Degradation and deactivation of a plasmid-encoded extracellular antibiotic resistance gene during separate and combined exposures to UV and radicals.

This study investigated the degradation and deactivation of an extracellular ampicillin resistance gene (amp) encoded in plasmid pUC19 during exposure to UV, OH (generated by UV/HO), and combined exposure to UV and OH (and/or SO) using UV/HO and UV/SO. The degradation rates of amp measured by quantitative polymerase chain reaction increased with increasing target amplicon length (192-851 bps). The rate constants for the degradation of pUC19 (2686 bps) were calculated as 0.26 cm/mJ for UV and 1.5 × 10 Ms for OH, based on the degradation rates of amp amplicons and assuming an equal sensitivity of DNA damage across the entire plasmid. DNA repair-proficient Escherichia coli (E. coli) AB1157 strain (wild-type) and its repair-deficient mutants including AB1886 (uvrA), AB2463 (recA), AB2480 (uvrA, recA), and DH5α (recA, endA) were applied as recipient cells in gene transformation assays. Results suggested that the elimination efficiency of transforming activity during UV and OH exposure was dependent on the type of DNA repair genes in recipient E. coli strains. Losses of transforming activity were slower than the degradation of pUC19 by a factor of up to ∼5 (for E. coli DH5α), highlighting the importance of DNA repair in recipient cells. The degradation rates of amp amplicons were much larger (by a factor of ∼4) in UV/HO and UV/SO than UV direct photolysis, indicating the significant contribution of OH and SO to the gene degradation. Not only UV and SO, but also OH contributed to the degradation of amp during UV/SO, which was attributed to the conversion of SO to OH and a 10-fold larger reactivity of OH towards amp as compared to SO. However, the enhanced gene degradation by radicals did not lead to a faster elimination of gene transforming activity during UV/HO and UV/SO, suggesting that UV- and radical-induced DNA damage were not additive in their contributions to losses of gene transforming activity. Wastewater effluent organic matter (EfOM) accelerated the degradation of amp during UV irradiation by means of reactive species production through indirect photolysis reactions, whereas EfOM mainly acted as a radical scavenger during UV/HO and UV/SO treatments.