Stimulants and donors promote megaplasmid pND6-2 horizontal gene transfer in activated sludge

The activated sludge process is characterized by high microbial density and diversity, both of which facilitate antibiotic resistance gene transfer. Many studies have suggested that antibiotic and non-antibiotic drugs at sub-inhibitory concentrations are major inducers of conjugative gene transfer. The self-transmissible plasmid pND6-2 is one of the endogenous plasmids harbored in Pseudomonas putida ND6, which can trigger the transfer of another co-occurring naphthalene-degrading plasmid pND6-1. Therefore, to illustrate the potential influence of stimulants on conjugative transfer of pND6-2, we evaluated the effects of four antibiotics (ampicillin, gentamycin, kanamycin, and tetracycline) and naphthalene, on the conjugal transfer efficiency of pND6-2 by filter-mating experiment. Our findings demon-strated that all stimulants within an optimal dose promoted conjugative transfer of pND6-2 from Pseudomonas putida GKND6 to P. putida KT2440, with tetracycline being the most ef-fective (100 mu WL and 10 mu g/L), as it enhanced pND6-2-mediated intra-genera transfer by approximately one hundred-fold. Subsequently, seven AS reactors were constructed with the addition of donors and different stimulants to further elucidate the conjugative behav-ior of pND6-2 in natural environment. The stimulants positively affected the conjugal pro-cess of pND6-2, while donors reshaped the host abundance in the sludge. This was likely because stimulant addition enhanced the expression levels of conjugation transfer-related genes. Furthermore, Blastocatella and Chitinimonas were identified as the potential receptors of plasmid pND6-2, which was not affected by donor types. These findings demonstrate the positive role of sub-inhibitory stimulant treatment on pND6-2 conjugal transfer and the function of donors in re-shaping the host spectrum of pND6-2. (c) 2022 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.