State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai, 200092, China; Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, China. Electronic address: [Email]
Recently, the dissemination of antibiotic resistance genes (ARGs) via plasmid-mediated conjugation has been reported to be facilitated by a series of contaminants. This has highlighted potential challenges to the effective control of this principal mode of horizontal transfer. In the present study, we found that low levels (<0.02 mgN/L) of free nitrous acid (FNA) remarkably inhibited (over 90%) the conjugative transfer of plasmid RP4, a model broad-host-range plasmid, between Escherichia coli. The antimicrobial role of FNA at the applied dosages was firstly ruled out, since no dramatic reductions in viabilities of donor or recipient were observed. Instead, FNA appeared to reduce the available intracellular free Mg2+, which was confirmed to be triggered by the liberation of intracellular Fe2+. These alterations in intracellular Mg2+ and Fe2+ concentrations were found to significantly limit the available energy for conjugative transfer through suppression of glycolysis by decreasing the activities of glycogen phosphorylase and glyceraldehyde-3-phosphate dehydrogenase and also by diverting the glycolytic flux into the pentose phosphate pathway via activation of glucose-6-phosphate dehydrogenase towards the generation of NADPH rather than ATP. Moreover, RP4-encoding genes responsible for DNA transfer and replication (traI, traJ and trfAp), coupling (traG) and mating pair formation (traF and trbBp) were all significantly down-regulated after FNA treatment, indicating that the transfer apparatus required for plasmid processing and delivery was deactivated. By validating the inhibitory effects of FNA on conjugation in real wastewater, this study highlights a promising method for controlling the dissemination of ARGs in systems such as wastewater treatment plants.