Construction of a nrdA::luxCDABE Fusion and Its Use in Escherichia coli as a DNA Damage Biosensor

Abstract

The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.

Tissue-Specific Salmonella Typhimurium Gene Expression during Persistence in Pigs

“…The ribonucleoside diphosphate reductase encoding nrdB is part of the nrdAB operon that is involved in DNA replication and repair by reducing ribonucleotides to deoxyribonucleotides. The E. coli nrdA promoter was found to respond strongly to DNA damaging chemicals and is suggested to function as a biosensor for DNA damage [36] . It is possible that this operon has a comparable function in Salmonella Typhimurium and that the operon is expressed upon DNA damage induced in the hostile porcine environment.…”

Ribonucleoside Diphosphate Reductase Plays an Important Role in Patulin Degradation by Enterobacter cloacae subsp. dissolvens

“…31,32 NrdA is involved in DNA biosynthesis and converts ribonucleotides to deoxyribonucleotides, acting as a DNA damage biosensor. 33 To verify the potential involvement of the two candidate proteins in patulin biodegradation, ahpC and nrdA knockout mutants of TT-09 were constructed via allele exchange, and their ability to degrade patulin was determined. Results demonstrated that ΔnrdA mutants completely lost the ability to degrade patulin, while ΔahpC mutants did not alter the ability of the mutant strain of TT-09 cells to degrade patulin (Figure 6C,D).…”

“…Of the two proteins, AhpC belongs to a large family of peroxidases and is typically induced by oxidative stress to protect cells against various abiotic stresses, such as heat, salt, pesticides (carbofuran), heavy metals, and UV-B. , NrdA is involved in DNA biosynthesis and converts ribonucleotides to deoxyribonucleotides, acting as a DNA damage biosensor . To verify the potential involvement of the two candidate proteins in patulin biodegradation, ahpC and nrdA knockout mutants of TT-09 were constructed via allele exchange, and their ability to degrade patulin was determined.…”

Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources?

“…Hwang et al [ 20 ] constructed strain BBT NrdA , which is an E. coli based lux strain that has the nrdA gene as the promoter. The nrdA gene is activated in DNA synthesis, but is not regulated by the SOS response.…”