DOI: 10.1128/aem.65.2.813-821.1999. Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual <i>gfp-luxAB</i> Marker System

Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System

10.1128/aem.65.2.813-821.1999

Crossref journal-article

American Society for Microbiology

Applied and Environmental Microbiology (235)

Abstract

ABSTRACT A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.

Bibliography

Unge, A., Tombolini, R., MÃ¸lbak, L., & Jansson, J. K. (1999). Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System. Applied and Environmental Microbiology, 65(2), 813â821.

Authors 4

Annika Unge (first)
Riccardo Tombolini (additional)
Lars Mølbak (additional)
Janet K. Jansson (additional)

References 32 Referenced 157

10.1111/j.1365-294X.1995.tb00276.x
Bakken L. R. Lindahl V. Recovery of bacteria cells from soil Nucleic acids in the environment: methods and applications. Trevors J. T. van Elsas J. D. 1995 9 27 Springer-Verlag Heidelberg Germany (10.1007/978-3-642-79050-8_2)
10.1128/aem.63.11.4543-4551.1997
10.1016/0006-291X(81)91599-0
10.1126/science.8303295
10.1128/aem.60.4.1308-1316.1994
10.1111/j.1574-6968.1993.tb06570.x
10.1128/aem.61.9.3494-3498.1995
10.1016/S0022-2836(83)80284-8
10.1073/pnas.91.26.12501
10.1128/JB.172.11.6557-6567.1990
10.1007/BF00273585
10.1128/aem.54.3.703-711.1988
10.1016/0958-1669(95)80048-4
10.1007/BF02761746
10.1111/j.1365-294X.1995.tb00256.x
10.1111/j.1574-6968.1992.tb05570.x
10.1016/0038-0717(94)90268-2
Möller A. Jansson J. K. Detection of firefly luciferase-tagged bacteria in environmental samples. Methods Mol. Biol. 102 1998 269 283 / Methods Mol. Biol. / Detection of firefly luciferase-tagged bacteria in environmental samples. by Möller A. (1998)
10.1128/AEM.64.2.721-732.1998
10.1126/science.273.5280.1392
10.1002/(SICI)1097-0320(19960201)23:2<91::AID-CYTO1>3.0.CO;2-O
10.1099/13500872-140-1-5
10.1002/cyto.990210402
Sambrook J. Fritsch E. F. Maniatis T. Molecular cloning: a laboratory manual 2nd ed. 1989 Cold Spring Harbor Laboratory Cold Spring Harbor N.Y
10.1016/S0378-1119(97)00197-2
Thompson I. P. Lilley A. K. Ellis R. J. Bramwell P. A. Bailey M. J. Survival, colonization and dispersal of genetically modified Pseudomonas fluorescens SBW25 in the phytosphere of field grown sugar beet. Bio/Technology 13 1995 1493 1497 / Bio/Technology / Survival, colonization and dispersal of genetically modified Pseudomonas fluorescens SBW25 in the phytosphere of field grown sugar beet. by Thompson I. P. (1995)
10.1111/j.1574-6941.1997.tb00352.x
Tombolini R. Jansson J. K. Monitoring of GFP tagged bacterial cells. Methods Mol. Biol. 102 1998 285 298 / Methods Mol. Biol. / Monitoring of GFP tagged bacterial cells. by Tombolini R. (1998)
Unge A. Tombolini R. Möller A. Jansson J. K. Optimization of GFP as a marker for detection of bacteria in environmental samples Bioluminescence and chemiluminescence: molecular reporting with photons. Hastings J. W. Kricka L. J. Stanley P. E. 1997 391 394 John Wiley & Sons Chichester United Kingdom
Unge A. Tombolini R. Davey M. E. de Bruijn F. J. Jansson J. K. GFP as a marker gene Molecular microbial ecology manual. Akkermans A. D. L. van Elsas J. D. de Bruijn F. J. 1998 6.1.13.1 6.1.13.16 Kluwer Academic Publishers Dordrecht The Netherlands
10.1038/nbt1096-1246

Dates

Type	When
Created	5 years, 8 months ago (Dec. 19, 2019, 2:47 p.m.)
Deposited	3 years, 6 months ago (Feb. 23, 2022, 3:04 a.m.)
Indexed	4 weeks, 2 days ago (Aug. 5, 2025, 8:48 a.m.)
Issued	26 years, 7 months ago (Feb. 1, 1999)
Published	26 years, 7 months ago (Feb. 1, 1999)
Published Print	26 years, 7 months ago (Feb. 1, 1999)

Funders 0

None

BibTeX

@article{Unge_1999, title={Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System}, volume={65}, ISSN={1098-5336}, url={http://dx.doi.org/10.1128/aem.65.2.813-821.1999}, DOI={10.1128/aem.65.2.813-821.1999}, number={2}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Unge, Annika and Tombolini, Riccardo and Mølbak, Lars and Jansson, Janet K.}, year={1999}, month=feb, pages={813–821} }

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