Characterisation of DNA Damage and Repair in Bacillus subtilis Spores after Treatment with Cold Technical Plasmas

Abstract

Contaminations with endospore-formers like Bacillus subtilis are hard to combat, because dormant spores are resistant to various environmental stresses and conventional sterilisation methods. Plasma sterilisation, based on sporicidal properties of ionised gas, has been suggested as alternative. Consisting of ions, reactive species and photons, plasma induces damage on nucleic acids and proteins, as well as eroding target surfaces by intrinsic photo desorption and etching. Effectiveness and mode of action in regard to structural properties and repair mechanisms of B. subtilis were analysed in this study by examining effects of plasma treatment on mutant spores with deficiencies in DNA protection and repair. The survivability of mutant spores lacking genes splB, uvrAB, ykoVU, sspA or recA, were compared to that of B. subtilis 168 wild type spores. Experiments were conducted with an atmospheric pressure plasma jet (μ-APPJ) or in a low-pressure, very high frequency capacitively-coupled plasma (VHF-CCP) reactor. UV radiation impacted spore inactivation strongly, but under atmospheric conditions carrier gas compositions should favour etching, because partial absorption of UV radiation led to high survival rates after μ-APPJ treatment. Wild type spores were more resistant to low-pressure VHF-CCP treatment than the tested mutants. The importance of RecA as mediator of the SOS-response and part of homologous recombination (HR) was confirmed by the low resistance of recA-deficient spores. In contrast, a lack of small acid-soluble proteins of subtype α or spore photoproduct lyase (SPL) could potentially be compensated by structurally similar molecules or alternative repair mechanisms. Spores lacking in nucleotide excision repair (NER) or SPL, both are involved in the repair of DNA-photoproducts, showed similar resistance capabilities. Spores with deficiencies for SPL and non-homologous end joining (NHEJ) displayed low survivability due to cumulative mutations.