Nitric Oxide: A Neglected Driver for the Conjugative Transfer of Antibiotic Resistance Genes among Wastewater Microbiota

Abstract

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.

Reductive Stress Boosts the Horizontal Transfer of Plasmid-Borne Antibiotic Resistance Genes: The Neglected Side of the Intracellular Redox Spectrum

“…Plasmid conjugation is a delicate process that requires the well-functioning of the encoding transfer apparatus, which bridges the inner and outer membranes of donor and transports processed DNA to potential recipients . In the case of plasmid RP4, its transfer apparatus consists of three major components: the DNA transfer and replication (Dtr) system, the mating pair formation (Mpf) system, and a coupler connecting these two systems. , Their encoding genes are mainly located in two distinct regions of plasmid RP4 (i.e., Tra1 and Tra2) and are synergistically regulated by global repressor proteins, KorA, KorB, and TrbA (Figure A) . To evaluate the responses of essential transfer functions to DTT exposure, we chose representative genes and assessed mRNA expression levels by RT-PCR.…”

“…To simulate this scenario, a widely applied inducer of reductive stress, that is, dithiothreitol (DTT), a membrane-permeating thiol donor, was applied in this study. The main procedures of conjugation experiments were according to our previous studies. , Briefly, the conjugation medium is the sterile phosphate-buffered saline (PBS, pH 7.4) containing the donor and corresponding recipient, whose concentrations were both around 10 8 CFU/mL. Certain dosages of freshly prepared DTT (1 M) were added into the conjugation mixture at the beginning of mating, resulting in the designed DTT concentrations (i.e., 0, 0.1, 1, and 5 mM).…”

“…Then, strains without/with DTT treatment were harvested by centrifugation (4500 g , 5 min), followed by washing with sterile PBS and freezing with liquid nitrogen. Details about RNA extraction, cDNA library construction, genome sequencing, and subsequent bioinformatic analysis can be seen in our previous study . TPM (transcripts per million reads) was applied to normalize the gene expression levels.…”

“…The concentrated strains were sonicated (20 kHz) for 5 min on an ice-bath, followed by centrifugation (12000 g , 4 °C) for 10 min to obtain the supernatant. High-performance liquid chromatography (1200 HPLC, Agilent) equipped with a Poroshell HPH-C18 column (3.0 × 150 mm, Agilent) as well as a fluorescence detector was applied to quantify glutamine in the supernatant . A pre-column derivatization coupled with gradient elution strategy was used.…”

“…The dissemination of antibiotic resistance genes (ARGs) is becoming a global challenge to the “One Health” concept. , Among diverse pathways for ARG propagation, plasmid-mediated conjugation, which transfers ARGs from the donor to diverse recipients, is the most noteworthy and alarming one because of the following: (1) conjugation is the main route for the horizontal transfer of ARGs; (2) ARGs carried by plasmids not only dominate the environmental resistomes , but also exhibit higher transcriptional activity as well as higher risk rank; , and (3) plasmids are able to transfer cross the interspecies barrier and even domains of life, − which may exacerbate the risk of ARGs spreading via the “pathogen–human” route, , endangering human health. These characteristics indicate that particular attention should be paid to dissect conjugative patterns among environmental bacteria.…”