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Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii

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Abstract

The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (ΔpyrF). A markerless gene disruption vector was constructed by incorporating a pyrF-sim R cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon–nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii.

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Acknowledgments

This work was supported by the National Basic Research Program of China “973” Grant 2014CB441503 and National Natural Science Foundation of China NSFC Grant 41376137. The authors would like to thank Tom Santangelo for providing the plasmid pTS535 and Douglas Bartlett for critical reading on the manuscript.

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Authors and Affiliations

  1. Institute of Oceanology, Shanghai Jiao Tong University, Minhang Dongchuan Road 800, Shanghai, 200240, China

    Xuegong Li, Ling Fu, Zhen Li, Xiaopan Ma, Xiang Xiao & Jun Xu

  2. State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Xiang Xiao & Jun Xu

  3. Deep-sea cellular microbiology, Department of Deep-Sea Science, Sanya Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences, Sanya, 572000, China

    Xuegong Li

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  1. Xuegong Li
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  2. Ling Fu
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  3. Zhen Li
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  4. Xiaopan Ma
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  5. Xiang Xiao
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  6. Jun Xu
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Correspondence to Jun Xu.

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Communicated by H. Atomi.

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Li, X., Fu, L., Li, Z. et al. Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii . Extremophiles 19, 59–67 (2015). https://doi.org/10.1007/s00792-014-0705-2

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  • DOI: https://doi.org/10.1007/s00792-014-0705-2

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