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Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer

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Abstract

Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical and X-ray crystallographic studies. We found that T. elongatus displays natural transformation, and we established a simple and efficient protocol for transferring exogenous DNAs into the organism’s genome. We obtained transformants directly on selective agar plates without having to amplify them prior to plating. We constructed several targeting vectors that enabled us to insert exogenous DNAs into specific sites without disrupting endogenous genes and operons. We also developed a new selectable marker gene for T. elongatus by optimizing the codons of the gene encoding a kanamycin nucleotidyltransferase derived from the thermophilic bacterium Bacillus stearothermophilus. This synthetic gene enabled us to select transformants as kanamycin-resistant colonies on agar plates at 52°C. Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7×103 transformants per μg of DNA. The exogenous DNAs were integrated stably into the targeted sites of the T. elongatus genome via homologous recombination by double crossovers.

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Acknowledgements

We thank Drs. Masahiro Takagi (Osaka University) and Tadayuki Imanaka (Kyoto University) for kind gift of the original kanamycin resistance gene from Bacillus stearothermophilus. We also thank Drs. Shigeru Itoh (Nagoya University), Hideo Shinagawa (Osaka University), and Hideaki Nakashima (Okayama University) for critical reading of this manuscript, and Dr. Miriam Bloom (SciWrite, Jackson, Miss.) for professional editing. This study was supported by grants to M.I. from the following sources: the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Naito Foundation (Tokyo), the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) promoted by BRAIN, Research for the Future: Novel Gene Function Involved in Higher-Order Regulation of Nutrition-Storage in Plants promoted by the Japan Society for the Promotion of Science, Ground-based Research for Space Utilization, promoted by the Japan Space Forum, and the National Project on Protein Structural and Function Analyses, supported by MEXT, and the Promoting Cooperative Research Project, sponsored by the Aichi Science and Technology Foundation. The Division of Biological Science, Graduate School of Science, Nagoya University is supported by a 21st COE grant from MEXT.

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Authors and Affiliations

  1. Center for Gene Research, Nagoya University, Furo, Chikusa, 464-8602, Nagoya, Japan

    K. Onai, M. Morishita & M. Ishiura

  2. Bio-oriented Technology Research Advancement Institution (BRAIN), Nagoya University, Furo, Chikusa, 464-8602, Nagoya, Japan

    K. Onai & M. Ishiura

  3. Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, 292-0818, Kisarazu, Chiba, Japan

    T. Kaneko & S. Tabata

  4. Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, 464-8602, Nagoya, Japan

    M. Ishiura

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  1. K. Onai
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  2. M. Morishita
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  3. T. Kaneko
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  4. S. Tabata
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  5. M. Ishiura
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Correspondence to M. Ishiura.

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Communicated by H. Ikeda

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Onai, K., Morishita, M., Kaneko, T. et al. Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer. Mol Genet Genomics 271, 50–59 (2004). https://doi.org/10.1007/s00438-003-0953-9

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  • DOI: https://doi.org/10.1007/s00438-003-0953-9

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