Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter

Background A bacterial biosensor refers to genetically engineered bacteria that produces an assessable signal in the presence of a physical or chemical agent in the environment. Methods We designed and evaluated a bacterial biosensor expressing a luciferase-reporting gene that is controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously Ralstonia metallidurans ) CH34 and pI258 plasmids of Staphylococcus aureus , respectively, for detection of heavy metals. In the present study, we produced biosensor plasmids designated pGL3-luc/pbr -biosensor and pGL3-luc/cad -biosensor, which were based on the expression of luc+ under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans . Results: We found that the biodegradable pGL3-luc/pbr -biosensor could measure lead concentrations between 1-100 μM in the presence of other metals such as zinc, cadmium, tin and nickel, in the presence of which gene expression of the reporter was not observed. The pGL3-luc/cad -biosensor was able to detect lead concentrations between 10 nM to 10 μM. Conclusions: This biosensor was found to be a specific sensor for identifying lead ions in both environmental and biological samples.

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

ABSTRACT Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se0). We have studied the kinetics of selenite (SeIV) and selenate (SeVI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se0 and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se0. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se0 was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. SeIV was detected as a transient species in the first 12 h after selenate introduction, Se0 also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.

The Chromosomally Encoded Cation Diffusion Facilitator Proteins DmeF and FieF from Wautersia metallidurans CH34 Are Transporters of Broad Metal Specificity

ABSTRACT Genomic sequencing of the β-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family. One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter. The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W. metallidurans or Escherichia coli. The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences. DmeF plays the central role in cobalt homeostasis in W. metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II). This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup.

Characteristics of Zinc Transport by Two Bacterial Cation Diffusion Facilitators from Ralstonia metallidurans CH34 and Escherichia coli

ABSTRACT CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 μM and a V max of 0.57 nmol of Zn2+ min−1 mg of protein−1. Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.