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Archives of Microbiology

, Volume 189, Issue 3, pp 227–238 | Cite as

Molecular characterization of Vibrio cholerae ΔrelA ΔspoT double mutants

  • Bhabatosh Das
  • Rupak K. BhadraEmail author
Original Paper

Abstract

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA + background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp0 strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae.

Keywords

Vibrio cholerae relA spoT (p)ppGpp Stringent response 

Notes

Acknowledgments

BD is grateful to Indian Council of Medical Research (ICMR), Government of India, for a research fellowship. The work was partially supported by the research grant (SMM003) from the Council of Scientific and Industrial Research, Government of India.

References

  1. Arvind L, Koonin EV (1998) The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends Biochem Sci 23:469–472CrossRefGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1989) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  3. Barker MM, Gaal T, Gourse RL (2001) Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP. J Mol Biol 305:689–702PubMedCrossRefGoogle Scholar
  4. Braeken K, Moris M, Daniels R, Vanderleyden J, Michiels J (2006) New horizons for (p)ppGpp in bacterial and plant physiology. Trends Microbiol 14:45–54PubMedCrossRefGoogle Scholar
  5. Bremer H, Dennis PP (1987) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, In: Neidhardt FC (ed) American Society for Microbiology, Washington, pp 1527–1542Google Scholar
  6. Cashel M (1969) The control of ribonucleic acid synthesis in Escherichia coli. J Biol Chem 244:3133–3141PubMedGoogle Scholar
  7. Cashel M, Gentry DR, Hernandes VJ, Vinella D (1996) The stringent response, In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, pp. 1458–1496Google Scholar
  8. Chatterji D, Ojha AK (2001) Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 4:160–165PubMedCrossRefGoogle Scholar
  9. Foti JJ, Persky NS, Ferullo DJ, Lovett ST (2007) Chromosome segregation control by Escherichia coli ObgE GTPase. Mol Microbiol 65:569–581PubMedCrossRefGoogle Scholar
  10. Fujita C, Maeda M, Fujii T, Iwamoto R, Ikehara K (2002) Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein. Biosci Biotechnol Biochem 66:2735–2738PubMedCrossRefGoogle Scholar
  11. Gentry DR, Cashel M (1996) Mutational analysis of the Escherechia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation. Mol Microbiol 19:1373–1384PubMedCrossRefGoogle Scholar
  12. Gropp M, Strausz Y, Gross M, Glaser G (2001) Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. J Bacteriol 183:570–579PubMedCrossRefGoogle Scholar
  13. Haralalka S, Nandi S, Bhadra RK (2003) Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors. J Bacteriol 185:4672–4682PubMedCrossRefGoogle Scholar
  14. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H, Richardson D, Ermolaeva MD, Vamathevan J, Bass S, Qin H, Dragoi I, Sellers P, McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O, Salzberg SL, Smith HO, Colwell RR, Mekalanos JJ, Venter JC, Fraser CM (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477–483PubMedCrossRefGoogle Scholar
  15. Hogg T, Mechold U, Malke H, Cashel M, Hilgenfeld R, (2004) Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response. Cell 117:57–68PubMedCrossRefGoogle Scholar
  16. Jiang M, Sullivan SM, Wout PK, Maddock JR (2007) G-protein control of the ribosome-associated stress response protein SpoT. J Bacteriol 189:6140–6147PubMedCrossRefGoogle Scholar
  17. Josaitis CA, Gaal T, Gourse RL (1995) Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements. Proc Natl Acad Sci USA 92:1117–1121PubMedCrossRefGoogle Scholar
  18. Maiti D, Das B, Saha A, Nandi RK, Nair GB, Bhadra RK (2006) Genetic organization of pre-CTX and CTX prophages in the genome of an environmental Vibrio cholerae non-O1, non-O139 strain. Microbiology 152:3633–3641PubMedCrossRefGoogle Scholar
  19. Mechold U, Malke H (1997) Characterization of the stringent and relaxed responses of Streptococcus equisimilis. J Bacteriol 179:2658–2667PubMedGoogle Scholar
  20. Mittenhuber G (2001) Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins). J Mol Microbiol Biotechnol 3:585–600PubMedGoogle Scholar
  21. Murray K, Bremer H (1996) Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli. J Mol Biol 259:41–57PubMedCrossRefGoogle Scholar
  22. Nandi S, Maiti D, Saha A, Bhadra RK (2003) Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of CTXΦ array and its position in the genome. Microbiology 149:89–97PubMedCrossRefGoogle Scholar
  23. Occhino DA, Wyckoff EE, Henderson DP, Wrona TJ, Payne SM (1998) Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes. Mol Microbiol 29:1493–1507PubMedCrossRefGoogle Scholar
  24. Ostling J, Flardh K, Kjelleberg S (1995) Isolation of a carbon starvation regulatory mutant in marine Vibrio strain. J Bacteriol 177:6978–6982PubMedGoogle Scholar
  25. Raskin DM, Judson N, Mekalanos JJ (2007) Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae. Proc Natl Acad Sci USA 104:4636–4641PubMedCrossRefGoogle Scholar
  26. Rudd KE, Bochner BR, Cashel M, Roth JR (1985) Mutation in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol 163:534–542PubMedGoogle Scholar
  27. Saha A, Haralalka S, Bhadra RK (2004) A naturally occurring point mutation in the 13-mer R repeat affects the oriC function of the large chromosome of Vibrio cholerae O1 classical biotype. Arch Microbiol 182:421–427PubMedCrossRefGoogle Scholar
  28. Schreiber G, Metzger S, Aizenman E, Roza S, Cashel M, Glaser G (1991) Overexpression of the relA gene in Escherichia coli. J Biol Chem 266:3760–3767PubMedGoogle Scholar
  29. Silva AJ, Benitez BA (2006) A Vibrio cholerae relaxed (relA) mutant expresses major virulence factors, exhibits biofilm formation and motility, and colonizes the suckling mouse intestine. J Bacteriol 188:794–800PubMedCrossRefGoogle Scholar
  30. Skorupski K, Taylor RK (1996) Positive selection vectors for allelic exchange. Gene 169:47–52PubMedCrossRefGoogle Scholar
  31. Uzan M, Danchin A (1976) A rapid test for the relA mutation in E. coli. Biochem Biophys Res commun 69:751:758PubMedCrossRefGoogle Scholar
  32. Xiao H, Kalman M, Ikehara K, Zamel S, Glaser G, Cashel M, (1991) Residual guanosine 3′, 5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266:5980PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Infectious Diseases and Immunology DivisionIndian Institute of Chemical BiologyJadavpur, KolkataIndia

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