Single-molecule fluorescence microscopy is allowing us to study low-abundance proteins inside live bacterial cells for the first time. We are using single-molecule imaging to study DNA polymerases in E. coli, in particular those that carry out translesion DNA synthesis on damage DNA. When induced, TLS polymerases increase mutation rates of cells dramatically and play a demonstrated role in the development of de novo antibiotic resistance mutations.

DNA polymerase V (UmuD’₂C) is tightly regulated in E. coli. It is produced and activated in response to DNA damage, but only after a time delay that allows error-free repair systems a chance to act first. Using single-molecule fluorescence microscopy we have discovered that pol V is also spatially regulated. We find that when first expressed, the UmuC subunit is sequestered on the inner cell membrane, repressing mutagenesis. If damage persists beyond the initial repair stage, UmuC is released gradually into the cytosol and activated to its mutagenic form, pol V Mut (UmuD’₂C-RecA-ATP). Cleavage of the UmuD protein acts not only as a biochemical switch that allows formation of active pol V Mut, but also a spatial switch that releases UmuC from the membrane. This spatial control mechanism, in which the membrane is used like a compartment, is unprecedented in bacteria.

Using single-molecule imaging we can also monitor the binding of pol V to DNA, allowing us to infer relative mutation rates in real time.  We find that pol V is induced and activated by several classes of antibiotics, in particular DNA gyrase inhibitors such as ciprofloxacin. We are developing novel flow cell devices that facilitate the rapid development of de novo resistance mutations, whilst also allowing us to monitor pol V activity. We aim to produce quantitative models that describe the contribution of pol V-induced mutagenesis to the development of antibiotic resistance.