NDLI: DNA Fragmentation in Microorganisms Assessed In Situ▿

Content Provider	PubMed Central
Author	Fernández, José Luis Cartelle, Mónica Muriel, Lourdes Santiso, Rebeca Tamayo, María Goyanes, Vicente Jaime, Gosálvez Bou, Germán
Abstract	Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.
Related Links	http://dx.doi.org/10.1128/aem.00318-08
Ending Page	5933
Page Count	9
Starting Page	5925
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	19
Volume Number	74
Language	English
Publisher	American Society for Microbiology (ASM)
Publisher Date	2008-10-01
Access Restriction	Open
Rights Holder	American Society for Microbiology (ASM)
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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