Resumo

Cupriavidus necator is a non-pathogenic, facultative chemolithoautotrophic beta-proteobacterium, which is considered as a model organism for polyhydroxyalkanoate (PHA) production. In the present work, we have looked for strong promoters with the purpose of optimizing heterologous proteins expression in C. necator . The mrgA promoter from Bacillus subtilis is known to be repressed by Mn(II), Fe(II), Co(II) and Cu(II). Nevertheless, in the gram-negative bacterium Cupriavidus metallidurans, the same promoter with a slight modification (pan) displayed a high constitutive activity. Moreover, a further increase in pan promoter activity could be achieved in the presence of 1 mM of Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II). In order to verify the behavior of the pan promoter in C. necator DSMZ 545, the Escherichia coli lac promoter (plac) and the pan promoter were cloned in a broad host-range plasmid (pBBR1MCS) carrying the coding sequence of the green fluorescent protein (pBB-EGFP). Heterologous expression of EGFP exhibits bright green fluorescence when exposed to blue light without any exogenous substrate or co-factors addition. The resulting recombinant plasmids were used to transform C. necator DSMZ545 and the promoter activity was monitored by flow citometry. The pan promoter was found to provide high constitutive expression of the reporter gene in C. necator in comparison to the plac promoter. Furthermore, the presence of Fe(II) or Mn(II) increased significantly the EGFP expression. Standard epifluorescence microscopy was also used to detect C. necator harboring pBB-lacEGFP or pBB-panEGFP. The fluorescence phenotype was observed to be much weaker for cells transformed with the egfp gene fused to the plac than to the pan promoter. In conclusion, the pan promoter proved to be a powerful tool to express recombinant proteins in C. necator DSMZ 545.