Effect of Plasmids on Entry into the VBNC State

To make the situation potentially worse, it now appears that our knowledge of what induces the VBNC state in various bacteria must be re-examined, taking into account whether or not the cells under investigation harbor extrachromosomal plasmids. We have reported10 that Pseudomonas fluorescens cells enter the VBNC state when incubated in sterile river water at 37°C, but not at 5° or 25°C (Fig. 1.8A). In rather remarkable contrast, when this same strain harbored a plasmid (pFAC510), the cells entered the VBNC state at 5°C, but not at 37°C (Fig. 1.8B). The same result was seen for P. syringae, with and without an entirely different plasmid (pQF70/19H). Most recently, we have observed similarly dramatic plasmid effects when P. fluorescens is incubated in soil microcosms (Fig. 1.9).

The fact that plasmids may have such a dramatic affect on culturability has obvious and significant implications for the monitoring of GMOs intended for environmental release. Indeed, the plasmid-induced "temperature reversal" observed in our studies suggests that such cells must be thoroughly characterized as to their plasmid content, with the realization that plasmid size and copy number may affect the temperature at which they enter the VBNC state.

Fig. 1.7. Randomly amplified polymorphic (RAPD) DNA analysis of V. vulnificus cells induced into the VBNC state by incubation at 5°C. Lanes 1, 5, 9, 11, and 12 contain a 123 bp ladder. Lanes 2-4 contain cells entering the VBNC state at 0, 1, and 2 days; lanes 6-8 contain cells entering the VBNC state at 3, 4, and 5 days; lane 10 contains cells entering the VBNC state at 7 days. Reprinted with permission from: Warner JM, Oliver JD. Appl Environ Microbiol 1998; 64:3025-3028.

Fig. 1.7. Randomly amplified polymorphic (RAPD) DNA analysis of V. vulnificus cells induced into the VBNC state by incubation at 5°C. Lanes 1, 5, 9, 11, and 12 contain a 123 bp ladder. Lanes 2-4 contain cells entering the VBNC state at 0, 1, and 2 days; lanes 6-8 contain cells entering the VBNC state at 3, 4, and 5 days; lane 10 contains cells entering the VBNC state at 7 days. Reprinted with permission from: Warner JM, Oliver JD. Appl Environ Microbiol 1998; 64:3025-3028.

Fig. 1.8. (opposite) Effect of plasmid FAC510 carriage in P. fluorescens on culturability in river water at different temperatures. The same P. fluorescens strain was without the plasmid (•, "parent"), with the plasmid (O, "plasmid"), or with the entire plasmid inserted into the chromosome (A, "chromosome"). Cells incubated at 5°C (A) or at 37°C (B). Plate counts were performed on L agar. Reprinted with permission from: Oliver JD, McDougald D, Barrett T et al. FEMS Microbiol Ecol 1995; 17:229-238.

TIME (days)
TIME (days)
Fig. 1.9. P. fluorescens placed into soil microcosms at 5°(B), 25°(0), and 37°C (•). Shown are plate counts for each temperature. S. Bunker and J.D. Oliver (unpublished).

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