SI Results Peculiarities in the consumption of ammonium and glucose by AmtB - strains. The AmtB - strain failed to consume all the ammonium in the medium under NH 3 -limiting conditions (.5 mm total ammonium at ph 5.5; Fig. S2A). Whereas residual ammonium left behind by the parental strain fell below our detection limit of 25 µm, that in the AmtB - strains remained 5 μm. We infer that at 2 μm internal ammonium [assuming an internal ph of 7. at an external ph of 5.5; ref. (13)], GS ceases to function in synthesis of glutamine (14). The AmtB - strains showed unusually high light scattering at the end of growth (~1% above the parental strain) and ~1% of the residual ammonium was unutilized. Moreover, at c = 18 and 89 nm (exps. 27 and 1), the molar C/N ratio in the biomass of the AmtB - strain was significantly elevated, rising from 4.3 to 4.9 and from 4.4 to 5.2 as ammonia in the growth medium was depleted. These values are sharply higher than those observed in all other experiments (C/N ~ 4., see Fig. S3 in Supplementary Information) and indicate a significant difference in cellular composition. We have not pursued the basis for increased light scattering by AmtB - strains, e. g. whether it is due to a difference in cell size or shape, nor have we explored the basis for changed cellular composition. There is precedent for changes in composition of the cell envelope (apart from proteins) in nutrient-limited, wild-type bacteria of various sorts (15 17). As the growth of AmtB - strains ceases but detectable ammonium is left behind in the medium, the cells begin to consume glucose rapidly, whereas this occurs in the wild-type strain only after stationary phase has been reached (Fig. S1B). The phenomenon is very striking. We do not know whether the reasons for it are non-specific i.e. somehow due to slow growth generally or specific. A particularly intriguing possibility is that the rapid consumption of glucose is due to spontaneous cyclization of γ-glutamyl-phosphate, the high energy intermediate in the GS reaction, to γ-glutamyl pyrrolidone (2-pyrrolidone 5-carboxylate) (17). If this compound is a dead end, its formation would require one mol of glucose per mol. SI Materials and Methods Strain Construction. NCM3722 (1) was the parental strain for all genetic mutant strains used in this work (Table 1). NCM4587 (ΔamtB::kan) was constructed by transducing NCM3722 to kanamycin resistance with P1 vir bacteriophage grown on Keio strain JWK441-1 (2, 3). Analogously, strains carrying ΔgdhA::kan (NCM4454) and gltd::kan (NCM4453) were obtained by transducing NCM3722 to kanamycin resistance with phage grown on JWK175-6 or a gltd::kan insertion strain from the collection of Kang et al., (4), respectively. The kanamycin insert in NCM4587 was removed by site-directed recombination (2) to yield NCM459 (ΔamtB). NCM471 (gltd::kan ΔamtB) was constructed by transducing NCM459 (ΔamtB) to kanamycin resistance with phage grown on strain NCM4453 (gltd::kan). NCM4199, which lacks the C-terminus of AmtB, was constructed as described (5, 6). amtb and gltd gene arrangement. The amtb gene is the second and last gene in the glnk amtb operon and the gdha gene is in an operon by itself. The gltd gene is the second gene in the gltbdf operon. The lesion in gltd may be polar on gltf but gltf has no known function under our growth conditions (7, 8). 1
Justification for gltd mutant. The gltd gene codes for the small subunit of GOGAT, which has an electron transfer function (9). Although the small subunit was thought to have the capacity for NH 3 - (but not glutamine-)dependent synthesis of glutamate (1), which is why we originally inactivated it rather than gltb, this seems unlikely in view of structural work on a related glutamate synthase (9). Although the gltb product (large subunit of GOGAT) may, in fact, have the capacity for NH 3 -dependent synthesis of glutamate, this capacity is low in vitro (11, 12), and we do not think it influenced our experiments because the gltd strain grew poorly at ph 7.4 on.5 mm NH 4 Cl and did not grow at all at ph 5.5, even at 5 mm NH 4 Cl (see Results). 2
SI References 1. Lyons E, Freeling M, Kustu S, Inwood W (211) Using genomic sequencing for classical genetics in E. coli K12. PloS ONE 6:1 16. 2. Baba T et al. (26) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:26.8. 3. Silhavy TJ, Berman ML, Enquist LW (1984) in Experiments with Gene Fusions (Cold Spring Harbor Laboratory Press, Cold Spring Harbor). 4. Kang Y et al. (24) Systematic mutagenesis of the Escherichia coli genome. J Bacteriol 186:4921 3. 5. Fong RN, Kim K-S, Yoshihara C, Inwood WB, Kustu S (27) The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion. Proc Natl Acad Sci USA 14:1876 11. 6. Inwood WB, Hall JA, Kim K-S, Fong R, Kustu S (29) Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB. Genetics 183:1341 55. 7. Goss TJ, Perez-Matos A, Bender RA (21) Roles of glutamate synthase, gltbd, and gltf in nitrogen metabolism of Escherichia coli and Klebsiella aerogenes. J Bacteriol 183:667 19. 8. Grassl G, Bufe B, Müller B, Rösel M, Kleiner D (1999) Characterization of the gltf gene product of Escherichia coli. FEMS Microbiol Lett 179:79 84. 9. Raushel FM, Thoden JB, Holden HM (23) Enzymes with molecular tunnels. Acc Chem Res 36:539 48. 1. Mäntsälä P, Zalkin H (1976) Active Subunits of Escherichia coli Glutamate Synthase. J Bacteriol 126:539 541. 11. Mäntsälä P, Zalkin H (1976) Glutamate synthase. Properties of the glutamine-dependent activity. J Biol Chem 251:3294 3299. 12. Mäntsälä P, Zalkin H (1976) Properties Apoglutamate Synthase and Comparison with Glutamate Dehydrogenase. J Biol Chem 251:33 335. 13. Wilks JC, Slonczewski JL (27) ph of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry. J Bacteriol 189:561 7. 3
14. Boogerd FC et al. (211) AmtB-mediated NH3 transport in prokaryotes must be active and as a consequence regulation of transport by GlnK is mandatory to limit futile cycling of NH4(+)/NH3. FEBS Lett 585:23 8. 15. Zimmer DP et al. (2) Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation. Proc Natl Acad Sci USA 97:14674 9. 16. Gyaneshwar P et al. (25) Sulfur and nitrogen limitation in Escherichia coli K-12: specific homeostatic responses. J Bacteriol 187:174 9. 17. Gamper H (1974) Enzyme organization in the proline biosynthetic pathway of Escherichia coli. Biochim Biophys Acta 354:75 87. 4
Figure Legends Fig. S1. Growth (A), ammonium consumption (B), glucose consumption (C), and (D) determination of ε b for assimilation of external ammonium (c = 7 µm NH 3,.5 mm NH 4 Cl,.1% glucose, ph 7.4) in GOGAT - strains (gltd::kan and gltd::kan ΔamtB). Fig. S2. Ammonium consumption (A) and glucose consumption (B) for wild-type and AmtB - strains (c =.89 µm NH 3,.5 mm total NH 4 Cl,.1% glucose, ph 5.5). Fig. S3. C/N molar ratio of biomass. Exp. 2-3, excluding exp. 27, were combined. AmtB - strains (exp. 1, exp. 27, c =.89 µm NH 3,.5 mm total NH 4 Cl,.1% glucose, ph 5.5) are shown individually. 5
A) 1 B) 5 4.1 M NH4Cl 3 2.1 gltd::kan amtb gltd::kan gltd::kan 1 1 2 3 Time (hrs).1.2.3.4 C) 2 18 D) 1 5 g/ml glucose 16 14 12 1 8 6 4 2 15 N -5-1 -15-2 -25-3.1.2.3.4-1 -.5 f/(1 - f)] lnf
A) M NH4Cl 5 4 3 2 1 B) 1 8 6 4 2 g/ml glucose.1.2.3.1.2.3
8. 7. 6. C/N 5. 4. 3. Exp. 1 2. Exp. 27 1....5 f 1.