Role of DNA Repair and Protective Components in Bacillus subtilis Spore Resistance to Inactivation by 400-nm-Wavelength Blue Light

Abstract

The high intrinsic decontamination resistance of Firmicutes spores, is important medically (disease) and commercially (food spoilage). Effective methods of spore eradication would be of considerable interest in the health care and medical products industries; particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light at ∼ 400 nm is one such treatment that has drawn significant interest. This work has determined the resistance of spores to blue light in an extensive panel of strains, including wild-type strains and mutants that: i) lack protective components such as the spore coat and its pigment (s) or the DNA protective α/β-type small acid-soluble spore proteins (SASP); ii) have an elevated spore core water content; or iii) lack enzymes involved in DNA repair, including homologous recombination and non-homologous end joining (HR and NHEJ), apurinic/apyrimidinic endonucleases, nucleotide- and base excision repair (NER& BER), translesion synthesis (TLS) by Y-family DNA polymerases and spore photoproduct (SP) removal by SP lyase (SPL). The most important factors in spore blue light resistance were determined to be: spore coats/pigmentation, α/β-type SASP, NER, BER TLS and SP repair. A major conclusion from this work is that blue light kills spores by DNA-damage, and the results in this work indicate at least some of the specific DNA-damage. It appears that high-intensity blue light could be a significant addition to the agents used to kill bacterial spores in applied settings.Effective methods of spore inactivation would be of considerable interest in the health care and medical products industries; particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light radiation is one such treatment that has drawn significant interest. In this work in a systematic fashion all known spore protective features, as well as universal and spore-specific DNA repair mechanisms were tested on their contribution to the resistance of spores to blue light radiation.

Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores

“…Bacillus strains used and spore preparation. The B. subtilis strains used were PS832, a wild-type laboratory derivative of B. subtilis 168 (36), and eight isogenic strains: (i) PS533, identical to PS832 but carrying plasmid pUB110 encoding resistance to kanamycin (10 g/ml) (37); (ii) PS4150, in which the cotE and gerE genes are replaced with tetracycline and spectinomycin resistance cassettes, respectively (19), resulting in spores that lack both the inner and most of the outer coat layer, although a thin layer of insoluble coat material remains and is even present after these spores are digested with lysozyme, DNase, and pronase and boiled in sodium dodecyl sulfate (SDS) (19,38); (iii) FB122, in which the spoVF operon and sleB gene have been deleted and replaced with spectinomycin and tetracycline resistance cassettes (this strain cannot make DPA in sporulation and its spores lack CaDPA, and although CaDPAless spores normally rapidly germinate spontaneously, the sleB mutation stabilizes CaDPA-less spores) (20, 21); (iv) 2066 (22), with a deletion of the dacB gene, which encodes an enzyme that modifies the structure of spore cortex PG; (v) PS2421 (22), with deletions of the dacB and dacF genes, which both encode enzymes that modify cortex PG structure; (vi) PS2307 (23), which lacks the cwlD gene, which encodes an enzyme essential for generation of the spore cortex PG-specific modification, muramic acid-␦-lactam; (vii) PS2422 (24), with deletions in both cwlD and dacB genes; and (viii) PS3738 (18), with a deletion of the safA gene, which encodes a protein important in spore coat assembly (16,38). In addition, four isogenic B. subtilis strains were used with the PY79 genetic background, including PS3483 (the wild-type strain), PE620 lacking the cotXYZ operon, PE2763 lacking spsI, and PE2916 lacking cgeB.…”

Observations on research with spores of Bacillales and Clostridiales species

“…Determining the mechanism(s) whereby spores resist inactivation by a new sporicidal agent is easiest with B. subtilis spores, as there are isogenic mutants available that lack most coat proteins, most α/β‐type SASP, or much DNA repair capacity, including missing individual DNA repair pathways (Young and Setlow ; Djouiai et al . ). There are also B. subtilis mutants that produce stable spores with elevated core water content, either because of a modified spore cortex or the complete absence of DPA (Popham et al .…”

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

“…With photoproducts generated by UV 222 in spore and cell DNA identified, it was then possible to examine the effects of the loss of a DNA repair protein important for the repair of the most abundant of the photoproducts formed in spores (2,31,32). The specific DNA repair protein targeted was Spl, which monomerizes SP in spore DNA.…”