Reviving the view: evidence that macromolecule synthesis fuels bacterial spore germination

Abstract

The Gram positive bacterium Bacillus subtilis and its relatives are capable of forming a durable dormant long-lasting spore. Although spores can remain dormant for years, they possess the remarkable capacity to rapidly resume life and convert into actively growing cells. This cellular transition initiates with a most enigmatic irreversible event, termed germination, lasting only for a few minutes. Germination is typified by a morphological conversion that culminates in loss of spore resilient properties. Yet, the molecular events occurring during this brief critical phase are largely unknown. The current widely accepted view considers germination to occur without the need for any macromolecule synthesis; however, accumulating data from our laboratory and others, highlighted here, provide evidence that both transcription and translation occur during germination and are required for its execution. We further underline numerous overlooked studies, conducted mainly during the 1960s-1970s, reinforcing this notion. We propose to revisit the fascinating process of spore germination and redefine it as a pathway involving macromolecule synthesis. We expect our perspective to shed new light on the awakening process of a variety of spore-forming environmental, commensal, and pathogenic bacteria and possibly be applicable to additional organisms displaying a quiescent life form.

Keywords: Bacillus subtilis; spore dormancy; spore germination; spore revival; sporulation.

Figure 1.

Evidence for the occurrence of transcription and translation during spore germination. (A) The morphological sequence of events occurring during spore revival is shown as captured by phase contrast images at the indicated time points [min]. Germination is visualized by a switch from a bright spore to a grey spore and subsequently to a fully germinated dark cell. The ripening period is not associated with an evident morphological change, and outgrowth is characterized by increasing cell length. (B) Incorporation of ¹⁴C-uracil and ¹⁴C-L-valine during Bacilli spore germination provides indication for transcription and translation during germination. Labels were added for clarity. Adopted from (Torriani and Levinthal 1967). Reprinted with permission from the ASM. (C) Spores were induced to germinate with the non-nutrient germinant Ca-DPA in the presence of methionine analogue (azidohomoalaine, AHA). Shown is a dot blot analysis of protein samples (in duplicates) that were collected from dormant and germinated spores. Samples were diluted (1:100 and 1:10) and spotted on a membrane that was subsequently probed with anti-biotin antibodies, indicating the presence of AHA. The marker (M) represents known amounts of biotinylated BSA. Adopted from (Sinai et al. 2015). (D)In vitro transcription reaction was carried out in whole extracts from dormant and germinated spores of WT and sigA* phospho-mutant strains, in transcription buffer supplemented with NTPs (ATP, CTP, GTP, UTP, [α-32P]-UTP). After 40 min of incubation the reaction was stopped, RNA was purified and radioactively labeled RNAs were analyzed in polyacrylamide gel. Adopted from (Zhou et al. 2019).

Figure 2.

A revised model for bacterial spore germination. (A) A revised view for bacterial spore germination. Following nutrient sensing, the germination receptors activate the Arg phosphatase YwlE (①). Consequently, SigA is dephosphorylated by active YwlE to reactivate the transcription machinery (②③④). Next, translation is reestablished by the YwlE-mediated dephosphorylation of Tig after DPA release (⑥⑦⑧). The source of the transcripts for protein synthesis during germination could be derived from the newly transcribed RNA (⑤). (B) Our revised model very much resembles that of Hansen et al. (Hansen et al. 1970) based on their results. Reprinted with permission from the AAAS.