Mechanistic insight into roles of α/β-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

Aim: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores.

Methods And Results: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/β-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm.

Conclusions: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.