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

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-pTet(r) 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-pTet(r) 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. 15N 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 N-2 associated with the host plants.