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Mutants of Escherichia coli defective in the degradation of guanosine 5′-triphosphate, 3′-diphosphate (pppGpp)

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Summary

A new class of mutants of E. coli exhibiting altered metabolism of ppGpp and pppGpp has been isolated, and mapped at a locus designated gpp, near min 83 on the genetic map. These mutants accumulate elevated levels of pppGpp during amino acid starvation or carbon source downshift, and exhibit a reduced rate of pppGpp degradation in vivo. The in vitro evidence suggests that the gpp mutants are defective in a 5′-nucleotidase, which specifically hydrolyzes pppGpp to ppGpp. Certain combinations of gpp and spoT mutations are inviable. A gpp spoT double mutant, constructed by employing a leaky spoT mutation, was found to have a slower rate of pppGpp degradation than the gpp mutant alone. This result indicates that spoT also participates in pppGpp degradation. The inviability of certain gpp spoT combinations is attributed to the inability of the double mutants to degrade pppGpp. This is supported by the observation that selection for increased growth rate on the double mutant results in the recovery of relA mutations. Various effects of the gpp mutation upon the pppGpp and ppGpp pools provide additional support for a scheme in which pppGpp is the major precursor of ppGpp.

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References

  1. Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K12. Bacteriol. Rev. 40, 116–167 (1976)

  2. Boer, H.A. de, Weyer, W.J., De Boer, J.G., Van Der Heide, S., Gruber, M.: Synthesis of guanosine 5′-diphosphate, 3′-diphosphate in spoT mutants of Escherichia coli. Biochim. Biophys. Acta 474, 165–172 (1977)

  3. Cashel, M.: Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes. Anal. Biochem. 57, 100–107 (1974)

  4. Cashel, M., Gallant, J.: Two compounds implicated in the function of the RC gene of E. coli. Nature 221, 838–841 (1969)

  5. Cashel, M., Lazzarini, R.A., Kalbacher, B.: An improved method for thin layer chromatography of nucleotide mixtures containing 32P labeled orthophosphate. J. Chromatogr. 40, 103–109 (1969)

  6. Cashel, M., Kalbacher, B.: The control of ribonucleic acid synthesis in Escherichia coli. V. Characterization of a nucleotide associated with the stringent response. J. Biol. Chem. 245, 2309–2318 (1970)

  7. Chaloner-Larsson, G., Yamazaki, H.: Synthesis of guanosine 5′-triphosphate, 3′-diphosphate in a spoT strain of Escherichia coli. Can. J. Biochem. 54, 935–940 (1976)

  8. Cochran, J.W., Byrne, R.W.: Isolation and properties of a ribosome bound factor required for ppGpp and pppGpp synthesis in Escherichia coli. J. Biol. Chem. 249, 353–360 (1974)

  9. Fiil, N.P., Willumsen, B.M., Friesen, J.D., von Meyenburg, K.: Interaction of alleles of the relA, relC, and spoT genes in Escherichia coli: Analysis of the interconversion of GTP, ppGpp, and pppGpp.Mol. Gen. Genet. 150, 87–101 (1977)

  10. Friesen, J.D., Fiil, N.P., von Meyenburg, K.: Synthesis and turnover of basal level guanosine tetraphosphate in Escherichia coli. J. Biol. Chem. 250, 304–309 (1975)

  11. Gallant, J., Margason, G., Finch, B.: On the turnover of ppGpp in Escherichia coli. J. Biol. Chem. 247, 6055–6058 (1972)

  12. Hamel, E., Cashel, M.: Role of guanosine nucleotides in protein synthesis: Elongation factor G and guanosine 5′-triphosphate, 3′-diphosphate. Proc. Natl. Acad. Sci. U.S.A. 70, 3250–3254 (1973)

  13. Hansen, M.T., Pato, M.L., Molin, S., Fiil, N.P., von Meyenburg, K.: Simple downshift and resulting lack of correlation between ppGpp pool size and ribonucleic acid accumulation. J. Bacteriol. 122, 585–591 (1975)

  14. Haseltine, W.A., Block, R., Gilbert, W., Weber, K.: MSI and MSII made on ribosome in idling step of protein synthesis. Nature 238, 381–385 (1972)

  15. Kaempfer, R.O., Magasanik, B.: Effect of infection with T-even phage on the inducible synthesis of β-galactosidase in E. coli J. Mol. Biol. 27, 453–468 (1967)

  16. Kari, C., Török, I., Travers, A.: ppGpp cycle in Escherichia coli. Mol. Gen. Genet. 150, 249–255 (1977)

  17. Laffler, T., Gallant, J.: spoT, a new genetic locus involved in the stringent response in E. coli. Cell 1, 27–30 (1974)

  18. Lazzarini, R., Cashel, M., Galant, J.: On the regulation of guanosine tetraphosphate levels in stringent and relaxed strains of Escherichia coli. J. Biol. Chem. 246, 4381–4385 (1971)

  19. Lennox, E.S., Yanofsky, C.: Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli. Virology 8, 425–447 (1959)

  20. Leung, K.L., Yamazaki, H.: Enzymatic preparation of adenosine 5′-[β-32P] triphosphate. Can. J. Biochem. 55, 223–226 (1977)

  21. Miller, J.H.: Experiments in molecular genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory 1972

  22. Roth, J.R.: Genetic techniques in studies of bacterial metabolism. In: Methods in enzymology (Tabor, H., Tabor, C.W., eds.), Vol. 17 (A), pp. 3–35. New York: Academic Press 1970

  23. Stamminger, G., Lazzarini, R.A.: Altered metabolism of the guanosine tetraphosphate, ppGpp, in mutants of Escherichia coli. Cell 1, 85–90 (1974)

  24. Sy, J.: In vitro degradation of ppGpp. Proc. Natl. Acad. Sci. U.S.A. 74, 5529–5533 (1977)

  25. Sy, J., Lipmann, F.: Identification of the synthesis of guanosine tetraphosphate (MSI) as an insertion of a pyrophosphoryl group into the 3′-position on guanosine 5′-diphosphate. Proc. Natl. Acad. Sci. U.S.A. 70, 306–309 (1973)

  26. Winslow, R.M.: A consequence of the rel gene during a glucose to lactate downshift in Escherichia coli. J. Biol. Chem. 246, 4872–4877 (1971)

  27. Yang, H-.L., Zubay, G., Urm, E., Reiness, G., Cashel, M.: Effects of guanosine tetraphosphate and β-γ-methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro. Proc. Natl. Acad. Sci. U.S.A. 71, 63–67 (1974)

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Correspondence to A. Ahmed.

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Communicated by G.A. O'Donovan

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Somerville, C.R., Ahmed, A. Mutants of Escherichia coli defective in the degradation of guanosine 5′-triphosphate, 3′-diphosphate (pppGpp). Molec. Gen. Genet. 169, 315–323 (1979). https://doi.org/10.1007/BF00382277

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Keywords

  • Growth Rate
  • Carbon Source
  • Elevated Level
  • Triphosphate
  • Slow Rate