Physiology

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation

Willie Taylor, Emily Camilleri, D. Levi Craft, George Korza, Maria Rocha Granados, Jaliyah Peterson, Renata Szczpaniak, Sandra K. Weller, Ralf Moeller, Thierry Douki, Wendy W. K. Mok, Peter Setlow
Donald W. Schaffner, Editor
DOI: 10.1128/AEM.03039-19

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    UV222 killing of spores of different species. Spores of different species were treated with UV222, and spore survival was measured, all as described in Materials and Methods. Symbols: ○, B. subtilis PS533 (wild type); ●, B. thuringiensis Al Hakam; △, B. cereus T; ▲, C. difficile 43593; □, C. difficile JIR8094. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated 3 times, and the same relative rates of killing of spores of different species were seen.

  • FIG 2
    FIG 2

    Killing of different concentrations of B. subtilis spores by UV222 and UV254. B. subtilis PS533 spores at an OD600 of 1 (● and ○), 0.1 (▲ and △), or 0.01 (■ and □) were irradiated with UV222 (●, ▲, and ■) or UV254 (○, △, and □), and duplicate samples of various dilutions were spotted onto L broth agar plates to determine spore survival as described in Materials and Methods. Data shown are averages of duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, with the same relative rates of killing of spores seen at different concentrations and with different wavelengths of UV radiation.

  • FIG 3
    FIG 3

    UV222 killing of spores of strains of B. subtilis with defects in possible protective components. Spores of isogenic B. subtilis strains were treated with UV222, and spore survival was measured as described in Materials and Methods. Symbols: ○, PS533 (wild type); ●, PS3328 (cotE); △, PS578 (α− β−); ▲, PS2318 (recA); ■, PS4150 (cotE gerE); □, FB122 (spoVF sleB), prepared with DPA. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, with the same relative rates of killing of spores of different species seen.

  • FIG 4
    FIG 4

    Effects of loss of DPA on B. subtilis and C. difficile spore UV resistance. (A) Purified spores of strain PS832 (wild type) (●) and DPA-less spores of strains FB122 (spoVAF sleB) (▲) and PS3406 (spoVA sleB) (■) were irradiated with UV222, and spore survival was determined as described in Materials and Methods. (B) Purified spores of C. difficile JIR8094, either the wild type (○ and ●), its dpaAB derivative (△ and ▲), or the complemented dpaAB derivative (□), were irradiated with UV222 (○, △, and □) or UV254 (● and ▲), and spore survival was determined as described in Materials and Methods. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, and the same relative rates of killing of spores of different species/strains were seen.

  • FIG 5
    FIG 5

    UV222 killing of log- and stationary-phase cells of various species and strains. Log-phase and stationary-phase cells of various species and strains were isolated and UV222 treated, and cell survival was measured, all as described in Materials and Methods. Symbols: ○, log-phase cells of B. subtilis PS533 (wild type); ●, stationary-phase cells of B. subtilis PS533; □, log-phase cells of B. subtilis PS2318 (recA); △, log-phase cells of S. aureus; ▲, stationary-phase cells of S. aureus. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, and the same relative rates of killing of cells of different species were seen.

  • FIG 6
    FIG 6

    UV222 and UV254 killing of wild-type B. subtilis spores and growing cells and HSV inactivation. Dormant spores and log-phase growing cells of wild-type PS533 B. subtilis (A) or HSV (B) was irradiated for various times, and spore and growing cell survival and HSV inactivation were measured as described in Materials and Methods. Results for HSV inactivation are averages from two independent experiments. Symbols: ○ and △, irradiated with UV222; ● and ▲, irradiated with UV254; ○ and ●, spores or HSV; △ and ▲, growing cells. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, and the same relative rates of killing of spores, cells, and virus, and at both wavelengths, were seen.

  • FIG 7
    FIG 7

    Agarose gel electrophoretic analysis of DNA from untreated or UV222-treated B. subtilis wild-type spores. Total DNA was purified from ∼6 mg (dry weight) of B. subtilis PS533 (wild-type) spores that were either untreated or UV222 irradiated, giving 98% spore killing, as described in Materials and Methods. Approximately 1 μg of each purified DNA was run on an agarose gel plus ethidium bromide alongside 2 μg of DNA size markers, with their sizes shown in kilobases, and the gel was photographed. The samples run in the various lanes are DNA size markers (M), untreated spores’ DNA, and UV222-treated spores’ DNA. The arrow to the right denotes the migration position of supercoiled plasmid pUB110.

  • FIG 8
    FIG 8

    UV222 and UV254 killing of wild-type and spl B. subtilis spores. Isogenic wild-type and spl spores were irradiated with UV222 and UV254, and spore survival at various times was determined as described in Materials and Methods. Symbols: △ and ○, wild type; ▲ and ●, spl; ○ and ●, UV254 irradiated; △ and ▲, UV222 irradiated. Data shown are averages from duplicate determinations ± standard deviations in one experiment. This experiment was repeated twice, and the same relative rates of killing of spores of all strains and at the two wavelengths were seen.

  • FIG 9
    FIG 9

    Germination of untreated and UV222-killed B. subtilis wild-type spores. Spores of B. subtilis PS533 (wild type), either without or after UV222 irradiation giving ∼99% killing, were germinated with l-valine, and spore germination was monitored by the fluorescence in relative fluorescence units (RFU) of released DPA with Tb3+. Symbols: ○, unirradiated spores; ●, UV222-irradiated spores. Analysis of the spores by phase-constant microscopy at the end of the experiment showed that >85% of both types of spores had germinated completely (data not shown). a.u., arbitrary units.

Tables

  • TABLE 1

    Mutagenesis and killing of Bacillus subtilis cells and spores by UV222a

    TABLE 1

    • ↵a Log-phase cells and dormant spores of B. subtilis strains were or were not UV222 irradiated, and levels of cell/spore killing were determined as described in Materials and Methods. Large numbers of colonies from survivors of the irradiation were obtained on L broth agar plates and toothpicked onto Spizizen’s minimal medium and sporulation plates for assessment of mutants that were auxotrophic (aux) or asporogenous (spo) or that had both mutations, all as described in Materials and Methods.

  • TABLE 2

    Levels of photoproducts in UV222-irradiated B. subtilis spores and growing cellsa

    TABLE 2

    • ↵a B. subtilis PS533 (wild-type [WT]) log-phase cells and PS533 and PS578 (α− β−) spores were or were not UV222 irradiated, DNA was isolated and hydrolyzed, and photoproducts were analyzed and quantitated as described in Materials and Methods.

    • ↵b ND, not detectable and <0.1 molecules/106 bases.

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KEYWORDS

Bacillus
decontamination
spores
ultraviolet radiation

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