The Use of Modified GFP as a Reporter for Metabolic Activity in Pseudomonas putida

Many bacteria are now known to enter into a "viable but nonculturable" (VBNC) state in response to various environmental stresses. In this state, the cells are no longer culturable on routine media, but retain viability and in many cases have been shown to be capable of resuscitating to the metabolically active and culturable state. There have been no simple means of measuring the metabolic activity of cells in the VBNC state. The use of green fluorescent protein (GFP) variants with short half-lives was examined in cells intended for environmental release to examine the potential of GFP as a reporter of metabolic activity. Unlike strains with the native (stable) GFP, Pseudomonas putida strains tagged with unstable GFP rapidly lost GFP fluorescence following exposure to starvation and VBNC-inducing conditions. Our results suggest that tagging cells with the modified GFP provides a method for determining metabolic activity in these cells.