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Updated: Oct 11, 2025
Measuring Microbial Mutation Rates with the Fluctuation Assay
Published on: November 28, 2019
Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay
Mei-Chen Liu1, Sebastian D Fugmann2,3,4,5
1Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Abstract:
Mutator enzymes alter the nucleotide sequences of DNA or RNA molecules; immune systems utilize them to destroy the integrity of pathogen genomes and to optimize immune mediators of the host. Their dysregulation has been linked to tumorigenesis in various tissues. Defining and comparing the activities of such mutator enzymes requires a robust versatile assay that is independent of their biological context as in vivo mutation rates are typically low. Here we provide detailed protocols for two widely used E. coli-based approaches that detect the activities of ectopically expressed cytidine deaminases on two distinct reporter genes: an extrachromosomal kanamycin-resistance gene or an endogenous chromosomal substrate, the rpoB gene-encoding RNA polymerase. The generation of mutations is in both cases measured in a colony formation assay. With appropriate modifications, these assays can be extended to study other mutator enzymes.
Insights
This study presents new E. coli-based assays to measure mutator enzyme activity, crucial for understanding DNA/RNA sequence alteration and its link to cancer. These versatile methods aid in defining enzyme functions outside their natural biological context.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Mutator enzymes modify DNA/RNA sequences, playing roles in immunity and host optimization.
- Dysregulation of these enzymes is implicated in various cancers.
- Accurate measurement of mutator enzyme activity requires context-independent assays due to low in vivo mutation rates.
Purpose of the Study:
- To provide detailed protocols for two E. coli-based assays to detect cytidine deaminase activity.
- To enable the study of mutator enzymes independent of their biological context.
- To offer a versatile platform for analyzing enzyme-driven mutations.
Main Methods:
- Utilized E. coli as a host system for ectopic expression of mutator enzymes.
- Employed two distinct reporter genes: an extrachromosomal kanamycin-resistance gene and the endogenous chromosomal rpoB gene.
- Quantified mutation generation using a colony formation assay.
Main Results:
- Successfully detected and quantified the activity of ectopically expressed cytidine deaminases.
- Demonstrated the utility of both reporter gene systems for measuring enzyme-induced mutations.
- Established robust and versatile assays for mutator enzyme characterization.
Conclusions:
- The developed E. coli-based assays provide a robust and versatile method for defining mutator enzyme activities.
- These assays are valuable for studying enzymes like cytidine deaminases and can be adapted for other mutator enzymes.
- This work facilitates research into the role of mutator enzymes in biological processes and disease.
