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Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli

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Summary

This work describes an approach towards analyzing the regulatory effects of variation of guanosine 3′,5′-bispyrophosphate (ppGpp) basal levels in Escherichia coli during steady state growth. A series of strains was derived by mutating the spoT gene (which encodes the major cellular ppGppase) so as to obtain systematic increments in ppGpp basal levels. These strains differ genetically at the spoT locus and, in some cases, also at the relA locus because of the severity of spoT mutant alleles. Measurements of ppGpp revealed a ten-fold range of basal levels during growth on minimal medium. The empirical relationship between ppGpp concentration and growth rate is a simple linear inverse correlation. Tandem rrnA ribosomal RNA promoters, present on a multicopy plasmid, are shown to be differentially regulated over this range of basal levels. The upstream P 1 promoter activity shows an inverse exponential relation to ppGpp concentration whereas the downstream P 2 promoter is only weakly affected. We conclude that there are systematic regulatory consequences associated with small changes in ppGpp basal levels during steady state growth that probably are part of a continuum with more dramatic effects observed during the stringent response to amino acid deprivation.

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References

  • Adams CW, Hatfield GW (1984) Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J Biol Chem 259:7399–7403

    Google Scholar 

  • Alfoldi L, Stent GS, Clowes RC (1962) The chromosomal site for the RNA control (R.C.) locus in Escherichia coli. J Mol Biol 5:348–355

    Google Scholar 

  • An G, Justesen J, Watson RJ, Friesen JD (1979) Cloning the spoT gene of Escherichia coli: identification of the spoT gene product. J Bacteriol 137:1100–1110

    Google Scholar 

  • Block R, Haseltine WA (1974) In vitro synthesis of ppGpp and pppGpp. In: Nomura M, Tissieres A, Lengyel P (eds) Ribosomes. Cold Spring, Harbor Laboratory, Cold Spring Harbor, NY, pp 747–761

    Google Scholar 

  • Bochner BR, Ames BN (1982) Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem 247:9759–9769

    Google Scholar 

  • Borek EJ, Rockenbach J, Ryan A (1956) Studies on a mutant of E. coli with unbalanced RNA synthesis. J Bacteriol 71:318–323

    Google Scholar 

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

    Google Scholar 

  • Cashel M, Rudd KE (1987) The stringent response. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol 2. ASM Press, Washington, DC, pp 1410–1438

    Google Scholar 

  • Chaloner-Larsson G, Yamazaki H (1975) Relationship between ppGpp levels and rates of protein and RNA synthesis in Escherichia coli. Can J Biochem 54:291–295

    Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Donini P (1972) Turnover of ribosomal RNA during the stringent response in Escherichia coli. J Mol Biol 72:553–569

    Google Scholar 

  • Ellwood M, Nomura M (1980) Deletion of a ribosomal ribonucleic acid operon in Escherichia coli. J Bacteriol 143:1077–1080

    Google Scholar 

  • Fiil NP, Mortensen U, Friesen JD (1976) Genes involved in magic spot metabolism. In: Kjeldgaard N, Maaloe O (eds) Control of ribosome synthesis. Alfred Benzon Symposium IX, Munksgaard, Copenhagen, pp 437–444

    Google Scholar 

  • Fiil NP, Willumsen BM, Friesen JD, von Meyenberg K (1977) 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

    Google Scholar 

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

    Google Scholar 

  • Gausing K (1977) Regulation of ribosome production in Escherichia coli: Synthesis and stability of ribosomal RNA and of ribosomal protein messenger RNA at different growth rates. J Mol Biol 115:335–354

    Google Scholar 

  • Gottesman S (1984) Bacterial regulation: global regulatory networks. Annu Rev Genet 18:415–441

    Google Scholar 

  • Gourse RL, Nomura M (1985) Levels of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA genes in Escherichia coli. J Bacteriol 160:1022–1026

    Google Scholar 

  • Gourse RL, Takebe Y, Sharrock RA, Nomura M (1985) Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc Natl Acad Sci USA 82:1069–1073

    Google Scholar 

  • Gourse RL, deBoer HA, Nomura M (1986) DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell 44:197–205

    Google Scholar 

  • Hecker M, Schroeter A, Mach F (1983) Replication of pBR322 DNA in stringent and relaxed strains of Escherichia coli. Mol Gen Genet 190:355–357

    Google Scholar 

  • Heinemeyer EA, Richter D (1977) In vitro degradation of guanosine tetraphosphate (ppGpp) by an enzyme associated with the ribosomal fraction from Escherichia coli. FEBS Lett 84:357–361

    Google Scholar 

  • Hoopes BC, McClure WR (1987) Strategies in regulation of transcription initiation. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol 2. ASM Press, Washington, DC, pp 1231–1240

    Google Scholar 

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

    Google Scholar 

  • Lagosky PA, Chang FN (1978) The extraction of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation. Biochem Biophys Res Commun 84:1016–1024

    Google Scholar 

  • Lagosky PA, Chang FN (1980) Influence of amino acid starvation on guanosine 5′-diphosphate, 3′-diphosphate basal-level synthesis in Escherichia coli. J Bacteriol 144:499–508

    Google Scholar 

  • Lagosky PA, Chang FN (1981) Correlation between RNA synthesis and basal level guanosine 5′-diphosphate, 3′-diphosphate in relaxed mutants of Escherichia coli. J Biol Chem 256:11651–11656

    Google Scholar 

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

    Google Scholar 

  • Lin-Chao S, Bremer H (1986) Effect of relA function on the replication of plasmid pBR322 in Escherichia coli. Mol Gen Genet 203:150–153

    Google Scholar 

  • Little R, Bremer H (1982) Quantitation of guanosine 5′,3′-bisdiphosphate in extracts from bacterial cells by ion-pair reversephase high-performance liquid chromatography. Anal Biochem 126:381–388

    Google Scholar 

  • Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1112

    Google Scholar 

  • Neidhardt FC (1987) Multigene systems and regulons. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol 2. ASM Press, Washington, DC, pp 1313–1317

    Google Scholar 

  • Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for Enterobacteria. J Bacteriol 119:737–747

    Google Scholar 

  • Nomura M, Gourse R, Baughman G (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem 53:75–117

    Google Scholar 

  • O'Farrell PH (1978) The suppression of defective translation by ppGpp and its role in the stringent response. Cell 14:545–557

    Google Scholar 

  • Pao CC, Gallant J (1979) A new nucleotide involved in the stringent response in Escherichia coli. J Biol Chem 254:688–692

    Google Scholar 

  • Pao CC, Dyess BT (1981) Stringent control of RNA synthesis in the absence of guanosine-5′-diphosphate-3′-diphosphate. J Biol Chem 256:2252–2257

    Google Scholar 

  • Parker J, Watson RW, Friesen JD (1976) A relaxed mutant with an altered ribosomal protein L11. Mol Gen Genet 144:111–113

    Google Scholar 

  • Riggs DL, Mueller RD, Kwan HS, Artz SW (1986) Promoter domain mediates guanosine tetraphosphate activation of the histidine operon. Proc Natl Acad Sci USA 83:9333–9337

    Google Scholar 

  • Rudd KE, Bochner BR, Cashel M, Roth JR (1985) Mutations in the spoT gene of Salmonella typhimurium. J Bacteriol 163:534–542

    Google Scholar 

  • Ryals J, Little R, Bremer H (1982) Control of rRNA and tRNA synthesis in Escherichia coli by guanosine tetraphosphate. J Bacteriol 151:1261–1268

    Google Scholar 

  • Sands MK, Roberts RB (1952) The effects of a tryptophan-histidine deficiency in a mutant of Escherichia coli. J Bacteriol 63:505–511

    Google Scholar 

  • Sarmientos P, Cashel M (1983) Carbon starvation and growth rate dependent regulation of the Escherichia coli ribosomal RNA promoters: differential control of dual promoters. Proc Natl Acad Sci USA 80:7010–7013

    Google Scholar 

  • Sarmientos P, Sylvester JE, Contente S, Cashel M (1983) Differential stringent control of the tandem E. coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids. Cell 32:1337–1346

    Google Scholar 

  • Spadaro A, Spena V, Santonastaso V, Doniui P (1981) Stringency without ppGpp accumulation. Nature 291:256–258

    Google Scholar 

  • Stamminger G, Lazzarini RA (1974) Altered metabolism of the guanosine tetraphosphate, ppGpp, in mutants of E. coli. Cell 1:85–90

    Google Scholar 

  • Stent GS, Brenner (1961) A genetic locus for the regulation of ribonucleic acid synthesis. Proc Natl Acad Sci USA 47:2005–2014

    Google Scholar 

  • Stephens JC, Artz SW, Ames BN (1975) Guanosine 5′-diphosphate, 3′-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino acid deficiency. Proc Natl Acad Sci USA 74:5524–5533

    Google Scholar 

  • Steuber D, Bujard H (1982) Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1:1399–1404

    Google Scholar 

  • Sy J (1977) In vitro degradation of guanosine 5′-diphosphate, 3′-diphosphate. Proc Natl Acad Sci USA 74:5529–5533

    Google Scholar 

  • Tomkins GM (1975) The metabolic code. Science 189:760–763

    Google Scholar 

  • VanBogelen RA, Kelly PM, Neidhardt FC (1987) Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J Bacteriol 169:26–32

    Google Scholar 

  • Yamagishi M, Cole Jr, Nomura M, Studier FW, Dunn JW (1987) Stringent control in Escherichia coli applies also to transcription by T7 RNA polymerase. J Biol Chem 262:3940–3943

    Google Scholar 

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Author notes

  1. Edoardo Sarubbi

    Present address: Centro Ricerche Lepetit, I-21040, Gerenzano (VA), Italy

Authors and Affiliations

  1. Section of Molecular Regulation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, 20892, Bethesda, MD, USA

    Edoardo Sarubbi, Kenneth E. Rudd & Michael Cashel

Authors
  1. Edoardo Sarubbi
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  2. Kenneth E. Rudd
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  3. Michael Cashel
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Communicated by D. Goldfard

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Sarubbi, E., Rudd, K.E. & Cashel, M. Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli . Mol Gen Genet 213, 214–222 (1988). https://doi.org/10.1007/BF00339584

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