Figure 1.
Schematic diagram of the plasmids engineered for this work.
(A) Plasmid pCHRGFP1: pPROBE-NT containing the chr promoter and chrB gene upstream of gfp. (B) Plasmid pCHRGFP2: pCHRGFP1 with an additional gentamicin resistance gene cloned into mob gene. Arrows indicate the direction of transcription. The restriction enzymes indicated were those used in this work.
Figure 2.
Time-dependent induction of pCHRGFP1 E. coli (A) and pCHRGFP2 O. tritici (B) reporters with chromate.
Cells were exposed to increasing concentrations of chromate, 0 nM (⋄), 10 nM (▪), 100 nM (▵), 1 µM (x), 5 µM (•) 10 µM (○), 25 µM (▴), 50 µM (□) e 100 µM (). The data are the mean values of three experiments with the standard deviations.
Figure 3.
Chromate-fluorescence response of pCHRGFP1 E. coli (A) and pCHRGFP2 O. tritici (B) reporters measured after different exposure periods.
0 h chromate incubation (⧫), 1 h chromate incubation (▪), 2 h chromate incubation (○), 3 h chromate incubation (x), 4 h chromate incubation (•), 5 h chromate incubation (□). The data are the mean values of three experiments with the standard deviations.
Table 1.
Induction of fluorescence in both bioreporters by several compounds.
Figure 4.
Selectivity of the pCHRGFP1 E. coli and pCHRGFP2 O. tritici reporters to different compounds.
Cells were treated individually with sulphate and phosphate in concentration of 1 mM and with arsenate and chromate in concentration of 0.1 mM or mixtures of the several compounds for 5 hours. Control refers to no-metal treatment reporter bacteria. The data are the mean values of three experiments with the standard deviations.
Figure 5.
Effect of growth phase and culture medium on efficiency of pCHRGFP1 E. coli reporter.
Exponential cells grown in TMM medium (⧫) or LB medium (□) and stationary cells grown in TMM medium (▴) or LB medium (○) were incubated with the indicated chromate concentrations for 5 hours. The data are the mean values of three experiments with the standard deviations.
Figure 6.
GFP reporter detection of chromate-contaminated environmental water samples.
A) Cultures of fresh exponential-growing cells or cryo-preserved exponential cells of pCHRGFP1 E. coli and pCHRGFP2 O. tritici reporters were exposed to Mondego river water (white bars), Mondego river water spiked with 1 µM chromate (black bars) and 10 µM chromate (dashed bars). The fluorescence was assayed after 5 hours of incubation. The data are the mean values of three experiments with the standard deviations. B) Exponential grown cells of pCHRGFP1 E. coli (upper panel) and of pCHRGFP2 O. tritici (lower panel) were incubated overnight to Mondego river water spiked with 10 µM of chromate, after which green fluorescent protein expression in bacterial cells was visualized by using epifluorescence microscope. Magnification, ×1000.
Figure 7.
Fluorescence of bioreporter strains exposed to different chromate-contaminated environmental waters.
Cultures of exponential-growing cells of pCHRGFP1 E. coli and pCHRGFP2 O. tritici were exposed to chromate spiked stream waters of Malhou, Braças and Carritos. These samples were contaminated with 1 µM chromate (black bars) and 10µM chromate (dashed bars). The fluorescence was assayed after 5 hours of incubation. The data are the mean values of three experiments with the standard deviations.
Table 2.
Chemical analyses of environmental water samples.