Properties of Aged Spores of Bacillus subtilis

Bacillus spores incubated on plates for 2-98 d at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich or poor medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal media and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content, and significantly higher wet heat and NaOCl resistance. In addition, 47 and 98 d spores had lost > 98% of intact 16 and 23S rRNA and 97-99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 d. Levels of 3-phosphoglyceric acid (3PGA) also fell 30-60% in the oldest spores but how the 3PGA was lost is not clear. These results indicate that: i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; ii) in at least sporulation for up to 91 d at 37°C, the RNA broken down generates minimal levels of mononucleotides; and iii) the lengths of time that spores are incubated in sporulation media should be considered when determining conditions for spore inactivation by wet heat, in particular in using spores to test for the efficacy of sterilization regimens. Importance We show that spores incubated at 37°C on sporulation plates for up to 98 d have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2 d spores and all these spores had identical viability. Thus, it is unlikely that spore mRNAs, rRNAs or protein synthesis are important in spore germination. Spores incubated for 47-98 d also had much higher wet heat resistance than 2 d spores, suggesting that spore “age” should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 d, complementing published data showing dry spore survival for years.