title: Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12.
abstract: Organisms that use ammonium as the sole nitrogen source discriminate between [(15)N] and [(14)N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥ 0.89 µM), discrimination against the heavy isotope by the WT strain (-19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4(+) to NH3 + H(+). NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤ 0.18 µM), discrimination reflects an isotope effect for the NH4(+) channel AmtB (-14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (-10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.
authors: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors: Jason
authors: J
authors: Vo
authors: William
authors: W
authors: Inwood
authors: John M
authors: JM
authors: Hayes
authors: Sydney
authors: S
authors: Kustu
mesh_terms: D027682:Cation Transport Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D009584:Nitrogen / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*
chemical_list: C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D000644:Quaternary Ammonium Compounds; D009584:Nitrogen
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