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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

Journal of Microbiology volume 52, pages 139–147 (2014)Cite this article

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.

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References

  • Bohnert, H.J., Nelson, D.E., and Jensen, R.G. 1995. Adaptations to environmental stresses. Plant Cell 7, 1099–1111.

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • Brown, A.D. 1976. Microbial water stress. Bacteriol. Rev. 40, 803–846.

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    PubMed  Article  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17, 368–376.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • Galinski, E.A. and Trüper, H.G. 1994. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev. 15, 95–108.

    CAS  Article  Google Scholar 

  • 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.

    PubMed  Article  Google Scholar 

  • 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.

    PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    CAS  PubMed  Article  Google Scholar 

  • Lee, S.J. and Gralla, J.D. 2004. Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol. Cell 14, 153–162.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

  • 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 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • Peters, P., Galinski, E.A., and Trüper, H.G. 1990. The biosynthesis of ectoine. FEMS Microbiol. Lett. 71, 157–162.

    CAS  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

    CAS  PubMed  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

  • 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.

    PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    PubMed Central  PubMed  Article  Google Scholar 

  • Ventosa, A., Nieto, J.J., and Oren, A. 1998. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544.

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    PubMed Central  PubMed  Article  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

  • 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.

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

  • 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.

    CAS  PubMed  Article  Google Scholar 

  • Zhang, L.H., Lang, Y.J., and Nagata, S. 2009. Efficient production of ectoine using ectoine-excreting strain. Extremophiles 13, 717–724.

    CAS  PubMed  Article  Google Scholar 

  • 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.

    CAS  Article  Google Scholar 

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Affiliations

  1. Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, 430079, P. R. China

    Derui Zhu, Jian Liu & Deli Liu

  2. Research center of Basic Medical Sciences, Qinghai University Medical College, Xining, 810016, P. R. China

    Derui Zhu, Guoping Shen, Qifu Long & Xiaoxing Wei

  3. Qinghai Academy of Agricultural Forestry Sciences, Qinghai University, Xining, 810016, P. R. China

    Rui Han

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  1. Derui Zhu
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Correspondence to Deli Liu.

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Zhu, D., Liu, J., Han, R. et al. 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. J Microbiol. 52, 139–147 (2014). https://doi.org/10.1007/s12275-014-3389-5

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  • DOI: https://doi.org/10.1007/s12275-014-3389-5

Keywords

  • Halomonas
  • compatible solutes
  • ectoine
  • ectABC gene cluster
  • cloning and expression