Hostname: page-component-7dd5485656-pnlb5 Total loading time: 0.001 Render date: 2025-10-30T09:33:21.716Z Has data issue: false hasContentIssue false
  • English
  • Français

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

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

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

Information

Type
Research Article
Information
Environmental Biosafety Research , Volume 3 , Issue 2 , April 2004 , pp. 83 - 90
Copyright
© ISBR, EDP Sciences, 2004

References

Amin-Hanjani, S, Meikle, A, Glover, LA, Prosser, JI, Killham, K (1993) Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil. Mol. Ecol . 2: 47–54 CrossRef
Bloemberg, GV, O’Toole, GA, Lugtenberg, BJJ, Kolter, R (1997) Green fluorescent protein as a marker for Pseudomonas spp. Appl. Environ. Microbiol. 63: 4543–4551
Bokman SH, Ward WW (1981) Renaturation of Aequorea green-fluorescent protein. Biochem. Biophys. Res. Commun. 101: 1372–1380
Chalfie, M, Tu, Y, Euskirchen, G, Ward, WW, Prasher, DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802–805 CrossRef
Cho, J, Kim, S (1999) Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples. J. Microbiol. Methods 36: 227–235 CrossRef
de Weger, LA, Dunbar, P, Mahafee, WF, Lugtenberg, BJJ, Sayler, GS (1991) Use of bioluminescence markers to detect Pseudomonas spp. in the rhizosphere. Appl. Environ. Microbiol. 57: 3641–3644
Duncan, S, Glover, LA, Killham, K, Prosser, JL (1994) Luminescence-based detection of activity of starved and viable but nonculturable bacteria. Appl. Environ. Microbiol. 60: 1308–1316
Grant, FA, Prosser, JI, Glover, LA, Killham, K (1992) Luminescence-based detection of Erwinia carotovora in the soil. Soil Biol. Biochem. 24: 961–967 CrossRef
Jansson J (1998) Marker genes as tags for monitoring microorganisms in nature. An opinion. European Commission Biotechnology Programme, DGXII, Centraltryckeriet AB, Borås, Sweden
Jansson J (1999) Monitoring methods for specific microorganisms and microbial communities in nature. An opinion. European Commission Biotechnology Programme, DGXII, Centraltryckeriet AB, Borås, Sweden
Kogure, K, Simidu, U, Taga, N (1979) A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25: 415–420 CrossRef
Leff, LG, Leff, AA (1996) Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments. Appl. Environ. Microbiol. 62: 3486–3488
Lleò, MM, Pierobon, S, Tafi, MC, Signoreto, C, Canepari, P (2000) mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol. 66: 4564–4567 CrossRef
Lilley, AK, Bailey, MJ (1997) The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environment conditions. Appl. Environ. Microbiol. 63: 1577–1583
Lindemann J, Suslow TV (1987) Competition between ice-nucleation active wild-type and ice nucleation-deficient deletion mutant strains of Pseudomonas syringae and P. fluorescens biovar I and biological control of frost injury on strawberry blossoms. Phytopathology 77: 882–886
Lowder, M, Oliver, JD (2001) The use of modified GFP as a reporter for metabolic activity in Pseudomonas putida. Microb. Ecol. 41: 310–313
Lowder, M, Maraha, N, Unge, A, Jansson, JK, Swiggett, J, Oliver, JD (2000) Effect of starvation and the-viable-but nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl. Environ. Microbiol. 66: 3160–3165 CrossRef
Meikle, A, Killham, K, Prosser, JI, Glover, LA (1992) Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil. FEMS Microbiol. Lett. 78: 217–220 CrossRef
Normander, B, Christensen, BB, Molin, S, Kroer, N (1998) Effect of bacterial distribution and activity on conjugal gene transfer on the phylloplane of the bush bean (Phaseolus vulgaris). Appl. Environ. Microbiol. 64: 1902–1909
Normander, B, Hendriksen, NB, Nybroe, O (1999) Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability and activity in the natural barley rhizosphere. Appl. Environ. Microbiol. 65: 4646–4651
Oliver JD (1993) Formation of viable but nonculturable cells. In Kjelleberg S, ed, Starvation in bacteria, Plenum Press, New York, pp 239–272
Oliver JD (2000a) Problems in detecting dormant (VBNC) cells, and the role of DNA elements in this response. In Jansson JK, van Elsas JD, Bailey M, eds, Tracking Genetically Engineered Microorganisms, Landes Bioscience, Georgetown, TX, pp 1–15
Oliver JD (2000b) Public health significance of viable but nonculturable bacteria. In Colwell RR, Grimes DJ, eds, Non-Culturable Microorganisms in the Environment, Amer. Soc. Microbiol. Press, Washington, DC, pp 277–300
Oliver, JD, McDougald, D, Barrett, T, Glover, LA, Prosser, JI (1995) Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release. FEMS Microbiol. Ecol. 17: 229–238 CrossRef
Ramos, JL, Duque, E, Ramos-Gonzalez M (1991) Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid. Appl. Environ. Microbiol. 57: 260–266
Saux, MF-L, Hervio-Heath, D, Loaec, S, Colwell, RR, Pommepuy, M (2002) Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater. Appl. Environ. Microbiol. 68: 5641–5646 CrossRef
Unge A, Tombolini R, Möller, Jansson JK (1997) Optimization of GFP as a marker for detection of bacteria in environmental samples. In Hastings JW, Kricka LJ, Stanley PE, eds, Bioluminescence and chemiluminescence: molecular reporting with photons, John Wiley & Sons, Sussex, UK, pp 391–394
Unge, A, Tombolini, R, Mølbak, L, Jansson, JK (1999) Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB system. Appl. Environ. Microbiol. 65: 813–821
Yaron S, Matthews K (2002) A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes. J. Appl. Microbiol. 92: 633–640