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Published on: December 22, 2010
Rapid immunoassays for detection of UV-induced cyclobutane pyrimidine dimers in whole bacterial cells
Jordan Peccia1, Mark Hernandez
1Department of Civil, Environmental, and Architectural Engineering, University of Colorado, Boulder, Colorado 80309, USA.
Abstract:
Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled ((125)I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.
Insights
New immunoassays detect DNA damage in UV-exposed bacteria. These methods quantify cyclobutane pyrimidine dimers in whole cells, revealing insights into bacterial DNA repair mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Ultraviolet (UV) radiation induces DNA damage, primarily cyclobutane pyrimidine dimers (CPDs), in bacterial cells.
- Quantifying intracellular DNA damage and repair responses is crucial for understanding bacterial UV resistance and survival.
- Existing methods for DNA damage assessment often require DNA extraction, which can be laborious and introduce artifacts.
Purpose of the Study:
- To develop and validate sensitive immunoassays for measuring DNA damage in UV-irradiated whole bacterial cells.
- To assess the utility of these assays for quantifying intracellular cyclobutane pyrimidine dimers (CPDs) and photoenzymatic repair (PER) in bacteria.
- To compare the sensitivity of whole-cell assays with traditional DNA extract-based methods.
Main Methods:
- Development of indirect whole-cell immunoassays using antibodies specific for cyclobutane pyrimidine dimers (CPDs).
- Application of fluorescent (Alexa Fluor 488) and radiolabeled ((125)I) secondary antibodies as reporters.
- Exposure of *Mycobacterium parafortuitum* and *Serratia marcescens* to controlled UV doses (254 nm) followed by immunofluorescent microscopy and comparison with immunoslot blot assays on DNA extracts.
Main Results:
- The developed whole-cell immunoassays were sensitive enough to detect intracellular UV photoproducts and photoenzymatic repair (PER) responses in *M. parafortuitum* and *S. marcescens*.
- Detection limits for fluorescent CPDs in whole-cell assays (immunofluorescent microscopy) were comparable to those in DNA extract-based assays (immunoslot blot).
- Quantification of intracellular CPDs was reliable even after a significant decrease in bacterial culturability (<0.5-log10).
- Immunofluorescent microscopy revealed non-uniform distribution of PER competence within UV-irradiated bacterial cultures.
Conclusions:
- Indirect whole-cell immunoassays provide a sensitive and reliable method for measuring intracellular DNA damage and repair in UV-exposed bacteria.
- These assays offer an alternative to DNA extraction-based methods, simplifying the assessment of bacterial UV responses.
- The findings highlight the heterogeneity of photoenzymatic repair capacity within bacterial populations.
