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The actinobacterium Microbacterium sp. 16SH accepts pBBR1-based pPROBE vectors, forms biofilms, invades roots, and fixes N2 associated with micropropagated sugarcane plants

  • Applied genetics and molecular biotechnology
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Abstract

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTetr plasmids were constructed by cloning the promoter region of the Tet r gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTetr plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. 15 N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N2 associated with the host plants.

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Acknowledgments

We thank for Prof. Steven E. Lindow of the University of California, Berkeley for providing the pPROBE plasmids. This work was supported by the Natural Science Foundation of China (Grant No. 30660085), Zhejiang Provincial Natural Science Foundation of China (Grant No. Y307143), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and Education Department of Zhejiang Province (Grant No. 20070018).

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Author notes

  1. Wei Guo

    Present address: Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore

  2. Xincheng Zhang

    Present address: MOE Key Laboratory of Environment Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, Zijingang Campus, Hangzhou, 310058, China

Authors and Affiliations

  1. Agricultural College, Guangxi University, Daxuedong Road 100, Nanning, 530004, China

    Li Lin, Yongxiu Xing, Xincheng Zhang & Yangrui Li

  2. Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Daxuedong Road 174, Nanning, 530007, China

    Li Lin, Yongxiu Xing & Yangrui Li

  3. Guangxi Key Laboratory of Sugarcane Genetic Improvement, Daxuedong Road 172, Nanning, 530007, China

    Song Li & Yangrui Li

  4. Microbiology Institute, Guangxi Academy of Agricultural Sciences, Daxuedong Road 174, Nanning, 530007, China

    Chunjin Hu

  5. Sugarcane Research Center, Chinese Academy of Agricultural Sciences, Daxuedong Road 172, Nanning, 530007, China

    Song Li & Yangrui Li

  6. Institute of Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou, 310058, China

    Wei Guo, Zhengyi Li & Qianli An

  7. Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Daxuedong Road 174, Nanning, 530007, China

    Yangrui Li

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Lin, L., Guo, W., Xing, Y. et al. The actinobacterium Microbacterium sp. 16SH accepts pBBR1-based pPROBE vectors, forms biofilms, invades roots, and fixes N2 associated with micropropagated sugarcane plants. Appl Microbiol Biotechnol 93, 1185–1195 (2012). https://doi.org/10.1007/s00253-011-3618-3

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