Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T

Abstract

A single copy of the green fluorescent protein (GFP)-encoding gene <i>gfp</i>-P64L/S65T under the control of the constitutive <i>nptII</i> promoter was introduced in a neutral region of the <i>Sinorhizobium meliloti</i> chromosome, between the genes <i>recA</i> and <i>alaS</i>. Within the same chromosomal region downstream of <i>gfp</i>-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 <i>gfp</i>-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 <i>gfp</i>-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The <i>gfp</i>-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 <i>S. meliloti</i> strains allowing their detection in biologically complex soil environments.