Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

Abstract

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695–707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination. IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Spore Germination of the Obligate Biotroph Spongospora subterranea: Transcriptome Analysis Reveals Germination Associated Genes

“…Metabolomics analysis of P. digitatum revealed that central carbon and energy metabolism were upregulated during germination of spores (Che et al, 2020). In line with previously reported works on gene expression during spore germination (Luo et al, 2020;Swarge et al, 2020), important cellular metabolic pathways including fatty acid biosynthesis and amino acid biosynthesis showed significant changes in the germinating spores in comparison with dormant spores (Figure 4 and Table 3). In the lipid fraction, there is a decrease in fatty acid synthase and trehalase activity (Table 2).…”

Bacterial Spore mRNA – What’s Up With That?

“…The same authors suggest that the observed increase in MalS-GFP fluorescence reflects a change in the environment of the germinating spore core -most likely partial ingress of water from the environment promoting enhanced fluorescence from existing MalS-GFP -and is not associated with de novo protein synthesis. The observation that malS mRNA sits outside the top 1,000 most abundant transcripts detected within spores, at levels indicative of being present in only a small sub-set of the population and with a copy number averaging much less than 1/spore (Nagler et al, 2016;Korza et al, 2019), further supports the conclusions reported by Swarge et al (2020b). Indeed, very recently published work conducted by the same group, this time combining an integrated transcriptomic and multi-faceted proteomic approach, permitting a hitherto unsurpassed level of resolution to these analyses in spores, robustly supports the idea that both transcription and protein synthesis start after the completion of germination (Swarge et al, 2020a).…”

“…Notably, the absence of nucleotide biosynthetic enzymes from spores and that these proteins are synthesized at various times in spore outgrowth have also recently been shown for B. subtilis spores in several proteomic studies (Swarge et al, 2018(Swarge et al, , 2020b. The latter findings were also made for amino acid biosynthetic enzymes as well.…”

“…The same authors suggested that the concentration of malate in dormant spores was relatively high, in contrast to earlier studies and a more recent one Scott and Ellar, 1978;Korza et al, 2017), providing a readily available substrate with which to generate ATP for the germinating spore. A more recent study, employing analogous strains and a range of sporulation conditions, reported a similar increase in fluorescence associated with MalS-GFP during the early stages of germination (Swarge et al, 2020b). However, in contrast to results of Sinai et al (2015), Western blot data in the Swarge et al report indicated that MalS-GFP abundance remained stationary throughout germination.…”

What’s new and notable in bacterial spore killing!

“…These changes result in water uptake by and expansion of the spore core such that core enzymes become active and metabolism and macromolecular biosynthesis resume. However, metabolism and macromolecular synthesis are most likely not required for complete germination (Setlow and Christie 2020 ; Swarge et al 2020a , b ). The order of CaDPA release first and then cortex PG hydrolysis is seen in germination of spores of Bacillales and some Clostridiales species, while in other Clostridiales, cortex PG hydrolysis precedes CaDPA release (Setlow et al 2017 ).…”