Journal of Microbiology

, Volume 52, Issue 2, pp 139–147 | Cite as

Identification and characterization of ectoine biosynthesis genes and heterologous expression of the ectABC gene cluster from Halomonas sp. QHL1, a moderately halophilic bacterium isolated from Qinghai Lake

  • Derui Zhu
  • Jian Liu
  • Rui Han
  • Guoping Shen
  • Qifu Long
  • Xiaoxing Wei
  • Deli Liu
Microbial Physiology and Biochemistry

Abstract

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ70-dependent promoter and a δ38-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

Keywords

Halomonas compatible solutes ectoine ectABC gene cluster cloning and expression 

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References

  1. Bohnert, H.J., Nelson, D.E., and Jensen, R.G. 1995. Adaptations to environmental stresses. Plant Cell 7, 1099–1111.PubMedCentralPubMedGoogle Scholar
  2. Bordo, D., Monfort, R.L., Pijning, T., Kalk, K.H., Reizer, J., Saier, M.H., and Dijkstra, B.W. 1998. The three dimensional structure of the nitrogen regulatory protein IIANtr from Escherichia coli. J. Mol. Biol. 279, 245–255.PubMedCrossRefGoogle Scholar
  3. Brown, A.D. 1976. Microbial water stress. Bacteriol. Rev. 40, 803–846.PubMedCentralPubMedGoogle Scholar
  4. Calderón, M.I., Vargas, C., Rojo, F., Iglesias, G.F., Csonka, L.N., Ventosa, A., and Nieto, J.J. 2004. Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T. Microbiology 150, 3051–3063.PubMedCrossRefGoogle Scholar
  5. Cánovas, D., Vargas, C., Calderón, M.I., Ventosa, A., and Nieto, J.J. 1998. Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043. System Appl. Microbiol. 21, 487–497.CrossRefGoogle Scholar
  6. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17, 368–376.PubMedCrossRefGoogle Scholar
  7. Galinski, E.A., Pfeiffer, H.P., and Trüper, H.G. 1985. 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid: a novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur. J. Biochem. 149, 135–139.PubMedCrossRefGoogle Scholar
  8. Galinski, E.A. and Trüper, H.G. 1994. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev. 15, 95–108.CrossRefGoogle Scholar
  9. Graf, R., Anzali, S., Buenger, J., Pfluecker, F., and Driller, H. 2008. The multifunctional role of ectoine as a natural cell protectant. Clin. Dermatol. 26, 326–333.PubMedCrossRefGoogle Scholar
  10. Guzmán, H., Van-Thuoc, D., Martín, J., Hatti-Kaul, R., and Quillaguamán, J. 2009. A process for the production of ectoine and poly (3-hydroxybutyrate) by Halomonas boliviensis. Appl. Microbiol. Biotechnol. 84, 1069–1077.PubMedCrossRefGoogle Scholar
  11. Kuhlmann, A.U. and Bremer, E. 2002. Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus spp. Appl. Environ. Microbiol. 68, 772–783.PubMedCentralPubMedCrossRefGoogle Scholar
  12. Kushner, D.J. and Kamekura, M. 1988. Physiology of halophilic bacteria, pp. 109–138. Halophilic bacteria, CRC Press Inc., Boca Raton, Florida, USA.Google Scholar
  13. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  14. Lee, S.J. and Gralla, J.D. 2004. Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol. Cell 14, 153–162.PubMedCrossRefGoogle Scholar
  15. Lo, C.C., Bonner, A.C., Xie, G., D’Souza, M., and Jensen, R.A. 2009. Cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways. Microbiol. Mol. Biol. Rev. 73, 594–651.PubMedCentralPubMedCrossRefGoogle Scholar
  16. Louis, P. and Galinski, E.A. 1997. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143, 1141–1149.PubMedCrossRefGoogle Scholar
  17. Maskow, T. and Babel, W. 2001. Calorimetrically obtained information about the efficiency of ectoine synthesis from glucose in Halomonas elongata. Biochim. Biophys. Acta. 1528, 60–70.CrossRefGoogle Scholar
  18. Miller, J.H. 1992. A laboratory manual and handbook for Escherichia coli and related bacteria, pp. 194–195. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y., USA.Google Scholar
  19. Mustakhimov, I.I., Reshetnikov, A.S., Glukhov, A.S., Khmelenina, V.N., Kalyuzhnaya, M.G., and Trotsenko, Y.A. 2010. Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. J. Bacteriol. 192, 410–417.PubMedCentralPubMedCrossRefGoogle Scholar
  20. Nagata, S., Maekawa, Y., Ikeuchi, T., Wang, Y.B., and Ishida, A. 2002. Effect of compatible solutes on the respiratory activity and growth of Escherichia coli K-12 under NaCl stress. J. Biosci. Bioeng. 94, 384–389.PubMedGoogle Scholar
  21. Oesterhelt, D. and Stoeckenius, W. 1974. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Meth. Enzymol. 31, 667–678.PubMedCrossRefGoogle Scholar
  22. Ono, H., Okuda, M., Tongpim, S., Imai, K., Shinmyo, A., Sakuda, S., Kaneko, Y., Murooka, Y., and Takano, M. 1998. Accumulation of compatible solutes, ectoine and hydroxyectoine, in a moderate halophile, Halomonas elongata KS3 isolated from dry salty land in Thailand. J. Ferm. Bioeng. 85, 362–368.CrossRefGoogle Scholar
  23. Onraedt, A.E., Walcarius, B.A., Soetaert, W.K., and Vandamme, E.J. 2005. Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis. Biotechnol. Prog. 21, 1206–1212.PubMedCrossRefGoogle Scholar
  24. Pastor, J.M., Salvador, M., Argandoña, M., Bernal, V., Reina-Bueno, M., Csonka, L.N., Iborraa, J.L., Vargasb, C., Nietob, J.J., and Cánovasa, M. 2010. Ectoines in cell stress protection: Uses and biotechnological production. Biotechnol. Adv. 28, 782–801.PubMedCrossRefGoogle Scholar
  25. Peters, P., Galinski, E.A., and Trüper, H.G. 1990. The biosynthesis of ectoine. FEMS Microbiol. Lett. 71, 157–162.CrossRefGoogle Scholar
  26. Reshetnikov, A.S., Khmelenina, V.N., Mustakhimov, I.I., Kalyuzhnaya, M., Lidstrom, M., and Trotsenko, Y.A. 2011. Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria. Extremophiles 15, 653–663.PubMedCrossRefGoogle Scholar
  27. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.PubMedGoogle Scholar
  28. Saum, S.H. and Müller, V. 2008. Growth phase-dependent switch in osmolyte strategy in a moderate halophile: ectoine is a minor osmolyte but major stationary phase solute in Halobacillus halophilus. Environ. Microbiol. 10, 716–726.PubMedCrossRefGoogle Scholar
  29. Schwibbert, K., Marin, S.A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S.C., Klenk, H., Pfeiffer, F., Oesterhelt, D., and Kunte, H.J. 2011. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environ. Microbiol. 13, 1973–1994.PubMedCentralPubMedCrossRefGoogle Scholar
  30. Seip, B., Galinski, E.A., and Kurz, M. 2011. Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster. Appl. Environ. Microbiol. 77, 1368–1374.PubMedCentralPubMedCrossRefGoogle Scholar
  31. Severin, J., Wohlfarth, A., and Galinski, E.A. 1992. The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol. 138, 1629–1638.CrossRefGoogle Scholar
  32. Shikuma, N.J., Davis, K.R., Fong, J.N., and Yildiz, F.H. 2012. The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae. Environ. Microbiol. 15, 1387–1399.PubMedCrossRefGoogle Scholar
  33. Springer, B., Kirschner, P., Rost-Meyer, G., Schröder, K.H., Kroppenstedt, R.M., and Böttger, E.C. 1993. Mycobacterium interjectum, a new species isolated from a patient with chronic lymphadenitis. J. Clin. Microbiol. 31, 3083–3089.PubMedCentralPubMedGoogle Scholar
  34. Tamura, K., Dudley, J., Nei, M., and Kumar, S. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software Version 4.0. Mol. Biol. Evol. 24, 1596–1599.PubMedCrossRefGoogle Scholar
  35. Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.PubMedCentralPubMedCrossRefGoogle Scholar
  36. Vargas, C., Argandona, M., Reina, B.M., Rodríguez, M.J., Fernández, A.C., and Nieto, J.J. 2008. Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance. Saline Syst. 4, 14.PubMedCentralPubMedCrossRefGoogle Scholar
  37. Ventosa, A., Nieto, J.J., and Oren, A. 1998. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544.PubMedCentralPubMedGoogle Scholar
  38. Wang, Z., Ye, S., Li, J., Zheng, B., Bao, M., and Ning, G. 2011. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 11, 109–121.PubMedCentralPubMedCrossRefGoogle Scholar
  39. Wei, Y.H., Yuan, F.W., Chen, W.C., and Chen, S.Y. 2011. Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment. J. Biol. Bioeng. 111, 336–342.CrossRefGoogle Scholar
  40. Woese, C.R., Gutell, R.R., Gupta, R., and Noller, H.F. 1983. Detailed analysis of the higher order structure of 16S-like ribosomal ribonucleic acids. Microbiol. Rev. 47, 621–669.PubMedCentralPubMedGoogle Scholar
  41. Wohlfarth, A., Severin, J., and Galinski, E.A. 1990. The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae. J. Gen. Microbiol. 136, 705–712.CrossRefGoogle Scholar
  42. Zhao, B., Lu, W., Yang, L., Zhang, B., Wang, L., and Yang, S.S. 2006. Cloning and characterization of the genes for biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halobacillus dabanensis D-8T. Curr. Microbiol. 53, 183–188.PubMedCrossRefGoogle Scholar
  43. Zhang, L.H., Lang, Y.J., and Nagata, S. 2009. Efficient production of ectoine using ectoine-excreting strain. Extremophiles 13, 717–724.PubMedCrossRefGoogle Scholar
  44. Zhu, D., Niu, L., Wang, C., and Nagata, S. 2007. Isolation and characterisation of moderately halophilic bacterium Halomonas ventosae DL7 synthesizing ectoine as compatible solute. Ann. Microbiol. 57, 401–406.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Derui Zhu
    • 1
    • 2
  • Jian Liu
    • 1
  • Rui Han
    • 3
  • Guoping Shen
    • 2
  • Qifu Long
    • 2
  • Xiaoxing Wei
    • 2
  • Deli Liu
    • 1
  1. 1.Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life ScienceCentral China Normal UniversityWuhanP. R. China
  2. 2.Research center of Basic Medical SciencesQinghai University Medical CollegeXiningP. R. China
  3. 3.Qinghai Academy of Agricultural Forestry SciencesQinghai UniversityXiningP. R. China

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