CO2 or shifted from air to high CO2 (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO2) or shifted from high CO2 to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO2.

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 ΔΨ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the ΔΨ 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 ΔΨ. At ΔΨ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a ΔΨ 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 ΔΨ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the ΔpH.