Some species in the Bacillales and Clostridiales orders form spores under unfavourable environmental conditions. These spores are metabolically dormant and highly resistant to extreme stress. The spore core—analogous to the protoplast of vegetative cells—contains only 25–45% water by wet weight, compared to ~80% in vegetative cells. Upon activation by small-molecule nutrients, spores germinate, restoring their core water content, restoring metabolism and becoming easy to kill and then progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA germinant receptor (GR), a membrane protein belonging to the Amino Acid–Polyamine–Organocation (APC) superfamily of transporters. It functions as the L-alanine sensor that initiates germination and was previously predicted, based on molecular dynamics (MD) simulations, to contain a putative water channel. Using MD simulations, we identified low amount of water permeating through GerAB (ranging from 1 to 121 water molecule/μs in 10 parallel MD simulations), thus revealing a water pathway in GerAB that diverges from the L-alanine binding pocket, suggesting that water transport may play roles in germination beyond facilitating ligand binding. Analysis of water–residue contact frequencies identified eight hydrophilic residues lining the water’s path. Individual substitution of high-contact residues with similarly sized non-polar residues impaired L-alanine germination and disrupted GerAB structural integrity as assessed by Western Blotting. These mutants also respond to the AGFK germinant mixture (L-asparagine, D-glucose, D-fructose and potassium) in slower, yet individually distinct kinetics compared to that of wild-type (wt) spores. These findings prove that water contact residues in GerAB predicted by MD simulations are crucial for the stability of this protein and thus the germinosome complex with all GRs.

Dormant Bacillus subtilis spores germinate through interaction of germinants with germinant receptors (GRs). Subsequently, GR signals are transduced to SpoVA protein channels, and opening these channels leads to calcium dipicolinic acid (CaDPA) release and completion of germination. Spores exhibit memory in germination, as spores given a short germinant pulse more readily respond to a second pulse. We developed a mathematical model to identify the minimal network crucial for germination kinetics leading to memory of germinant exposure, and reproducing experimental double germinant pulse germination curves. Analysis of the reconstructed network indicates that a minimal set of inactive and active GRs and a SpoVA channel in three states - closed inactive, closed active and open - is needed to reproduce memory. Spore germination memory is introduced in the network by GR’s activation and deactivation rates, and the interplay between activation of closed SpoVA channels and their rates of opening and closing.

New effectors of spore properties have been identified in two Bacillus subtilis strains-bacillithiol, the major low-molecular-weight thiol in B. subtilis, and sporulene, a multi-ring compound synthesized from curcumene in spores' outer layers. The absence of either bacillithiol or sporulene caused faster germination of spores of one wild-type B. subtilis strain, PS832, with two germinant receptors (GR)-dependent germinants, L-valine for the GerA GR, and the AGFK mixture for the cooperative action of the GerB and GerK GRs, as well as the GR-independent germinant dodecylamine. However, these effects on PS832 spore germination were due to the presence of the antibiotic marker used to replace the genes for bacillithiol or sporulene synthesis. The absence of bacillithiol also caused reduced spore resistance to wet heat but not to hydrogen peroxide or UV radiation, while sporulenes' absence had no effect on spore wet heat resistance, but reduced spore resistance to hydrogen peroxide as found previously (T. Bosak, R. M. Losick, and A. Pearson, Proc Natl Acad Sci USA 105:6725-6729, 2008, https://doi.org/10.1073/pnas.0800199105) and to UV radiation, but the presence of the antibiotic markers in these mutant strains was not responsible for these effects. Notably, spores lacking sporulene were much whiter than wild-type (wt) spores, suggesting that sporulene and its precursor curcumene contribute to spore pigmentation that can absorb UV, thus reducing spores' UV resistance. Analyses of reasons for the effects of bacillithiol's or sporulene's absence on spore resistance, specifically core water content and/or altered spore inner membrane (IM) permeability, found no major differences from values in wt spores. However, other analyses showed that sporulene's absence led to slight changes in spore IM fluidity.IMPORTANCEThe work in this paper has identified two new factors, bacillithiol and sporulene, as modulators of the resistance and germination of spores of two Bacillus subtilis strains, and by extension, probably spores of other Bacillota. Since spores of some species can give rise to cells that can cause food spoilage and/or disease, new knowledge about spore resistance and germination could have applied utility.

Bacterial endospores are metabolically dormant yet can rapidly return to vegetative growth upon exposure to nutrients through the process of germination. Spore germination is triggered by specific chemical nutrients binding to cognate germinant receptors (GRs) in spores' inner membrane. These GRs function as ligand-gated ion channels and are composed of clusters of at least three subunits. Given their central role in germinant recognition and discrimination, elucidating 3D structures of GR subunits is a key part of efforts to understand the mechanism(s) of spore germination. Here, we present the crystal structure of the N-terminal domain of the Bacillus cereus GerIA protein (GerIANTD), a component of the inosine-responsive GerI GR. GerIANTD adopts a conformation homologous to substrate-binding proteins in bacterial ABC transporters. NMR chemical shift perturbation and site-directed mutagenesis identified GerIANTD residues potentially involved in inosine binding or critical for germinosome assembly in B. cereus spores, modification of which abrogated inosine-induced germination. Molecular modeling and mutagenesis additionally identified residues in the GerIB subunit forming germinant and cation-binding sites. GerQ, the second GR that contributes to inosine germination in B. cereus spores, was capable of complementing hypomorphic gerI alleles in several instances, demonstrating cooperative restoration of function despite being incapable of initiating germination to inosine in gerI null spores. Collectively, our results provide new insights into GR subunit function and the molecular basis of the B. cereus germinative response to inosine.IMPORTANCEMany bacteria in the order Bacillota form spores that survive antibacterial treatments, including antibiotics. However, once these spores germinate and return to growth, they become vulnerable to antibiotics and other treatments. Notably, growing cells of some of these species cause food spoilage or serious diseases. Thus, there is much interest in spore germination, as stimulating this process would allow for easy spore eradication. This study has investigated precisely how spores' germinant receptors (GRs) recognize and respond to triggers of spore germination, such as inosine and L-alanine. Using a combination of structural biology, computational modeling, and functional assays with targeted GR mutations, our work uncovered new insights into GR function and the initiation of germination. These findings not only advance our understanding of a critical biological process but also provide new directions for spore control strategies.

Previous work (V. K. Juneja, M. Osoria, E. G. Altuntas, N. K. Taneja, et al., Food Res Int 177:113904, 2024, https://doi.org/10.1016/j.foodres.2023.113904) showed that less well-purified spores of Bacillus cereus and Bacillus subtilis were more wet heat resistant than more well-purified spores. Extending the previous work, the current work used (i) B. subtilis spores identical to the one used previously but lacking a plasmid providing kanamycin resistance and (ii) spores of a different B. cereus strain, and with spores made on both plates and in liquid. There were larger differences in spores' levels of purity in the new work than previously, and differences in spore purity were visible in micrographs of spore preparations purified differently. With spores made on plates, the least purified B. subtilis and B. cereus spores were more wet heat resistant than those purified further, but less so with spores prepared in liquid. These findings suggest that it may be important to consider the purity of spores prepared as indicators for sterilization assurance, especially since large-scale spore preparations are often not extensively purified. However, resistance to UV radiation, hydrogen peroxide, or sodium hypochlorite was relatively similar with the B. cereus or B. subtilis spores purified to different degrees. Thus, these methods for spore inactivation appear not be affected significantly by spore purity. Spores' purity also had minimal effects on rates of germinant-receptor (GR) dependent spore germination with physiological germinants, or with dodecylamine that opens a channel protein leading to release of spore cores' large pool of Ca-dipicolinic acid (CaDPA). In addition, contents of water in cores of B. cereus or B. subtilis spores purified to different degrees were almost identical, as were CaDPA levels. All these results do not yet allow obvious assignment of causes for the higher wet heat resistance of cruder B. subtilis or B. cereus spores. However, it was notable that levels of soluble protein in suspensions of spores purified differently were the highest in the crude spores and lowest in the most highly purified spores. IMPORTANCE:Spores of many Bacillota give rise to cells and spores, some of which cause food spoilage, food poisoning, and serious human disease. Consequently, there is applied interest in spore killing, something that is difficult because of spores' resistance to killing regimens including by wet heat, radiation, and toxic chemicals and the many causes of this extreme resistance. The current work shows that for spores of two Bacillus species made on plates or in liquid, spore purity plays no role in the ability of spores to germinate or their resistance to agents other than wet heat. However, the least purified B. subtilis or B. cereus spores, especially those made on plates, were significantly more wet heat resistant than more purified spores. While precise causes of the effects of spore purity on spore wet heat resistance are not clear, this seems to be an additional variable to consider when preparing spores for various uses, including as in sterilization assurance.

Spores of the Bacillota phylum, which includes several bacterial species of significant economic and public health concern, are among the most durable and recalcitrant observed in nature. Their properties of extreme resistance to a raft of physicochemical regimens that are otherwise cidal to their planktonic counterparts are conferred by a unique cellular architecture and physiology that renders them metabolically dormant but primed to rapidly return to the vegetative and potentially deleterious state. This review considers contemporary approaches and developments aimed at killing or eradicating spores with a focus on techniques that may be applicable to the food industry. These include innovations with chemical sporicides and physical approaches aimed at circumventing traditional thermal-based approaches to killing spores and their associated negative effects on product properties. Despite progress in both of these broad areas, we posit that the “germinate to eradicate” approach is perhaps best placed for wider implementation in the food industry, building upon recent progress in understanding germination at the molecular level, and offering a plausible route to structure-led design of small molecules that can efficiently trigger germination across all Bacillota spores, sensitizing them to killing by mild heat treatment.

Some Bacillales and Clostridiales bacteria form spores in unfavorable environments. These spores are dormant but can rapidly resume metabolism in germination. This process can be initiated by a variety of low molecular weight nutrients termed germinants. Structural modeling and mutagenesis studies showed that GerAB, an inner membrane (IM) protein of the Bacillus subtilis spore germinant receptor (GR) GerA, is involved in L-alanine-initiated spore germination. A previous molecular simulation study also suggested there is a water channel in GerAB. In the current work, Steered Molecular Dynamics (SMD) simulations were employed to force a single water molecule through GerAB, identifying three key amino acid residues, Y97, L199 and F342, that interfere with water passage. When these residues were altered to alanine, L-alanine germination no longer occurred in spores with L199A, F342A and triA (Y97A, L199A and F342A triple mutant), while Y97A mutant spores germinated ∼61%. Additionally, except for Y97A, all other mutants showed compromised germination triggered by the AGFK mixture (L-asparagine, D-glucose, D-fructose and K+ ion). Western blotting found reduced levels of the GerA GR in the Y97A mutant, and an absence of the GerA GR in all other mutants. This proves that all three identified residues are crucial to the structural integrity of the GerA germinant receptor and also suggests they are essential for the formation of a fully functional GR complex, the germinosome.

This study aimed to determine reasons that longer sporulation times in liquid or on plates have large effects on Bacillus subtilis spore germination and resistance. B. subtilis spores were prepared for 3/30/60 days in liquid or on plates and their germination, resistance, levels of core water and Ca2+-dipicolinic acid (CaDPA), and inner membrane (IM) fluidity and permeability were measured. Liquid spores of 3/30/60 days had no differences in wet heat resistance, while plate spores of 30/60 days had higher wet heat resistance than 3-day plate spores. There were minimal increases in 3- to 60-day liquid spores' resistance to UV radiation and chemicals, while plate spores of 30/60 days had increased resistance to chemicals. Germinant receptor (GR) germination with L-valine was identical with 3- to 60-day liquid or plate spores, while plate spores of 30/60 days germinated faster than 3-day spores with the L-asparagine, D-glucose, D-fructose, and KCl mixture. Three-day liquid or plate spores germinated faster than 30- and 60-day spores with the GR-independent germinant, dodecylamine, and 30-day plate spores had lower IM permeability and higher IM rigidity than 3-day plate spores or 30-day liquid spores. However, levels of two spore core components' modulating spore wet heat resistance, water, and CaDPA were identical in spores of 3/30/60 days. Thus, increased IM rigidity and decreased IM permeability appear to be major factors in increased resistance of spores prepared on plates for long periods. However, precisely how these longer times cause increased IM rigidity and lower permeability, but not with spores prepared in liquid, is not clear.IMPORTANCECells of some Bacillota cause food spoilage and human diseases. These organisms' ability to do this is exacerbated by forming hard-to-kill dormant spores because of their resistance and ability to come "back to life" in germination. We examined two Bacillus subtilis sporulation parameters, liquid versus solid media and sporulation time, measuring effects on spore resistance and germination. We found (i) an effector of spore heat resistance, core water content, is not changed by different sporulation media or times; and (ii) spores' IM becomes more rigid and less permeable in spores made on solid media and for longer times. This knowledge may influence how spores are prepared as probiotics or as standards for analyses of autoclave function.

The mechanism by which ohmic heating (OH) accelerates bacterial spore killing compared to conventional heating (CH) is unclear. This study used genetically modified Bacillus subtilis spores to investigate OH’s impact on specific components. Flow cytometry assessed membrane integrity, and molecular dynamics (MD) simulations examined the DNA-SASP complex under an electric field. Among the inner membrane (IM) proteins (YetF, YdfS, and YkjA) tested for its resistance against OH and CH, YeTF was found to be the most significant contributor to spore resistance for both treatments. SASP, SpoVA proteins, and Ca-DPA interacted with the field, showing specific effects at certain temperature and field intensity combinations. Flow cytometry showed spore staining with propidium iodide (PI), which increased with higher field intensities, indicating significant IM damage. MD simulations showed that the electric field caused the SASP-DNA complex to dissociate, with greater separation at higher field intensities. Thus, OH accelerates spore killing by affecting key IM and core molecules.

Bacterial spores formed upon metabolic stress have minimal metabolic activity and can remain dormant for years. Nevertheless, they can sense the environment and germinate quickly upon exposure to various germinants. Germinated spores can then outgrow into vegetative cells. Germination of spores of some anaerobes, especially Clostridioides difficile, is triggered by cholic acid and taurocholic acid. Elevated levels of these bile acids are thought to correlate with a perturbed gut microbiome, which cannot efficiently convert primary bile acids into secondary bile acids. That bile acids are germination-triggers suggests these bacteria have a life cycle taking place partially in the mammalian digestive tract where bile acids are plentiful; notably bile acids can be made by all vertebrates. Thus, spores survive in the environment until taken up by a host where they encounter an environment suitable for germination and then proliferate in the largely anaerobic large intestine; some ultimately sporulate there, regenerating environmentally resistant spores in the C. difficile life cycle. This review summarizes current literature on the effects of bile acids and their metabolites on spore germination in the gut and evidence that adaptation to bile acids as germinants is a consequence of a life cycle both inside and outside the digestive tract.

Spores of bacteria are metabolically dormant, resistant to microbicides, and vectors of food spoilage and diseases and survive for years in their dormant state. Upon exposure to nutrient germinants, spores can rapidly return to life through germination, losing their resistance and becoming easy to kill. Despite extensive research on germination heterogeneity, commitment, and memory, many mechanisms underlying germination of Bacillota spores remain unclear, as a comprehensive mathematical model describing germination characteristics of individual spores is lacking. Woese et al. (PNAS, 59:869, 1968) developed a simple model predicting that time-to-germination of a spore with n active enzymes is proportional to 1/n. Here, we present a novel approach inspired by artificial neural networks to model spore germination, treating it as a decision-making process upon the activation produced by binding of germinants to germinant receptors. Major findings include the following: (i) using a sigmoid activation function to model germination thresholds allows predictions of distributions in time-to-commitment and kinetic germination to be well fitted to experimental observations; (ii) modeling spore commitment and memory after two separate germinant pulses fits well to experimental data of Bacillus spores germinated with L-alanine, and a zero fraction of germinated spores by a second pulse is predicted by loss of memory; and (iii) modeling kinetic CaDPA release from individual spores through SpoVA channels and fitting experimental data of Bacillus cereus spores. This work enhances our understanding of unexplored biophysical intricacies of spore germination, and the use of the model may generate new data.IMPORTANCESpore germination is a crucial process through which spores of bacteria return to life when triggered by germinants, and some spore species cause food spoilage, human diseases, and bioterrorism. Understanding and theoretical predictions of spore germination could facilitate the development of "germinate to kill" strategies as spores lose their resistance upon germination. Here, we developed a novel mathematical model to describe the characteristics of spore germination including heterogeneity, commitment, memory, and kinetic CaDPA release using an artificial neural network. This model predicts new aspects of germination such as the retention and loss of memory and the effect of GRs' distribution on germination rate and could be useful in data-driven discoveries to enhance our understanding of germination's biophysical intricacies.

Developing spores (forespores) of Bacillus subtilis lack TCA cycle and amino acid and ribonucleotide biosynthetic enzymes but still carry out much macromolecular synthesis to make a spore-but how and why? Work by many showed that the mother cell supplies ATP and metabolites to the forespore via a feeding tube. Two recent studies in this issue of Genes & Development, by Massoni and colleagues (doi:10.1101/gad.352498.124) and Riley and colleagues (doi:10.1101/gad.352535.124), now show that specific metabolic enzymes disappear early in forespore development via proteolysis by ClpCP and a forespore-specific activator termed MdfA. Future work may clarify how this proteolysis recognizes specific metabolic enzymes and determine the advantages of this overall process for spores.

ABSTRACT Spores of Bacillus subtilis have been found to germinate when incubated with LiCl, but not with other monovalent or divalent metal cations. Bacillus megaterium spores also germinated with LiCl, but B. cereus spores did not. In B. subtilis , the LiCl germination was via the activation of spores’ GerA germinant receptor (GR), and in B. megaterium, it was the GerU GR. Notably, LiCl germination was much slower than normal physiological germinant triggered GR germination. In B. subtilis spores, rates of LiCl germination were increased in spores with a more fluid IM and decreased in spores with a less fluid IM. Analyses of the GerA germinant binding site suggested that Li + could bind in a specific site in the B. subtilis GerAB subunit where normally a Na + likely binds. Importantly, NaCl strongly inhibited LiCl germination of B. subtilis spores, much more so than the larger cation in KCl, although neither salt inhibited L-alanine germination via the GerA GR. These findings increase the understanding of features of mechanisms of germination of Bacillus spores. IMPORTANCE The ability of some bacteria to form spores upon nutrient starvation confers properties of metabolic dormancy and enhanced resistance to environmental stressors that would otherwise kill vegetative cells. Since spore-forming bacteria include several notable pathogens and economically significant spoilage organisms, insight into how spores are stimulated to germinate and form new vegetative cells is important. Here, we reveal that relatively high concentrations of the inorganic salt lithium chloride trigger the germination of Bacillus subtilis and Bacillus megaterium spores by stimulating one of the spores of each species cohort of nutrient germinant receptors. This is significant since novel germinants and increased knowledge of the germination process should provide opportunities for improved control of spores in healthcare, food, and environmental sectors.

Spores of Bacillus species are dormant and resistant to heat and chemicals but "return to life" in germination, and cells of some species/strains can cause food spoilage or disease. Recently, two members of a family of five spore integral inner membrane (IM) small protein homologs were found to be important in B. subtilis spore resistance and germination. Among these IM proteins, YetF is the most abundant. In the current work, we show that loss of any of these five homologs decreased spore resistance to heat and chemicals, with greater decreases when multiple homologs were absent. In addition, B. subtilis spores' loss of YetF and its homologs decreased the rates of spore germination, whereas loss of the second most abundant homolog, YrbG, increased germination rates. Surprisingly, B. subtilis spores lacking YetF and YrbG germinated spontaneously early in sporulation. Although this spontaneous germination did not involve normal germinant receptors or cortex peptidoglycan lytic enzymes, it was accelerated by overexpression of the IM channel for CaDPA release in germination. Loss of various homologs increased IM fluidity significantly, perhaps important in the effects of these proteins on spore resistance and germination. Notably, a functional YetF-GFP fusion was located in 5-7 IM spots in wild-type B. subtilis spores and in spores lacking the coat and outer membrane, although the function of these YetF spots is not clear. Similar spots were observed in Bacillus megaterium YetF-GFP spores, whereas ydfS null spores showed decreased wet heat resistance. Clearly, these novel proteins may have more surprises in store!IMPORTANCESpores of Bacillota are vectors for food spoilage and disease, and are hard to kill, as B. subtilis spores are killed only slowly by wet heat at 90°C. Multiple factors contribute to spores' wet heat resistance, including low spore core water content and DNA-protective proteins. Recently, a group of spore-specific inner membrane (IM) proteins was identified as increasing IM rigidity and spore wet heat resistance. B. subtilis has five of these proteins, with multiple homologs in all Bacillus and Clostridium species. These proteins increase IM rigidity, which increases spore wet heat resistance and can either increase or decrease the rates of spore germination, with similar effects on B. megaterium spores. These proteins are thus a new factor important in spore properties.

Ohmic heating (OH) achieves superior spore inactivation compared to conventional heating (CH) under identical temperature conditions. This study examined the impact of different frequencies (60 Hz, 1 kHz, and 5 kHz) and temperatures (95 degrees C, 105 degrees C, and 115 degrees C) at various applied field strengths (30 V/cm, 40 V/cm, and 50 V/cm) during OH on spores of Bacillus subtilis. To gain more insight on which spore components might interact with the field, we tested spore strains 533 (wild type), 578 (SASP-deficient), and 2318 (RecA-deficient), in comparison with CH. A specialized apparatus enabled matching of temperature profiles between OH and CH, with all experiments conducted while holding electric field strengths constant while allowing temperature to rise linearly to a final set point without a holding time. Spore inactivation was independent of frequency under conditions of lowest (low field strength-temperature combinations) and highest (high field strength-temperature combinations) lethality; however differentiation between frequencies became apparent at intermediate levels of frequency. Where such differences could be observed, 1 kHz yielded the highest inactivation. Removal of SASP or RecA components did not alter this general trend. Differences between frequencies could neither be correlated to power inputs nor to calculated molecular displacements. More rigorous modeling approaches will be needed to more accurately delineate frequency effects.

ATP is one of the signature molecules of life. As the primary energy currency of cells, the debate concerning its involvement, if any, in driving the earliest biophysical events associated with germination of dormant Bacillota spores has continued without resolution for several decades. With a view to framing the debate, this article presents a synopsis of fundamental aspects of spore physiology coupled with key experimental observations in the context of bioenergetics and macromolecular synthesis. Evidently, neither the spore core nor the inner membrane present sub-cellular environments conducive to significant oxidative- or substrate-level phosphorylation, gene transcription, or protein translation activities. Additionally, neither the precursors of numerous critical macromolecules, nor the cellular apparatus required to synthesize these precursors, are present in dormant spores. Even if these might somehow be generated within localized micro-environments, the phosphorylation potential associated with the negligible quantities of ATP present in spores is severely reduced relative to actively metabolizing cells as a result of spores' sub-optimal adenylate energy charge. Thus, the scope for significant macromolecular synthesis is thermodynamically improbable. Looking ahead, clarity in the field of spore bioenergetics and metabolism will only be achieved by studies that unambiguously encompass the physiological constraints imposed by these most resolute cells.

Aim Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores.Methods and results We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' alpha/beta-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm.Conclusions In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

In response to extreme conditions, Bacillus subtilis generates highly resilient spores characterized by a unique multilayered structure. This confers resistance against various chemicals and enzymes yet adding complexity to the analysis of the spore proteome. As the first step in bottom-up proteomics, sample preparation poses a significant challenge. We assessed how an optimized protocol for sample preparation by easy extraction and digestion (SPEED) performed compared to previously established methods “One-pot” (OP) and single-pot, solid phase-enhanced sample-preparation (SP3) for the proteomic analysis of B. subtilis cell and spore samples. We found that SPEED outperformed both OP and SP3 in terms of peptides and proteins identified, moreover SPEED highly reproducibly quantified over 1000 proteins in limited input samples as low as 1 OD600 of B. subtilis cells and spores. SPEED was applied to analyze spore samples of different purity by applying sequential purification following harvesting of spores. Comparison of the differential abundance of proteins revealed clusters likely partially stemming from remaining vegetative cells in less purified spore samples. We show that ranking of absolute protein abundance in cellular and spore samples further enables us to rationally differentiate integral spore proteins from vegetative remnants. This is of importance in applications and organisms where highly homogenous spore samples are difficult to obtain. A deep proteomic analysis of spore and vegetative cell samples with the new approach led to the identification of 2447 proteins, 2273 of which were further quantified and compared between B. subtilis spores and cells. Our findings indicate that pathways related to peptidoglycan biosynthesis, glycolysis, carbon metabolism, and biosynthesis of secondary metabolites are shared between cells and spores. This corroborates and extends earlier work stressing that despite marked differences in their physiological states, spores preserve vegetative cell (core) proteins, essential for revival under conditions conducive to growth. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores.