Construction of green fluorescent protein (GFP)-marked strains of Bradyrhizobium for ecological studies

Abstract

Aim: To introduce the gfp gene encoding green fluorescent protein (GFP) into bradyrhizobia for their identification in nodules, soil and carrier-based inoculants. Methods and Results: Bradyrhizobium sp. strains M29 and GN7, which nodulate mungbean (Vigna radiata), were conjugated with Escherichia coli S17-1 carrying plasmid EDS 15 (a suicide plasmid carrying a promoterless gfp gene fused with Tn5). The GFP-marked strain expressed the gfp gene from a Bradyrhizobium promoter and gave green fluorescence when observed under an epifluorescent microscope or u.v. transilluminater. All the GFP-marked strains were able to nodulate mungbean and fix nitrogen. The GFP-marked bradyrhizobia were recovered at a frequency of 90-100% and 16-63% from nodules formed under sterilized and unsterilized conditions, respectively. The GFP-marked bradyrhizobia were identified from soil and from charcoal-based inoculants on the basis of green fluorescence. Conclusions: The GFP-marked Bradyrhizobium was successfully identified on the basis of green fluorescence to study its competition and survival in the soil and in charcoal-based inoculants. Significance and Impact of the Study: Introduction of the gfp gene into Bradyrhizobium provides a simple, specific and cost-effective method of strain identification for ecological studies.