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Effects of temperature on detection of plasmidor chromosomally encoded gfp- and lux-labeled Pseudomonas fluorescens in soil

Published online by Cambridge University Press:  15 September 2004

Stephen T. Bunker ,
Tonya C. Bates  and
James D. Oliver
Stephen T. Bunker
Affiliation:
Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
Tonya C. Bates
Affiliation:
Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
James D. Oliver
Affiliation:
Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA

Abstract

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Pseudomonas fluorescens is a normal inhabitant of the soil rhizosphere. The use of genetically altered strains of P. fluorescens in bioremediation has led to the need for effective monitoring of such cells released into the environment. In this study, we present data on the persistence in soil of P. fluorescens harboring gfp (green fluorescent protein) or lux (bioluminescence) genes. Comparisons were made between strains marked chromosomally and strains carrying these markers on a plasmid. Overall effects of plasmid carriage on culturability were also examined. Sterile soil microcosms were inoculated with washed cells to a final concentration of ca. 106 CFU.g–1 and placed at 5, 23, and 35–37 °C. Samples were taken periodically and examined for culturability and viability, using the substrate responsiveness assay. Our results indicated no significant loss of culturability at 5 and 23 °C for a period of over one year. In contrast, cells of P. fluorescens incubated at 35–37 °C entered the viable but nonculturable state within 7 days. All cells labeled with gfp retained fluorescence regardless of culturability, suggesting that the green fluorescent protein can be of value in monitoring the presence of cells following their release to the environment. Because fluorescence was maintained regardless of the cells’ physiological state, this protein may also be an indicator of cell viability.

Keywords

GFPPseudomonas fluorescensviable but nonculturable (VBNC)starvation
Type
Research Article
Information
Environmental Biosafety Research , Volume 3 , Issue 2 , April 2004 , pp. 83 - 90
Copyright
© ISBR, EDP Sciences, 2004

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