Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper (original) (raw)
Related papers
World Journal of Microbiology and Biotechnology, 2012
A number of human activities result in environmental contamination with copper compounds that can cause severe detrimental effects on the ecosystem as well as human health. The physico-chemical methods of metal detection have limitations such as inability to distinguish between total versus bio-available metals and differences in metal uptake in different organisms. The heavy metal resistance-encoding genetic systems of certain bacteria provide critical tools for development of biosensors for these purposes. This study reports a copper biosensor utilizing the cop operon of the heavy metal resistant bacterial isolate, Achromobacter sp. AO22, isolated from a contaminated site in Australia. A section located between the divergently transcribed putative response regulator gene copR and multicopper oxidase gene copA that included a palindromic cop box was identified as a copper-responsive promoter using a lacZ reporter construct, pCOPRP, in E. coli. The expression was found to be enhanced by inclusion of copR. Another engineered strain, AO22(pCOPRP), showed stronger induction, and the lacZ expression in both backgrounds was enhanced significantly (250-400 fold) by copper but minimally by other metals. The construct in Achromobacter sp. AO22 thus has a high potential as biosensor for detecting copper bioavailability (hence potential toxicity) in a soil bacterial background, while the construct in E. coli is ideal for laboratory-based testing.
Genes involved in copper homeostasis in Escherichia coli
Journal of Bacteriology, 2001
Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E. coli.
Escherichia colimechanisms of copper homeostasis in a changing environment
FEMS Microbiology Reviews, 2003
Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.
Harnessing the Potential of Bacillus altitudinis MT422188 for Copper Bioremediation
Frontiers in Microbiology, 2022
The contamination of heavy metals is a cause of environmental concern across the globe, as their increasing levels can pose a significant risk to our natural ecosystems and public health. The present study was aimed to evaluate the ability of a copper (Cu)-resistant bacterium, characterized as Bacillus altitudinis MT422188, to remove Cu from contaminated industrial wastewater. Optimum growth was observed at 37°C, pH 7, and 1 mm phosphate, respectively. Effective concentration 50 (EC 50), minimum inhibitory concentration (MIC), and cross-heavy metal resistance pattern were observed at 5.56 mm, 20 mm, and Ni > Zn > Cr > Pb > Ag > Hg, respectively. Biosorption of Cu by live and dead bacterial cells in its presence and inhibitors 1 and 2 (DNP and DCCD) was suggestive of an ATP-independent efflux system. B. altitudinis MT422188 was also able to remove 73 mg/l and 82 mg/l of Cu at 4th and 8th day intervals from wastewater, respectively. The presence of Cu resulted in increased GR (0.004 ± 0.002 Ug −1 FW), SOD (0.160 ± 0.005 Ug −1 FW), and POX (0.061 ± 0.004 Ug −1 FW) activity. Positive motility (swimming, swarming, twitching) and chemotactic behavior demonstrated Cu as a chemoattractant for the cells. Metallothionein (MT) expression in the presence of Cu was also observed by SDS-PAGE. Adsorption isotherm and pseudo-kinetic-order studies suggested Cu biosorption to follow Freundlich isotherm as well as second-order kinetic model, respectively. Thermodynamic parameters such as Gibbs free energy (∆G°), change in enthalpy (∆H° = 10.431 kJ/mol), and entropy (∆S° = 0.0006 kJ/mol/K) depicted the biosorption process to a feasible, endothermic reaction. Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Energy-Dispersive X-Ray Spectroscopy (EDX) analyses revealed the physiochemical and morphological changes in the bacterial cell after biosorption, indicating interaction of Cu ions with its functional groups. Therefore, these features suggest the potentially effective role of B. altitudinis MT422188 in Cu bioremediation.
Mechanisms of copper homeostasis in bacteria
Frontiers in Cellular and Infection Microbiology, 2013
Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu + transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.
JBIC Journal of Biological Inorganic Chemistry, 2009
A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are 64 Cu, 67 Cu, 187 W, 99 Mo, 69 Zn, 56 Mn, 65 Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native-native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope 64 Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins.
International Journal of Molecular Sciences
The aim of this research was to investigate the bioremediation conditions of copper in synthetic water. In the present study, copper ions accumulation efficiency was determined using various genetically modified strains of Saccharomyces cerevisiae (EBY100, INVSc1, BJ5465, and GRF18), Pichia pastoris (X-33, KM71H), Escherichia coli (XL10 Gold, DH5α, and six types of BL21 (DE3)), and Escherichia coli BL21 (DE3) OverExpress expressing two different peroxidases. Viability tests of yeast and bacterial strains showed that bacteria are viable at copper concentrations up to 2.5 mM and yeasts up to 10 mM. Optical emission spectrometry with inductively coupled plasma analysis showed that the tolerance of bacterial strains on media containing 1 mM copper was lower than the tolerance of yeast strains at the same copper concentration. The E. coli BL21 RIL strain had the best copper accumulation efficiency (4.79 mg/L of culture normalized at an optical density of 1.00), which was 1250 times more ...