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Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana

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Abstract

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.

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Acknowledgments

We are grateful to Dr. Robert M. Blumenthal at the University of Toledo College of Medicine for insightful discussions and critical reading of the manuscript. This work was supported by the BGSU internal funds to ZX.

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Authors and Affiliations

  1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403-0208, USA

    Zhaohui Xu, Dongmei Han, Jingjing Cao & Uksha Saini

  2. Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH, 43403-0208, USA

    Zhaohui Xu

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  1. Zhaohui Xu
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  2. Dongmei Han
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  3. Jingjing Cao
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  4. Uksha Saini
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Correspondence to Zhaohui Xu.

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Communicated by L. Huang.

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Xu, Z., Han, D., Cao, J. et al. Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana . Extremophiles 15, 665 (2011). https://doi.org/10.1007/s00792-011-0397-9

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  • DOI: https://doi.org/10.1007/s00792-011-0397-9

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