A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation.
Quadros OF. Construção de sistemas bacterianos para a detecção de metais pesados em amostras ambientais. [tese (Doutorado em Biotecnologia)]. São Paulo (Brasil): Instituto de Ciências Biomédicas da Universidade de São Paulo; 2011. Para a obtenção de biossensores bacterianos para os metais mercúrio, arsênio e chumbo, fragmentos dos operons mer, ars e pbr foram amplificados por PCR, a partir do DNA da bactéria Cupriavidus metallidurans CH34. Estes fragmentos de DNA foram inseridos à montante do gene EGFP, no plasmídeo pBB-EGFP, obtendo-se os plasmídeos pGHg, pGAs e pGPb, que foram clonados em C. metallidurans CH34 e Escherichia coli DH5α. As linhagens recombinantes foram incubadas em diferentes concentrações dos metais e analisadas quanto à emissão de fluorescência empregando-se microscopia e citometria de fluxo. As linhagens recombinantes C. metallidurans CH34 / pGHg e E. coli DH5α / pGHg, C. metallidurans CH34 / pGAs e C. metallidurans CH34 / pGPb permitiram a determinação de diferentes concentrações de mercúrio, arsênio e chumbo, respectivamente. Os resultados obtidos indicam forte potencial de aplicação para rápidos diagnósticos de águas contaminadas com estes metais pesados tóxicos. Palavras-chave: Bactérias. Bioluminescência. Biotecnologia. Meio ambiente. Metais pesados. Regulação gênica.
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