Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

Abstract

ABSTRACTrRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139 -114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium. Incubation of spores of these two species for ϳ20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10-to 30-fold, to <0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination. Spores of Bacillus species formed in sporulation are metabolically dormant and extremely resistant to many severe treatments, including heat, radiation, and toxic chemicals (1-3). Although these spores can survive in this dormant resistant state for many years, if given the appropriate stimulus, generally small molecules that are found in environments that are appropriate for the growth of these bacteria, the spores can rapidly break dormancy in the process of spore germination and then outgrowth (1, 4, 5). Notably, spores' extreme resistance properties are lost in the process of germination and early outgrowth, and thus, germinated spores are much easier to kill than dormant spores. This property has significant applied implications, as dormant spores are the vectors for much food spoilage, as well as some foodborne and other human diseases. Consequently, there is significant interest in mechanisms that determine rates of spore germination and how sporulation conditions, as well as storage conditions, modify spores' resistance properties and their rates of germination.In addition to the applied interest in spore resistance and germination, there has also long been much basic interest in the mechanism of spore germination, in particular, the signal transduction pathways operating during the process (4, 5). The major proteins involved in Bacillus spore germination are as follows. (i) Germinant receptors (GRs) in spores' inner membrane (IM) recognize and respond to the physiological germinants specific for spores of each species/strain. (ii)...

Commentary: Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

“…A recent paper published by the Setlow laboratory (Korza et al, 2016) presumably challenges our findings, claiming that protein synthesis is not required for germination. This discrepancy was highlighted by a commentary written by Boone and Driks (2016), thoroughly comparing the two papers.…”

“…However, even with these methodological weaknesses, when we closely examined and quantified their data, we could clearly see an increase in rRNA levels at time points corresponding to germination [Figure 8B (5 min) for B. megaterium ; Figure 9B (30 min) for B. subtilis . The latter is apparent upon proper scaling and quantification of the gel image, data not shown] (Korza et al, 2016). …”

“…(B) Phase contrast images of a field of B. subtilis spores before (left: Pre-treatment) and after (right: Post-treatment) incubation for 18 h at 80°C. (C) Spores were incubated for 18 h at 80°C (treated), and then purified with Histodenz gradient as described by Korza et al (2016). Untreated spores (untreated) were heat activated for 30 min at 75°C.…”

“…The authors used DPA release as a measure of germination (Korza et al, 2016: Figures 5–6). However, although not indicated by the authors, we have shown that DPA release precedes and is independent of protein synthesis (Sinai et al, 2015).…”

Protein Synthesis during Germination: Shedding New Light on a Classical Question

“…Since the work of Sinai et al, it is well recognized in the field that the general question of what, if any, cellular processes transpire during dormancy or germination needs further investigation. In the present issue, Korza et al (4) do exactly this by addressing the question of whether protein synthesis is required for germination in a different way than Sinai et al Korza et al inhibited protein synthesis during germination by depleting spores of rRNA. They found that rRNA depletion did not significantly reduce the rate of germination, strongly suggesting that protein synthesis is not required for germination.…”