Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Abstract

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.

FIG. 1

Spore resistance to 5% H₂O₂. The strains were assayed for H₂O₂ resistance as described in the text. LD₉₀ are expressed as averages ± standard deviations (n ≥ 3). The asterisks indicate LD₉₀ that were significantly different than the LD₉₀ for wild-type PY79 spores, as determined by ANOVA (P ≤ 0.05). w.t., wild type.

FIG. 2

Spore resistance to 254-nm UV-C radiation. The strains were irradiated and levels of survival were determined as described in the text. LD₉₀ are expressed as averages ± standard deviations (n = 3 for AD17, AD28, and chemically decoated PY79; n = 7 for PY79 and AD142). The asterisk indicates an LD₉₀ that was significantly different than the LD₉₀ for wild-type spores, as determined by ANOVA (P ≤ 0.05). w.t., wild type.

FIG. 3

Spore resistance to UV-B radiation. The strains were irradiated and levels of survival were determined as described in the text. LD₉₀ are expressed as averages ± standard deviations (n ≥ 3). The asterisk indicate LD₉₀ that were significantly different than the LD₉₀ for wild-type PY79, as determined by ANOVA (P ≤ 0.05). w.t., wild type.

FIG. 4

Spore resistance to full-spectrum solar radiation. The strains were irradiated and levels of survival were determined as described in the text. LD₉₀ are expressed as averages ± standard errors (n ≥ 3). The asterisks indicate LD₉₀ that were significantly different than the LD₉₀ for wild-type WN515 spores, as determined by ANOVA (P ≤ 0.05). w.t., wild type.

FIG. 5

Spore resistance to solar UV-A radiation. The strains were exposed and levels of survival were determined as described in the text. LD₉₀ are expressed as averages ± standard errors (n ≥ 3). The asterisks indicate LD₉₀ that were significantly different than the LD₉₀ for wild-type WN515 spores, as determined by ANOVA (P ≤ 0.05). w.t., wild type.

FIG. 6

Summary of the UV resistance of spores of coat mutant strains normalized to the resistance of the wild-type strain. The asterisks indicate LD₉₀ that were significantly different than the LD₉₀ for wild-type spores within a treatment group, as determined by ANOVA (P ≤ 0.05). w.t., wild type.