부산대학교 도서관

Organisms that use ammonium as the sole nitrogen source discriminate between [[sup.15N]] and [[sup.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 N[H.sub.3] (achieved by controlling total N[H.sub.4] CI and pH of the medium). At high N[H.sub.3] ([greater than or equal to ]0.89 [micro]M), discrimination against the heavy isotope by the WT strain (-19.2%) can be accounted for by the equilibrium isotope effect for dissociation of N[H.sub.4.sup.+] to N[H.sub.3] + [H.sup.+.] N[H.sub.3] equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low N[H.sub.3] ([[less than or equal to ]0.18 [micro]M), discrimination reflects an isotope effect for the N[H.sub.4.sup.+] channel AmtB (-14.1[per thousand]). 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 [per thousand]). Likewise, by making unmediated diffusion of N[H.sub.3] 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 N[H.sub.3] across the membrane (-10.9[per thousand]). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them. doi: 10.1073/pnas.1216683110