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Applied and Environmental Microbiology, May 2002, p. 2542-2549, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2542-2549.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rapid Immunoassays for Detection of UV-Induced Cyclobutane Pyrimidine Dimers in Whole Bacterial Cells

Jordan Peccia and Mark Hernandez*

Department of Civil, Environmental, and Architectural Engineering, University of Colorado, Boulder, Colorado 80309

Received 18 September 2001/ Accepted 31 January 2002

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 (125I) 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.

* Corresponding author. Mailing address: Department of Civil, Environmental, and Architectural Engineering, University of Colorado at Boulder, Boulder, CO 80309. Phone: (303) 492-5991. Fax: (303) 492-7317. E-mail: Mark.Hernandez{at}Colorado.edu.

Applied and Environmental Microbiology, May 2002, p. 2542-2549, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2542-2549.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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