Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of both community- and hospital-acquired urinary tract infections (UTI). The treatment of UPEC-mediated UTI has become more challenging due to the rapid increase in antimicrobial resistance. In particular, the blaNDM-1 gene is of significant concern as it encodes a metallo-β-lactamase that is able to inactivate carbapenem antibiotics, a last line treatment against UPEC and many other Gram-negative pathogens. In UPEC, the blaNDM-1 gene is most commonly encoded on conjugative incA/C plasmids. Hyper-saturated transposon mutagenesis in combination with high throughput transposon directed insertion-site sequencing (TraDIS) was employed to identify genes required for the replication, maintenance and conjugation of pNDM20A, a blaNDM-1-containing incA/C plasmid from an ST648 UPEC strain. The results demonstrated an essential role in the stable maintenance of pNDM20A for the repA replication gene, a putative toxin-antitoxin system and a putative ParAB-like partitioning system. Sequence analysis supports their essential role showing that these genes are 100% conserved in all completely sequenced incA/C plasmids, with the exception of parA (80/81 plasmids, 99%). Twenty-nine genes were identified to be involved in conjugative transfer, including nine novel genes not previously linked to plasmid transfer. The novel genes were further investigated by targeted mutagenesis using λ-Red-mediated homologous recombination and mutations in five genes were confirmed to have significantly lower conjugation rates. Particularly, mutation of the dsbP gene encoding a distinct disulfide isomerase led to approximately 100-fold reduction in mating efficiency, demonstrating a crucial role of disulfide bond formation in incA/C conjugation. Two previously unknown genes were also confirmed to be required for conjugation, one of which is likely a part of a central regulatory circuit of incA/C conjugation. Overall, these findings demonstrate the effective application of TraDIS to study plasmid biology, and provide new insights into the poorly understood incA/C plasmid type.