Construction of green fluorescent protein (GFP)-marked strains of Bradyrhizobium for ecological studies.
OBJECTIVE
To introduce the gfp gene encoding green fluorescent protein (GFP) into bradyrhizobia for their identification in nodules, soil and carrier-based inoculants.


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.


CONCLUSIONS
Introduction of the gfp gene into Bradyrhizobium provides a simple, specific and cost-effective method of strain identification for ecological studies.
Bhatia
R
R
Department of Microbiology, CCS Haryana Agricultural University, Hisar, India.
Dogra
R C
RC
Sharma
P K
PK
D020369:Bradyrhizobium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002606:Charcoal / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004463:Ecology; D004783:Environmental Microbiology; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics; D010641:Phenotype; D018517:Plant Roots / Q000378:metabolism / Q000382:microbiology*; D013559:Symbiosis
D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002606:Charcoal