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

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.The response of prokaryotes to UV radiation is important for understanding how UV influences population dynamics in natural systems (11,21) and the performance of engineered disinfection systems (10,27,30). UV performance in engineered systems has commonly been assessed on the basis of culturability changes using conventional enrichment techniques (7,16,19). While these assays provide a surrogate measure of the ability of UV radiation to render culturable bacteria nonculturable, only a small fraction of microorganisms in natural systems or engineered treatment systems are culturable (1). Additionally, conventional culturing assays cannot provide specific information regarding the types of DNA damage individual cells retain, the rate(s) at which DNA damage has been incurred, and the subsequent intracellular genetic repair (if any) that occurs in aquatic or aerosol environments.The literature cites the cyclobutane pyrimidine dimer (CPD), the 6-4 photoproducts, and the 5-thyminyl-5-dihydrothymine (spore photoproducts) as the most common type of UV-induced DNA damage observed in UV-irradiated prokaryotic cells (18,32,39). In bacteria, the detection and measurement of UV-induced DNA photoproducts has predominantly relied either on chromatographic separation techniques using radiolabeled DNA bases or on indirect assays. Both techniques require the extraction and purification of DNA. To measure DNA lesions by chromatography, 14 C or 3 H must be added to microbiological enrichments to produce organisms with radiolabeled thymine bases incorporated into their DNA. Following UV exposure, DNA is extracted from irradiated cells and hydrolyzed to cleave its phosphodiester bonds, which produces individual nucleotides and UV-associated dimers; UV photoproducts are typically quantified by high-performance liquid chrom...

UV-C inactivation in Escherichia coli is affected by growth conditions preceding irradiation, in particular by the specific growth rate

“…This indicates that the constitutive (not DNA damage‐induced) repair capacity varies with growth phase and, therefore, most likely also with specific growth rate. Nevertheless, we have preliminary data from antibody‐based detection of CPDs in E. coli K12 directly after UV‐C irradiation according to the method of Peccia and Hernandez (2002) indicating the accumulation of less DNA damage in stationary‐phase cells than in cells cultivated in the chemostat at D = 0·2 h −1 (data not shown). Thus, in addition to enhanced repair capacity, also shielding of the DNA might explain the higher resistance to UV‐C light in stationary‐phase bacteria.…”

Direct Effects of UV-B Radiation on the Freshwater Heterotrophic NanoflagellateParaphysomonassp

“…Although PER is widespread taxonomically (16), its importance relative to dark repair is highly variable for different organisms. PER has been shown to be a particularly important mechanism of DNA repair in several species of amphibians (2), zooplankton (7,12,14), protists (26,31), and bacteria (22). Zenoff et al (39) found several strains of aquatic bacteria with various degrees of reliance on PER and no obvious use of NER, although Cytophaga sp.…”

Special Issue: Abstracts from the 9th European Workshop on Astrobiology, Brussels, October 12–14, 2009

“…In this paper, we discuss current status of exposure/capture experiments of microorganisms developed for the TANPOPO mission. (Peccia and Hernandez, 2002), analysis of a cell's membrane potential or membrane damage using the fluorescent dyes BacLight Bacterial Membrane Potential Kit and LIVE/DEAD kit (Fendrihan et al 2009), and heat exposure of Hcc dombrowskii for 1 h. The irradiation and immunoassay experiments were also performed with Halobacterium salinarum NRC-1 which was used as a reference strain. The immunoassay for detecting the CPDs has never before been performed with haloarchaea, therefore some changes had to be made to the protocol, e.g.…”