Single Molecule Imaging for Detecting DNA Damage in Bacteria

Abstract

As antibiotic resistance increases, there is a growing interest in developing new strategies and targets for antibiotics to combat this issue. However, the mechanisms of action for antibiotics, particularly their effect on bacterial DNA, are often not fully understood. This lack of knowledge can hinder the application of DNA damaging agents as antibiotics. In this project, a fluorescence microscopy-based method was developed to analyze bacterial DNA damage caused by stressors such as chemicals and antibiotics. The method relied on enzymes that are part of the base excision repair system for single-strand DNA damage. These enzymes were responsible for removing different types of damage. To visualize the DNA damage, fluorescently labeled nucleotides were incorporated and observed using fluorescence microscopy. This method had not been previously employed to analyze DNA damage in bacteria. During the optimization process, several factors were identified as crucial for ensuring DNA quality and detecting DNA damage. The purity of chemicals used and the quantity of DNA were observed to be important. Additionally, it was discovered that the act of repeated temperature shock of B. subtilis potentially caused DNA damage. Although progress has been made, further studies are required to develop a more reproducible and robust method for detecting DNA damage caused by different antibiotics.