Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

Abstract

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.

Physiology and transcriptome of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. LH128 after long-term starvation

“…entrance of the cells into a viable but non-culturable (VBNC) state, rather than to cell death. Entrance into a VBNC state upon complete nutrient starvation was also shown for P. fluorescens A506 (Lowder et al, 2000). As for P. fluorescens A506, our conclusion is based on the observations that a high fraction (80 % in the case of LH128) of cells still showed GFP fluorescence after 7 months of starvation.…”

“…other soil bacteria but all were members of the genus Pseudomonas, for example Pseudomonas fluorescens R2f after carbon starvation for 72 days (van Overbeek et al, 1995), P. fluorescens A506 after complete nutrient starvation for 21 days (Lowder et al, 2000), and P. putida KT2442 after nitrogen and carbon starvation for 90 days (Givskov et al, 1994). The number of c.f.u.…”

“…numbers. Since GFP has been proposed as a marker for cell viability (Lowder et al, 2000), GFP fluorescent cells were counted by means of epifluorescence microscopy in a separate but identically set-up experiment.…”

Bioluminescent bacteria: lux genes as environmental biosensors

“…The stability of the GFP protein can be both an advantage and disadvantage for environmental studies. It permits the detection of organisms found under starved conditions or in a viable-but-nonculturable state but can also detect tagged cells regardless of their viability (30). In spite of the limitations associated with bacterial bioluminescent systems, their usefulness as biosensors in laboratory and field conditions have been proven.…”

“…In addition, it is possible to use a biosensor to assess changes in effluent toxicity (8,45) rather than monitoring exact toxin concentrations through measurement of bioluminescence quenching. Although new applications for bacterial whole-cell biosensors are expanding, there is growing recognition of their limitations, thereby stimulating the search for new biomarker systems (30).…”