The dissemination of plasmid-borne
antibiotic resistance genes
(ARGs) in wastewater is becoming an urgent concern. Previous studies
mainly focused on the effects of coexisting contaminants on plasmid
conjugation, but ignored the potential contribution of some byproducts
inevitably released from wastewater treatment processes. Herein, we
demonstrate for the first time that nitric oxide (NO), an intermediate
of the wastewater nitrogen cycle, can significantly boost the conjugative
transfer of plasmid RP4 from Escherichia coli K12 to different recipients (E. coli HB101, Salmonella typhimurium, and
wastewater microbiota). Phenotypic and genotypic tests confirmed that
NO-induced promotion was not attributed to the SOS response, a well-recognized
driver for horizontal gene transfer. Instead, NO exposure increased
the outer membrane permeability of both the donor and recipient by
inhibiting the expression of key genes involved in lipopolysaccharide
biosynthesis (such as waaJ), thereby lowering the
membrane barrier for conjugation. On the other hand, NO exposure not
only resulted in the accumulation of intracellular tryptophan but
also triggered the deficiency of intracellular methionine, both of
which were validated to play key roles in regulating the global regulatory
genes (korA, korB, and trbA) of plasmid RP4, activating its encoding transfer apparatus (represented
by trfAp and trbBp). Overall, our
findings highlighted the risks of NO in spreading ARGs among wastewater
microbiota and updated the regulation mechanism of plasmid conjugation.
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