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 bio availability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT 21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5 alpha and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degreesC). In op timized conditions the half maximal velocity of reaction was achieved at ac idic pH using a D-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5 alpha. When using a luci ferin concentration (150 mu) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 muM gave a stable reading between about 20 min and 3 h. The ability of t he 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 stra in used is an isolate from pine rhizosphere in oil and heavy metal contamin ated soil. It is also a good humus soil colonizer and is therefore a good c andidate for measuring soil heavy metal bioavailability.