Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T.
A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS. Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted. Both markers were very stable during at least 40 bacterial generations without any selective pressure. Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules. The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain. The labelling system described here can be used for the stable fluorescent tagging of S. meliloti strains allowing their detection in biologically complex soil environments.
D002876:Chromosomes, Bacterial; D005453:Fluorescence; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D009586:Nitrogen Fixation; D010641:Phenotype; D016962:Sinorhizobium meliloti / Q000235:genetics*; D012988:Soil Microbiology; D013559:Symbiosis
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins