Potential application of a recombinant bacterial strain carrying a groEL promoter as a whole-cell microbial biosensor for detecting bioavailable cadmium

In this study, we investigated the response to heavy metals of recombinant E. coli NovaBlue strains carrying plasmids harboring different Cupriavidus taiwanensis LMG19424-derived groEL promoter fragments fused to the lacZ reporter gene. Four recombinant plasmids (designated G14, G26, G27 and G28) were transformed into E. coli and screened for the ability to indicate the presence of heavy metals on Luria–Bertani (LB) agar. Only the G28 biosensor exhibited reproducible results, with an observed limit of Cd detection of 3 μg/mL. In LB broth, the NovaBlue host strain was able to grow and tolerate in the presence of 50 μg/mL As, Cd, Co, Ni, Pb and Zn but was susceptible to 6.2 μg/mL Cr. The β-galactosidase (β-gal) reporter activity was reduced when the concentration of heavy metals increased, with observed detection limits, as indicated by the blue color, of 25 μg/mL for CdCl2, CdSO4, and CdCO3; 50 μg/mL for As (III) and ZnSO4; and 100 μg/mL for CoCl2 and NiSO4. The specificity of the induction ratio and metal concentration was highest for Cd, with R2 values of 0.9196 and 0.9164 for CdCl2 and CdSO4, respectively. When the ratio of β-gal activity and growth was compared between NovaBlue strains carrying G28 or pETBlue-2 (containing an E. coli promoter), the G28 biosensor exhibited a significantly higher ratio than the strain carrying pETBlue-2 at 0.8–6.3 μg/mL CdCl2. The results demonstrated that the certain concentration range corresponded to Cd induction of G28. The G28 biosensor were immobilized on paper discs and tested using the most toxic and soluble Cd compound (CdCl2) at 0–0.8 μg/mL on LB agar. The G28 biosensor developed a blue color only in the absence of CdCl2. However, when testing soil extracts contaminated with low, medium or high levels of total Cd or those spiked with CdCl2 (up to 40 μg/mL), all the G28 biosensor discs showed a similar growth pattern with a spotted blue color compared with the even blue color of the control disc. Due to the complexity of environmental samples, several factors may interfere with the metal response of the G28 biosensor. However, the G28 biosensor exhibited different behaviors in the presence of specific types and concentrations of metals. Thus, combinations of G28 and more specific promoters should be further investigated to improve the sensitivity and specificity of the groEL promoter for detecting Cd and other heavy metals in contaminated samples.