A single nucleotide change in the polC DNA polymerase III in Clostridium thermocellum is sufficient to create a hypermutator phenotype

Clostridium thermocellum is a thermophilic, anaerobic, bacterium that natively ferments cellulose to ethanol, and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene. Here we reintroduce this mutation using recently-developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 50-fold increase in the mutation rate. This mutation appears to function by interfering with metal ion coordination in the PHP domain responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments.