Additionally to their primary targets within bacteria, many antibiotics have a secondary killing mechanism that results from the increased production of reactive oxygen species (ROS). ROS damage DNA, both by directly promoting strand breakage and by oxidising the nucleotide pool, leading to increased incorporation of oxidised nucleotides. Ultimately, ROS-mediated DNA damage potentiates the effectiveness of a wide variety of antibiotics, a phenomenon known as the common killing mechanism.
In bacteria, DNA damage induces the SOS response which upregulates specialised DNA polymerases. These polymerases can catalyse DNA synthesis over lesions on the template DNA (translesion DNA synthesis, TLS) but induce mutations much more frequently than the replicative polymerase. Beyond this, one of the three TLS polymerases in Escherichia coli, DNA polymerase IV (pol IV), is thought to incorporate oxidised nucleotides into the DNA. This activity is proposed to promote strand breakage and thus contribute to killing.
To gain further insight we are using single-molecule fluorescence microscopy to visualise pol IV during antibiotic treatments. Upon treatment with the DNA gyrase inhibitor ciprofloxacin, punctate foci form as the polymerase binds to DNA. Most of these foci form away from replisomes. Interestingly, the addition of reactive oxygen scavengers during ciprofloxacin treatment eliminates the majority of the non-replisomal pol IV foci. Different effects are observed after trimethoprim treatment, a drug which depletes the nucleotide pool. There, pol IV predominantly forms foci at replisomes which persist for several seconds. Contrary to the ciprofloxacin result, reactive oxygen scavengers during trimethoprim treatment have little effect on the colocalisation of pol IV with replisomes. Instead the lifetimes of pol IV foci are greatly reduced. These observations indicate that pol IV activity is strongly dependent on ROS-mediated changes to the DNA and/or the nucleotide pool and are consistent with the proposed role for pol IV in the common killing mechanism.