Green fluorescent protein-labeled monitoring tool to quantify conjugative plasmid transfer between Gram-positive and Gram-negative bacteria

Abstract

On the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positive Enterococcus faecalis and Gram-negative Escherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown from E. faecalis OG1X into the clinical isolate E. faecalis T9.

Fig 1

The pIP501 oriT region was subcloned into plasmid pJP_relGFP via HindIII. The gfp-oriT cassette was then cloned into plasmid pMSP3535VA via XmaI and XbaI sites, followed by insertion of the t₀ termination sequence via XbaI and XhoI, generating pVA-gfp-oriT.

Fig 2

Dot blot hybridization of nes (A), traE (B), traG (C), and traK (D). PCR products were generated with digoxigenin-labeled pSK41-derived probes. Spot 1, negative control (no DNA template was applied to the PCR); spot 2, PCR product of S. aureus(pSK41) as a positive control, spot 3, PCR product of E. faecalis T9.