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

ABSTRACT rRNAs 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 ∼20 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. IMPORTANCE A recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486–3495, 2015, doi: http://dx.doi.org/10.1016/j.molcel.2014.12.019 ) suggested that protein synthesis is essential for early steps in the germination of dormant spores of Bacillus subtilis . If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores of Bacillus megaterium or B. subtilis , and these rRNA-depleted spores are viable and germinate as well as or better than spores with normal rRNA levels. Thus, protein synthesis is not required in the process of spore germination.