Properties of Aged Spores of Bacillus subtilis

Abstract

Bacillus spores incubated on plates for 2 to 98 days 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 medium, 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-day spores had lost Ͼ98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 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 sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium 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 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day 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 ϳ100 days, complementing published data showing dry-spore survival for years.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return

“…S3). However, as noted in the text, the spore H-Mn(Pi) component of the spectrum includes contributions not only from Mn complexed with inorganic phosphate (Pi) but also from the similar spectra of Mn complexed with the large amounts of 3-phosphoglyceric acid found in spores of all Bacillus species examined, in some cases with levels even higher than Pi (Camilleri et al, 2019 ; Ghosh et al, 2011 ). ( C ) Survival of B. megaterium (QMB1551) spores and vegetative cells irradiated as in (A).…”

“…S2, left). Taking into consideration the high abundance of dipicolinic acid (DPA) in spores, but not in vegetative cells (Granger et al, 2011 ; Camilleri et al, 2019 ), we treated, for consistency, the H-Mn signal in the spores as a weighted sum of Mn-Pi and Mn-DPA spectra, but in the vegetative cells as a sum of Mn-Pi and Mn-Imi spectra, while emphasizing that reversal of the procedure showed that the choice has no influence on any conclusions (see Fig. S3).…”

Bacterial Spore mRNA – What’s Up With That?

“…Ruling this out is most likely impossible. However, a simple experiment of leaving spores to sporulate for different times, and then analyzing their mRNA levels provided strong evidence that spore mRNAs, more specifically intact spore mRNAs, are not essential for at least B. subtilis spore germination and outgrowth (Camilleri et al, 2019). Thus RNA-Seq analysis on B. subtilis spores harvested after 2 days of sporulation gave approximately the same ~50 spore mRNAs at ≥1 molecule/ spore, but spores allowed to sporulate on plates for 45-90 days at 37°C gave RNA from which levels of all spore mRNAs were decreased ≥95-fold, and rRNAs were significantly fragmented.…”

“…The second major evidence against any essential involvement of macromolecular synthesis in spore germination is that ATP levels are almost non-existent in dormant spores, and appear to increase only after germination is complete (Setlow and Kornberg, 1970a;Scott and Ellar, 1978). While this result is potentially definitive in ruling out macromolecular synthesis' involvement in spore germination, this experiment can be confounded to some extent by at least three factors -(i) the heterogeneity in germination rates between individual spores in populations (Setlow et al, 2017), so that curves for both germination and ATP accumulation are broadened significantly, (ii) completion of germination after CaDPA release requires hydrolysis of the spore cortex peptidoglycan, which leads to an increase in core water content to 80% of wet wt allowing full protein mobility in spores and thus metabolic activity; however, the latter increase in core water is not rapid and takes 10-15 min in B. subtilis (Camilleri et al, 2019), and perhaps there could be metabolic activity when core water content reaches 60% of wet wt, and (iii) analyses of the accumulation of ATP by germinating spores has always been measured by extraction of small molecules and subsequent analyses and it would be ideal to measure molecules such as ATP, NADH, etc., in individual germinating spores, as this would give much higher resolution for analyses of when ATP and other high energy compounds were generated in spore germination. Indeed, certainly our understanding of spore germination has been significantly increased by examining the germination of multiple individual spores (Setlow et al, 2017).…”

“…An obvious alternative is that the mother cell mRNAs are only fragments, which might even facilitate their passage through the feeding tube. Indeed, many spore mRNAs appear to be significantly fragmented as determined by both analysis of the coverage of reads along a gene (Camilleri et al, 2019;Korza et al, 2019), as well as isolation of 5'-modified regions of a few B. subtilis spore mRNAs and demonstration that 3'-regions of these mRNAs were not isolated with 5' ends (Craft et al, 2020). This analysis, as well as the mechanism of mRNA (as well as rRNA) hydrolysis in spores and which does not give rise to mononucleotides (Korza et al, 2016) are also subjects for further work, and will almost certainly give new and interesting information on what is going on inside developing spores, and even dormant spores themselves.…”