Resistance properties and the role of the inner membrane and coat of Bacillus subtilis spores with extreme wet heat resistance

Aims A protein termed 2Duf greatly increases wet heat resistance of Bacillus subtilis spores. The current work examines the effects of 2Duf on spore resistance to other sporicides, including chemicals that act on or must cross spores’ inner membrane (IM), where 2Duf is likely present. The overall aim was to gain a deeper understanding of how 2Duf affects spore resistance, and of spore resistance itself. Methods and Results 2Duf's presence increased spore resistance to chemicals that damage or must cross the IM to kill spores. Spore coat removal decreased 2Duf‐spore resistance to chemicals and wet heat, and 2Duf‐spores made at higher temperatures were more resistant to wet heat and chemicals. 2Duf‐less spores lacking coats and Ca‐dipicolinic acid were also extremely sensitive to wet heat and chemicals that transit the IM to kill spores. Conclusions The new work plus previous results lead to a number of important conclusions as follows. (1) 2Duf may influence spore resistance by decreasing the permeability of and lipid mobility in spores’ IM. (2) Since wet heat‐killed spores that germinate do not accumulate ATP, wet heat may inactivate some spore IM protein essential in ATP production which is stabilized in a more rigid IM. (3) Both Ca‐dipicolinic acid and the spore coat play an important role in the permeability of the spore IM, and thus in many spore resistance properties. Significance and Impact of the Study The work in this manuscript gives a new insight into mechanisms of spore resistance to chemicals and wet heat, to the understanding of spore wet heat killing, and the role of Ca‐dipicolinic acid and the coat in spore resistance.