Isotopic remembrance of metabolism past

Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12

“…The equality of « b for the ΔghdA and WT strains at all external concentrations of NH 3 confirms the finding of Yuan et al (24,25) that the GOGAT cycle is the major means for assimilation of NH 3 by E. coli K12 at not only low but also, high concentrations. Although the GOGAT cycle is widespread in bacteria and archaea (8,42,43), whereas the occurrence of biosynthetic GDH seems to be more restricted (44), important examples of organisms naturally lacking GOGAT have recently come to light (45).…”

“…At high NH 3 (≥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 NH 4 + to NH 3 + H + . NH 3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH 3 (≤0.18 μM), discrimination reflects an isotope effect for the NH 4 + channel AmtB (−14.1‰).…”

“…Specifically, 18 experiments yielding « b ≅ « h could then be explained only if (i) g, « GS , and « at happened in all cases to have values satisfying the relationship g = « GS /(« GS − « at ) and (ii) g was independent of the external concentration of NH 3 . Additionally, a well-documented experimental study of nitrogen and carbon isotope effects associated with glutamine synthetase from E. coli found a near-zero nitrogen isotope effect of −0.7 ± 0.6‰ (mean and SD, n = 7) (35).…”

“…To study effects of these proteins on the in vivo fractionation of ammonium N, we used WT and genetic mutant strains in which one or more was lacking or defective and studied these strains at high or low external concentrations of NH 3 . To decrease the concentration of external NH 3 and still achieve significant cell yield, we lowered both the total concentration of NH 4 Cl and the pH of the medium. Although E. coli lives in the human gut, which is nitrogenrich, it also survives in fresh and brackish water, in which supplies of available ammonium can be limited (9,10).…”

“…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 NH 3 (achieved by controlling total NH 4 Cl and pH of the medium). At high NH 3 (≥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 NH 4 + to NH 3 + H + .…”