Transposon insertion sequencing reveals T4SS as the major genetic trait for conjugation transfer of multi-drug resistance pEIB202 from Edwardsiella

Yang Liu, Yanan Gao, Xiaohong Liu, Qin Liu, Yuanxing Zhang, Qiyao Wang, Jingfan Xiao

Conjugation is a major type of horizontal transmission of genes that involves transfer of a plasmid into a recipient using specific conjugation machinery, which results in an extended spectrum of bacterial antibiotics resistance. However, there is inadequate knowledge about the regulator and mechanisms that control the conjugation processes, especially in an aquaculture environment where a cocktail of antibiotics may be present. Here, we investigated these with pEIB202, a typical multi-drug resistant IncP plasmid encoding tetracycline, streptomycin, sulfonamide and chloramphenicol resistance in fish pathogen Edwardsiella piscicida strain EIB202. We used transposon insertion sequencing (TIS) to identify genes that are responsible for conjugation transfer of pEIB202. All ten of the plasmid-borne type IV secretion system (T4SS) genes and a putative lipoprotein p007 were identified to play an important role in pEIB202 horizontal transfer. Antibiotics appear to modulate conjugation frequencies by repressing T4SS gene expression. In addition, we identified topA gene, which encodes topoisomerase I, as an inhibitor of pEIB202 transfer. Furthermore, the RNA-seq analysis of the response regulator EsrB encoded on the chromosome also revealed its essential role in facilitating the conjugation by upregulating the T4SS genes. Collectively, our screens unraveled the genetic basis of the conjugation transfer of pEIB202 and the influence of horizontally acquired EsrB on this process. Our results will improve the understanding of the mechanism of plasmid conjugation processes that facilitate dissemination of antibiotic resistance especially in aquaculture industries.