Using transposition to introduce eGFP fusions in Sinorhizobium meliloti: A tool to analyze protein localization...

Conventional methods used for the in vivo analysis of subcellular protein localizations and their spatio-temporal dynamics in prokaryotes are based on either the engineering of N(amino)- or C(carboxy)-terminal fusions of fluorescent proteins with the protein of interest, or involved probing internal sites for tag integration. In addition, the use of inducible or constitutive promoters for the expression of fluorescent fusion proteins can lead to overexpression and result in localization artifacts. Here, we describe a method for the synthesis of fluorescent fusion proteins using transposable elements, which can randomly integrate in the internal sections of the protein coding sequence to produce full-length fluorescent fusion proteins expressed at endogenous levels. The established method was used for investigating subcellular localization of proteins in the soil bacterium and plant symbiont Sinorhizobium meliloti. Two constructs for transposition-based insertion of the enhanced green fluorescent protein (eGFP), as well as for in vivo excision of the selection marker for the production of full-length proteins were engineered. Conjugation with pHB14 plasmid and induction of the transposition in S. meliloti produced approx. 3.22×10⁴ transconjugant colonies harboring the fluorescent marker with the transposition efficiency of 0.8%. Sixteen randomly targeted proteins of diverse functions, fused to the eGFP were identified and analyzed in living cells by epifluorescence microscopy, demonstrating the suitability of the novel tool for massive, random production of fluorescent proteins and for following of these proteins with different localizations inside the prokaryotic cell.