A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans.
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.
Ribeiro-dos-Santos
Gabriela
G
Instituto de Ciências Biomédicas, Centro de Pesquisas em Biotecnologia, Universidade de São Paulo, São Paulo, SP, Brazil.
Biondo
Ronaldo
R
Quadros
Oeber de Freitas
Ode F
Vicente
Elisabete José
EJ
Schenberg
Ana Clara Guerrini
AC
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D049921:Cupriavidus / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005434:Flow Cytometry; D005453:Fluorescence; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007763:Lac Operon; D008670:Metals / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008670:Metals; C095506:MrgA protein, Bacillus subtilis; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins