Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans
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ORIGINAL PAPER
Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans Lawrance Anbu Rajan · Toms C. Joseph · Nirmala Thampuran · Roswin James · Viswanathan Chinnusamy · Kailash C. Bansal
Received: 11 December 2007 / Revised: 13 April 2008 / Accepted: 9 June 2008 / Published online: 16 July 2008 © Springer-Verlag 2008
Abstract Ectoine, a cyclic tetrahydropyrimidine (2methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was conWrmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved. Keywords B. halodurans · Compatible solutes · Salt stress · Ectoine genes · Osmoregulation
Communicated by Ercko Stackebrandt. L. A. Rajan · T. C. Joseph (&) · N. Thampuran · R. James Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Technology, Matsyapuri P.O., Cochin 682029, Kerala, India e-mail: [email protected] V. Chinnusamy Water Technology Centre, Indian Agricultural Research Institute, New Delhi 110012, India K. C. Bansal National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India
Introduction Halophilic eubacteria cope with hyperosmotic shifts by accumulating low molecular weight, highly water-soluble organic solutes, the so-called compatible solutes (Brown 1976). In saline environments, these solutes adjust the intracellular turgor pressure of the microorganism by counteracting the dehydration caused by the eZux of water (Nuno and Costa 2006). The intracellular accumulation of compatible solutes in hyperosmotic shifts is evolutionarily well conserved in bacteria, archaea, and eukaryotes (Burg 1997; Da Costa et al. 1998; Chen and Murata 2002; Roberts 2005). Compatible solutes also play a major role in protection of cells and its components from freezing, desiccation and temperature stress (Welsh 2000; Santos and Da Coasta 2002; Muller et al. 2005). Besides these functions in living systems, the compatible solutes can also be used as enhancers in PCR, cryoprotectants of microorganisms and in cosmetics (Roberts 2005; Lentzen and Schwarz 2006). Among the compatible solutes, ectoine is the most extensively studied and has been found to be distributed in many chemoheterotrophic halophilic eubacteria (Lippert and Galinski 1992; Severin et al. 1992; Galinski and Truper 1994; Louis and Galinski 1997; Ono et al. 1999; Vargas et al. 2006). The biosynthetic pathway for ectoine consists of three steps and has been well characterized in many halophiles (Peters et al. 1990; Ga
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