Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea vi ctoria is being used as a reporter system for gene expression and as a mark er for tracking prokaryotes and eukaryotes. Cells that have been geneticall y altered with the gfp gene produce a protein that fluoresces when it is ex cited by UV light. This unique phenotype allows gth-tagged cells to be spec ifically monitored by nondestructive means, In this study we determined whe ther a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturab le (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrof luorometry were used to measure fluorescence intensity in starved, VBNC, an d dead or dying cells. Results obtained by using how cytometry indicated th at microcosms containing VBNC cells, which were obtained by incubation unde r stress conditions (starvation at 37.5 degrees C), fluoresced at an intens ity that mas at least 80% of the intensity of nonstressed cultures, Similar ly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstres sed cells. VBNC cells remained fluorescent during the entire 6-month incuba tion period. in addition, cells starved at 5 or 30 degrees C remained fluor escent for at least 11 months. Treatment of the cells with UV light or incu bation at 39 or 50 degrees C resulted in a loss of GFP from the cells. Ther e was a strong correlation between cell death and leakage of GFP from the c ells, although the extent of leakage varied depending on the treatment, Mos t dead cells were not GFP fluorescent, but a small proportion of the dead c ells retained some GFP at a lower concentration than the concentration in l ive cells, Our results suggest that gfp-tagged cells remain fluorescent fol lowing starvation and entry into the VBNC state but that fluorescence is lo st when the cells die, presumably because membrane integrity is lost.