A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype

Lanahan, Anthony; Zakowicz, Kamila; Tian, Liang; Olson, Daniel G.; Lynd, Lee R.

doi:10.1128/aem.01531-21

Title: A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype

Journal Article · 10 January 2022 · Applied and Environmental Microbiology

DOI: https://doi.org/10.1128/aem.01531-21 · OSTI ID:1845126

Lanahan, Anthony ^[1]; Zakowicz, Kamila ^[2]; Tian, Liang ^[3];

^[3]; Lynd, Lee R. ^[3]

Dartmouth College, Hanover, NH (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Dartmouth College
Dartmouth College, Hanover, NH (United States)
Dartmouth College, Hanover, NH (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

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, which encodes a DNA polymerase III enzyme. Here, we reintroduced 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 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. Here, the ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments.

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Research Organization:: Dartmouth College, Hanover, NH (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1845126

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 1 Vol. 88; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Title: A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype

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