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Recombineering-Mediated Sinorhizobium meliloti Rm1021 Gene Deletion

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Abstract

Sinorhizobium meliloti Rm1021 (S. meliloti Rm1021) is a Gram-negative, soil-dwelling α-proteobacterium which serves as a model microorganism for the studies of symbiotic nitrogen fixation. The S. meliloti Rm1021 genome consists of one chromosome and two megaplasmids, pSymA and pSymB. Gene deletion is an essential tool for the elucidation of gene function and generation of mutants with improved properties. However, only two gene deletion methods, counterselectable marker sacB-based and FLP/FRT, Cre/loxP site-specific recombination, have been reported for S. meliloti Rm1021 gene deletion. Both methods require time-consuming and tedious gene cloning and conjugation steps. Herein, a λ Red recombineering-mediated gene deletion strategy is reported. The mutant was obtained via electroporating overlap-extension PCR-generated linear targeting DNA into Red-proficient cells. One gene each from the S. meliloti Rm1021 chromosome, megaplasmid SymA and pSymB was deleted, with deletion efficiency up to 100%. The straightforward and highly efficient recombineering procedure holds the promise to be a general gene manipulation method for S. meliloti Rm1021.

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Acknowledgements

We thank Dr. Sharon Long (Stanford University, USA), Dr. Stephen Farrand (University of Illinois at Urbana-Champaign, USA), Dr. Alan Collmer (Cornell University, USA), Dr. Barry Wanner (Purdue University, USA), and Dr. Michael Kovach (Louisiana State University, USA) for the generous gifts of strains and plasmids used in this research.

Funding

This research was generously supported by the National Natural Science Foundation of China (NSFC 82073742).

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Authors and Affiliations

  1. Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, No. 1 Wenyuan Rd., Xixia District, Nanjing, 210023, Jiangsu, People’s Republic of China

    Jun Yang, Qiong Zhang, Guoyi Zhang & Guangdong Shang

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  1. Jun Yang
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  2. Qiong Zhang
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  3. Guoyi Zhang
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  4. Guangdong Shang
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Contributions

GS conceived and designed the study. JY, QZ, and GZ conducted the experiments. All authors contributed to data interpretation and manuscript preparation. All authors read and approved final version of the manuscript.

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Correspondence to Guangdong Shang.

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Yang, J., Zhang, Q., Zhang, G. et al. Recombineering-Mediated Sinorhizobium meliloti Rm1021 Gene Deletion. Curr Microbiol 80, 76 (2023). https://doi.org/10.1007/s00284-023-03188-1

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