| Abstract |
Tetracycline (TC)-sensing bioreporters using green fluorescent protein (GFP) were generated in Escherichia coli and solvent-tolerant Acinetobacter oleivorans. A TC-inducible promoter, tetH promoter, and a TetR repressor of the pAST2 plasmid recovered from sludge were used to construct plasmid-based and chromosome-based bioreporters. Two host plasmids with a broad range, pRK415 and pBBR1MCS2, and three randomly chosen chromosomal sites were used to create the reporter strains. Although the copy numbers of the two plasmids in A. oleivorans were greater than those in E. coli, GFP expression from the tetH promoter and growth under TC were significantly higher in E. coli. Thus, the E. coli bioreporter had higher GFP expression driven by TC, and the two plasmids differed in terms of their sensitivity. Our data reflected mosaic evolution of the constructed plasmids, suggesting that the plasmid replication efficiency and the tetH promoter strength differed in the two different hosts. Among the tested TC compounds, doxycycline (DC) was the most effective in promoting GFP expression. qRT-PCR data confirmed that the expression of the tetH promoter in the original pAST2 plasmid produced the most rapid response to DC. E. coli- and A. oleivorans-based plasmid reporters could detect 5 and 30 nM DC, respectively. Insertion of the GFP reporter into different positions of the A. oleivorans chromosome resulted in variations of GFP expression. Our stable A. oleivorans chromosomal bioreporter was functional in the presence of toxic organic solvents. Furthermore, the field test showed that strain A. oleivorans DR1-Tet1 could act as a sensitive bioreporter in activated sludge for DC detection.
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