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Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes

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Journal of Industrial Microbiology & Biotechnology

Abstract

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.

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Acknowledgments

We thank Sidney Kushner for his generosity in providing strains, materials, and advice throughout the course of this work; Estefania Olivar for technical assistance; Scott Hamilton-Brehm and Jim Elkins for C. obsidiansis; Sara Blumer-Schuette and Bob Kelly for C. saccharolyticus DSM 8903, C. hydrothermalis DSM 18901, C. kristjanssonii DSM 12137, C. kronotskyensis DSM 18902, and C. lactoaceticus DSM 9545, and Mike Adams for critical review of the manuscript. This work was supported by a grant to J.W. from the BioEnergy Science Center (DE-PS02-06ER64304) administered by Oak Ridge National Laboratory and by the Office of Biological and Environmental Research (FG02-08ER64690) in the DOE Office of Science.

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Authors and Affiliations

  1. Department of Genetics, University of Georgia, Athens, GA, 30602, USA

    Dae-Hwan Chung, Jennifer R. Huddleston, Joel Farkas & Janet Westpheling

  2. BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA

    Dae-Hwan Chung, Jennifer R. Huddleston, Joel Farkas & Janet Westpheling

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  1. Dae-Hwan Chung
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  2. Jennifer R. Huddleston
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  4. Janet Westpheling
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Correspondence to Janet Westpheling.

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Chung, DH., Huddleston, J.R., Farkas, J. et al. Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes. J Ind Microbiol Biotechnol 38, 1867–1877 (2011). https://doi.org/10.1007/s10295-011-0976-x

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  • DOI: https://doi.org/10.1007/s10295-011-0976-x

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