Molecular Biology Reports

, Volume 41, Issue 3, pp 1639–1644 | Cite as

Construction of Geobacillus thermoglucosidasius cDNA library and analysis of genes expressed in response to heat stress



Thermophiles exhibit various kinds of molecular mechanisms to survive in extreme environment, but their behavioral responses to long duration stress is poorly understood until date. In the present study, we have prospected for the genes differentially expressed in response to long duration heat stress in thermophilic bacteria. A cDNA library was constructed from Geobacillus thermoglucosidasius grown with a temperature upshift of 10 °C from optimum growth temperature of 45 °C for 16 h. A total of 451 clones from the library were sequenced with accurate base calling that generated 257 high quality sequences with an average read length of 350 bp. We queried our collection of single pass sequences against the NCBI non-redundant database using the BLASTX algorithm and obtained sequences that showed significant similarity (>60 %) with heat shock proteins, metabolic proteins and hypothetical proteins. The expressed sequence tags (ESTs) expressed in response to heat stress were annotated that further commuted a strong interaction network among one another. The ESTs based on the best hits were validated by RT-PCR. Di- and tri-nucleotide repeat motifs were also found to be associated with 17 genes involved in heat shock response, metabolism, transport and transcriptional regulation. The present results provide the novel identification of the putative genes responsible for imparting tolerance to bacteria under heat stress and unveil their role for survival of life in environmental extremes.


Geobacillus thermoglucosidasius Heat stress cDNA library Expressed sequence tags 



This project was supported by grants from National Agricultural Innovation Project, ICAR.

Supplementary material

11033_2013_3011_MOESM1_ESM.docx (417 kb)
Supplementary material 1 (DOCX 416 kb)
11033_2013_3011_MOESM2_ESM.docx (13 kb)
Supplementary material 2 (DOCX 13 kb)


  1. 1.
    Gottesman S, Wickner S, Maurizi MR (1997) Protein quality control: triage by chaperones and proteases. Genes Dev 11:815–823PubMedCrossRefGoogle Scholar
  2. 2.
    Bonham-Smith PC, Kapoor M, Bewley JD (1987) Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis. Plant Physiol 85:575–580PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Jozefczuk S, Klie S, Catchpole G, Szymanski J, Inostroza AC, Steinhauser D, Selbig J, Willmitzer L (2010) Metabolomic and transcriptomic stress response of Escherichia coli. Mol Syst Biol 6:1–15CrossRefGoogle Scholar
  4. 4.
    Feder ME, Hofmann GE (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 61:243–282PubMedCrossRefGoogle Scholar
  5. 5.
    Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV (2001) Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneusgen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillusas the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51:433–446PubMedGoogle Scholar
  6. 6.
    Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF et al (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252(5013):1651–1656PubMedCrossRefGoogle Scholar
  7. 7.
    Gibson UE, Heid CA, Williams PM (1996) A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001PubMedCrossRefGoogle Scholar
  8. 8.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Mol Endocrinol J 25(2):169–193CrossRefGoogle Scholar
  9. 9.
    Babu M, Diaz-Mejia JJ, Vlasblom J, Gagarinova A, Phanse S et al (2011) Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways. PLoS Genet 7(11):e1002377PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Field D, Wills C (1996) Long, polymorphic microsatellites in simple organisms. Proc Royal Acad London B 263(1367):209–215CrossRefGoogle Scholar
  11. 11.
    Toth G, Gaspari Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10(7):967–981PubMedCrossRefGoogle Scholar
  12. 12.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159PubMedCrossRefGoogle Scholar
  13. 13.
    Moriya Y, Itoh M, Okuda S, Yoshizawa A, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35:W182–W185PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Kocabas AM et al (2002) Expression profile of the channel catfish spleen: analysis of genes involved in immune functions. Mar Biotechnol 4:526–536PubMedCrossRefGoogle Scholar
  15. 15.
    Singh A, Sood N, Chauhan UK, Mohindra V (2012) EST-based identification of immune-relevant genes from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758). Gene 502:53–59PubMedCrossRefGoogle Scholar
  16. 16.
    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Livak KJ, Smittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods-Elseveir Sci (USA) 25:402–408Google Scholar
  18. 18.
    Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, Doerks T, Stark M, Muller J, Bork P, Jensen LJ, Mering CV (2011) The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39:D561–D568PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch SR (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res 11:1441–1452PubMedCrossRefGoogle Scholar
  20. 20.
    Zhong X, Luo J, Stevens E Jr (1997) Isolation of full length RNA from a thermophilic Cyanobacterium. Biotechniques 23:904–910Google Scholar
  21. 21.
    Zhang Y, Ohashi N, Rikihisa Y (1998) Cloning of the heat shock protein 70 (HSP70) gene of Ehrlichia sennetsu and differential expression of HSP70 and HSP60 mRNA after temperature upshift. Infect Immun 66:3106–3112PubMedCentralPubMedGoogle Scholar
  22. 22.
    Imboden P, Schoolnik GK (1998) Construction and characterization of a partial Mycobacterium tuberculosis cDNA library of genes expressed at reduced oxygen tension. Gene 213(1–2):107–117PubMedCrossRefGoogle Scholar
  23. 23.
    Persson B, Hedlund J, Jornvall H (2008) The MDR superfamily. Cell Mol Life Sci 65:3879–3894PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Mandelstam J (1963) Protein turnover and its function in economy of cell. Ann N Y Acad Sci 102:621CrossRefGoogle Scholar
  25. 25.
    Willetts NS (1967) Intracellular protein breakdown in non-growing cells of Escherichia coli. Biochem J 103:453PubMedGoogle Scholar
  26. 26.
    Gottesman S, Maurizi MR (1992) Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev 56:592–621PubMedCentralPubMedGoogle Scholar
  27. 27.
    Kanemori M, Nishihara K, Yanagi H, Yura T (1997) Synergistic roles of HslVU and other ATP-dependent proteases in controlling in vivo turnover of σ32 and abnormal proteins in Escherichia coli. Bacteriol J 179:7219–7225Google Scholar
  28. 28.
    Szappanos B, Kovacs K, Szamecz B, Honti F, Costanzo M, Baryshnikova A, Gelius-Dietrich G, Lercher MJ, Jelasity M, Myers CL, Andrews BJ, Boone C, Oliver SG, Pál C, Papp B (2012) An integrated approach to characterize genetic interaction networks in yeast metabolism. Nat Genet 43(7):656–662CrossRefGoogle Scholar
  29. 29.
    Moxon ER, Rainey PB, Nowak MA, Lenski RE (1994) Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr Biol 4:24–33PubMedCrossRefGoogle Scholar
  30. 30.
    Varshney RK, Chabane K, Hendre PS, Aggarwal RK, Graner A (2007) Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys. Plant Sci 173:638–649CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Microbiology Unit, Division of Fish Health ManagementCentral Institute of Freshwater AquacultureBhubaneswarIndia

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