Conformation-dependent UV inactivation efficiency of a conjugative, multi-drug resistant plasmid

Abstract

A conjugative, multi-drug-resistant plasmid was irradiated in-vivo and in-vitro with a 265-nm UV-light emitting diode (UV-LED) to investigate the gene inactivation efficiency of a plasmid deoxyribonucleic acid (pDNA) carrying DNA transfer and replication genes. The clinical-isolate 60 kb RP4 plasmid of the IncPα group containing the traG gene, was irradiated intracellularly in E. coli DH5α and extracellularly in a water medium at pH 8.5. Real-time quantitative polymerase chain reaction (RT-qPCR) measurements of the UV-fluence response gene inactivation rate constants revealed a decreasing pattern via a pseudo-1st-order inactivation kinetics in all forms examined. Our findings showed that the intracellular-supercoiled conformation, with k = 6.1 × 10-3 cm2/mJ, has the lowest UV susceptibility (lowest inactivation rate). UV absorbance measurements and a computational approach showed that the host's RNA provides the photo-shielding, demonstrating this high UV resistance. When UV exposure was measured in-vitro, the condensed DNA exhibited a self-shielding effect over supercoiled and denatured DNA due to the hypochromic-hyperchromic effects. This study has shown that large-sized conjugative plasmids with conformation-dependent UV/UV-LED-based gene inactivation play a significant role in preventing the spread of antibiotic resistance.