DNA fragmentation in microorganisms assessed in situ

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• DNA fragmentation is a known outcome of cell death in eukaryotes, but is less studied in microorganisms.
• Assessing DNA fragmentation in bacteria and yeasts is crucial for understanding cell death pathways and responses to stimuli.

Purpose of the Study:

• To develop and validate a simple, rapid diffusion-based assay for detecting fragmented chromosomal DNA in microorganisms.
• To demonstrate the assay's utility in identifying spontaneous and induced DNA fragmentation in various microbial species.

Main Methods:

• An adapted diffusion-based assay involving embedding cells in agarose microgel, partial lysis, and SYBR Gold staining.
• Utilizing peripheral DNA fragment diffusion to distinguish between fragmented and intact DNA within microbial cells.
• Confirmation of DNA fragmentation detection using fluoroquinolone treatment and DNA breakage detection-fluorescence in situ hybridization.

Main Results:

• The assay successfully detected fragmented DNA in Gram-negative and Gram-positive bacteria, including Proteus mirabilis and Escherichia coli.
• Fragmented DNA was also identified in Saccharomyces cerevisiae after treatment with amphotericin B.
• The assay distinguished between cells with fragmented DNA and those with intact DNA fiber loops.

Conclusions:

• The developed diffusion-based assay provides a simple and rapid method for evaluating DNA damage and repair in microorganisms.
• This assay is valuable for assessing spontaneous or induced cell death, including responses to antimicrobial agents and environmental factors.