The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems

Abstract

The potential of the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria as a visible marker for tracking lactic acid bacteria introduced into complex anaerobic ecosystems was examined. A red-shifted mutant gfp gene was placed downstream of the constitutive Lactococcus lactis P32 promoter and fluorescence resulting from green fluorescent protein (GFP) expression was detected in Escherichia coli and L. lactis. The construct (pKPSPsgfp) was also introduced into Enterococcus faecalis, Streptococcus gordonii and Streptococcus bovis by mobilisation with pVA797. In all cases fluorescence was detectable at the level of individual cells, and isolated colonies. Although oxygen is required for GFP to fluoresce, colonies or cells grown anaerobically were able to fluoresce upon subsequent exposure to air. A combination of selective plating and colony fluorescence allowed a gfp marked strain of E. faecalis to be enumerated after addition to an anaerobic continuous culture of human faecal flora.