Construction and use of broad host range mercury and arsenite sensor plasmids in the soil bacterium Pseudomonas fluorescens OS8
We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5α and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22°C). In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5α. When using a luciferin concentration (150 μM) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 μM gave a stable reading between about 20 min and 3 h. The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis. The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil. It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability.
KeywordsHeavy Metal Arsenite Pseudomonas Fluorescens Luciferin Graphite Furnace Atomic Absorption Spectrometry
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- 2.Beckman Instruments (1982) Beckman Microtox Operating Manual. Microbics, Carlsbad, CA, ppGoogle Scholar
- 17.Misra TK, Brown NL, Fritzinger D, Pridmore R, Barnes W, Silver S (1984) Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: The beginning of the operon including the regulatory region and the first two structural genes. Proc Natl Acad Sci USA 81:5975–5979PubMedCrossRefGoogle Scholar
- 23.Renzoni A, Zino F, Franchi E (1998) Mercury levels along the food chain and risk for exposed populations. Environ Res Section A 77:68–72Google Scholar
- 25.Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ppGoogle Scholar
- 26.SanFrancisco MJD, Hope CL, Owolabi JB, Tisa LS, Rosen BP (1990) Identification of the metalloregulatory element of plasmid—encoded arsenical resistance operon. Nucl Acid Res 18:619–624Google Scholar
- 27.Sarand I, Haario H, Jørgensen KS, Romantschuk M (2000) Effect of inoculation of a TOL plasmid containing mycorrhizophere bacterium on development of Scots pine seedlings, their mycorrhizophere and the microbial flora in m-toluateamended soil. FEMS Microbiol Ecol 31:127–141PubMedCrossRefGoogle Scholar
- 31.Steinnes E (1990) Mercury. In: Alloway BJ (ed) Heavy Metals in Soils. Blackie and Son Ltd, Glasgow and London, pp 222–236Google Scholar
- 37.Wood KV, DeLuca M (1987) Photographic detection of luminescence in Escherichia coli containg the gene for firefly luciferase. Anal Chem 161:501–507Google Scholar