The Molecular Timeline of a Reviving Bacterial Spore (2024)

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  We reveal the identity of the newly synthesized proteins throughout spore revival

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  We define the timeline of molecular events occurring during spore revival

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  Protein synthesis occurs during germination and is essential for its execution

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  RpmE and Tig are required for protein synthesis during germination

Determining the Temporal Landscape of the Newly Synthesized Proteins during Spore Revival

The remarkable transition from a dormant spore to a fully active cell offers a unique opportunity to follow the molecular events required to build a cell. To explore this transition, we attempted to determine the proteomic landscape of a reviving spore throughout germination, ripening, and outgrowth. To differentiate the newly synthesized proteins during revival from the pre-existing spore protein pool, we employed the BONCAT (BioOrthogonal Non-Canonical Amino-acid Tagging) protein tagging technique (Figure1A; see Experimental Procedures) (Dieterich etal., 2007). BONCAT allows the specific labeling and identification of newly translated proteins due toincorporation of an azide-bearing artificial amino acid termed azidohom*oalanine (AHA), which is a substitute for methionine.

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Figure1

Identifying the Newly Synthesized Proteins in the Course of Spore Revival

(A) Schematic description of the BONCAT strategy (Dieterich etal., 2007) for labeling, detection, and identification of the newly synthesized proteins tagged with AHA during spore revival. The general flow of the procedure (left) and detailed illustration of specific steps (right) are shown. Spores of LS5 (ΔmetE) strain are incubated in revival medium containing AHA (red hexagons), and the newly synthesized proteins (red curved lines) are separated from the existing spore proteins (blue curved lines). Spore proteins are extracted and tagged with TAP tag (green circles) using click chemistry (1, right). Next, newly produced proteins are isolated using avidin beads and identified by mass spectrometry (2, right). Scale bar represents 1μm.

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(B) Spores of LS5 (ΔmetE) strain were incubated in revival medium in which methionine was replaced by AHA. The sequence of events during revival is shown as captured by phase contrast images: germination is visualized as the loss of spore brightness (red line), the ripening period in which nomorphological change is evident (orange line), and outgrowth is characterized by increasing cell length (green line). The average length of the reviving spores is presented below. Scale bar represents 1μm.

(C) Spores of LS5 (ΔmetE) strain were incubated in revival medium and optical density (OD₆₀₀) was measured at the indicated time points. Data are presented as a fraction of the initial OD₆₀₀ of the phase-bright spores. Decreasing OD₆₀₀ signifies spore germination, and increasing OD₆₀₀ indicates spore outgrowth (Moir and Smith, 1990).

(D) Dot blot analysis of protein samples (in duplicates) collected throughout revival of LS5 (ΔmetE) spores at the indicated time points. Samples werediluted (1:100 and 1:200) and spotted on amembrane that was subsequently probed withanti-biotin antibodies. The obtained signal wascompared with known amounts of biotinylatedBSA.

Since B.subtilis is capable of synthesizing methionine, we constructed an auxotrophic strain to enhance AHA uptake by deleting the metE gene. Cells were induced to sporulate in the presence of methionine, and the resulting spores were purified. Spores were then subjected to revival in minimal medium containing the germination triggering molecules AGFK and l-alanine, supplemented with amino acids excluding methionine, which was replaced by AHA. The auxotrophic B.subtilis spores revived normally in the presence of AHA, likely due to sufficient amounts of endogenous methionine contained within spores (Setlow, 1988). As can be seen, germination, determined as the transition from a phase-bright spore to a phase-dark cell (Figure1B), wasaccompanied by a drop in OD₆₀₀ and took place from 0 to 15min (Figure1C). The subsequent ripening period, in which no morphological changes were evident, extended from 15 to 60min (Figures 1B and 1C), and finally outgrowth, characterized by increase in cell length and OD₆₀₀, occurred between 60 and 150min (Figures 1B and 1C). Proteins were extracted from dormant spores (t= 0) and at 30min intervals during revival until the first vegetative division took place at t= 150min (Figure1B). The extracted protein samples were then incubated with an alkyne-bearing biotin-Flag tag (TAP tag) to covalently label the newly synthesized proteins harboring AHA (Figure1A). The amount of tagged proteins was estimated using dot blot analysis with antibody against biotin. Synthesis of labeled proteins was readily detected at t= 30min, increasing gradually with time (Figure1D). To enrich for newly synthesized proteins, samples were incubated with NeutrAvidin beads, and the captured proteins were cleaved with trypsin and identified by mass spectrometry (Figure1A). At least two valid peptides per locus or one peptide containing an AHA-derived modification served as the minimal requirement to classify a detected protein as translated during the AHA-labeling step (Dieterich etal., 2007).

Utilizing this approach, a total of 653 proteins were identified as translated during the transition from dormant state to vegetative growth (Table S1 available online). Notably, 217 newly synthesized proteins were detected as early as 30min into revival (Table S1). The synthesis of these proteins was largely dependent on de novo transcription, since when spores were subjected to revival in the presence of the two transcriptional inhibitors rifampicin and actinomycin D, only 12 proteins were detected at the same time point (Table S2). Additional 158 newly identified proteins were monitored at t= 60min, when spores were at the ripening phase. At t= 90min, when outgrowth was evident, 176 new proteins were detected. Finally, about 50 proteins were added to the pool at each subsequent time point during the transition to vegetative growth (Table S1).

To analyze the awakening pattern of molecular processes upon revival, we categorized the identified proteins into distinct groups according to their molecular function, using various B.subtilis genomic databases (Subtiwiki, GenoList, Uniport) (Table S1). A global view of this transition is presented in Figure2. The activated cellular processes display different patterns, with several having all protein components detected at the first timepoint (e.g., glycolysis, ATP synthesis), while others exhibit a gradual awakening pattern (e.g., utilization of amino acids, rRNA and tRNA processing). Further, some pathways, in which all components were initially dormant, displayed synchronized synthesis at a given time point (e.g., fatty acid and teichoic acid biosyntheses). To corroborate our BONCAT-based data, representative proteins from different categories were fused to GFP, and their production during revival was followed. Indeed, fluorescence from GFP precisely coincided with the time of protein detection by BONCAT (Figure3).

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Figure2

The Temporal Landscape of the Newly Synthesized Proteins during Spore Revival

Spores of LS5 (ΔmetE) strain were incubated in revival medium in which methionine was replaced by AHA. Samples were collected at the indicated time points and processed as described in Figure1A. The newly synthesized proteins were categorized into groups representing different molecular processes. The total number of proteins related to each group is indicated in parenthesis (data extracted from Table S1). The colors (see colors bar) and numbers (in white) indicate the relative fraction of proteins that was detected at a specific time point out of the total identified proteins for each group throughout revival. The numbers below summarize the amount of newly detected proteins out of the total detected proteins at each time point. Shown above are corresponding phase contrast images of a single reviving spore. Scale bar represents 1μm.

See also FigureS1.

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Figure3

Monitoring Fluorescence of Representative Proteins Fused to GFP throughout Spore Revival

Representative proteins from the indicated functional groups (derived from Table S1 and Figure2) were fused to GFP and monitored for time of production during spore revival using fluorescence microscopy. GFP fluorescence images acquired at the indicated time points are shown. Upper panels show phase-contrast images of a strain harboring malS-gfp. For each strain, a representative experiment of three independent biological repeats is shown. Quantification of the fluorescence signal in arbitrary units (a.u.) is shown on the right. Fluorescence from at least 300 cells from three different fields was measured and averaged for each time point. The intensity of a WT (PY79) strain, lacking the gfp gene, was subtracted from the net average fluorescence intensity. Red arrows indicate the earliest time point at which a representative protein was identified by BONCAT. Scale bar represents 1μm.

The Early Spore Revival Proteome

During the first 30min of the revival process, the spore undergoes germination (after 15min, approximately 80% of the spores complete germination) and enters the ripening period (Figures 1B and 1C). During these initial stages, 217 proteins were detected, which corresponds to 33% of the spore revival proteome, demonstrating rapid acquisition of metabolism. Accordingly, the core components of the transcriptional and translational machineries were produced along with a group of chaperones required for proper protein folding. Energy generation machineries, including all ATP synthesis, glycolysis, and pyruvate dehydrogenase (PDH) components, were readily detected at this time, providing fuel to the ongoing revival process (Figures 2 and ​and3;3; Table S1).

Notably, the metabolic pathway components required for malate utilization, including all four malic enzymes (MaeA, MalS, MleA, and YtsJ) that convert malate into pyruvate through oxidative decarboxylation (Meyer and Stülke, 2013), were produced at this early stage (Figures 2 and ​and3;3; Table S1). Malate, which is a preferred carbon source for B.subtilis (Meyer and Stülke, 2013), was not supplemented into the medium, implying that the spore stores malate as a key carbon source to energize revival. Consistent with this view, spores compared with vegetative cells were found to harbor large amounts of malate (FigureS1A). Furthermore, a strain deleted of all four genes encoding malic enzymes showed no detectable growth defect but displayed an extended ripening period in comparison to reviving WT spores (Figures S1B–S1D). Additionally, spores of the quadro mutant contained malate levels similar to those of WT spores (FigureS1A), indicating that the observed ripening defect is due to deficiency in malate utilization.

The Progression of the Ripening Period

No morphological change was evident 60min into spore revival, indicating that the spore was still at the ripening period, although a substantial group of 158 additional proteins was produced at this time point (Figure2; Table S1). The pentose phosphate pathway was among the most prominent fully activated processes. This pathway is required to generate pentoses and NADPH; the former is essential for nucleotide biogenesis, whereas the latter is required for fatty acid biosynthesis (Zamboni etal., 2004). In line with this finding, processes related to lipid biosynthesis were fully activated after 60min, including fatty acid and phospholipid formation and production of malonyl-CoA, indicating that membrane construction was initiated (Figure2; Table S1).

A manifested activation was also observed for the pyrimidine biosynthesis pathway, in which all 14 proteins involved where detected at t= 60min. Notably, most of the purine biosynthesis proteins were already detected 30min into revival (Figure2; Table S1). These observations are consistent with the premise that the pur genes are induced in response to glucose contained in the revival medium, whereas the pyr genes are known to be controlled by pyrimidine availability (Blencke etal., 2003). This proteomic profile supports a model whereby RNA transcription is initially based on the spore pre-existing nucleotide reservoir, and only during ripening newly generated nucleotides are utilized. This model is in accord with the observation of Setlow and Kornberg (1970a) that de novo nucleotide biosynthesis initiates early during revival and relies on re-establishment of translation, as well as with our earlier finding that during ripening the spore is engaged in synthesizing large amounts of rRNA required to build up new ribosomes (Segev etal., 2013).

Along with membrane and nucleotide biosynthesis, several pathways related to cofactor production were induced at this time, including proteins involved in NAD, riboflavin, pyridoxal-phosphate, folate and coenzyme A production, which are required for numerous enzymatic activities. In parallel, general and oxidative stress proteins were highly induced (Figure2; Table S1). This activation might be associated with the hydration of the spore core and activation of metabolism, events likely to generate oxidative stress (Ibarra etal., 2008).

From Outgrowth into Vegetative Growth

At 90min into revival, spore size was increased (Figures 1B and 1C), indicating the initiation of outgrowth. Accordingly, processes related to cell wall synthesis and cell elongation were fully activated, as evidenced by the production of proteins involved in peptidoglycan (PG) biosynthesis and cell shape determination (Figure2; Table S1). Furthermore, the main energy producing cellular pathways, tricarboxylic acid cycle (TCA) and respiration, were entirely induced to facilitate spore growth (Figure2; Table S1). At this point, the spore was capable of generating the complete set of building molecules, essential for cell outgrowth and elongation. In light of this increased metabolic activity, the spore produced DNA metabolic proteins required to repair the damaged genome and to carry out replication in preparation for the upcoming division (Figures 2 and ​and3;3; Table S1).

A more significant increase in size of the reviving spore was apparent at t= 120min, with the spore accomplishing the conversion into a rod shape vegetative cell by t= 150min (Figure1B). Approximately 100 proteins were added to the revival proteome during these later phases, with the majority being components associated with ongoing processes. Nevertheless, three major pathways, including cell division, biosynthesis of teichoic and lipoteichoic acids, and protein secretion, were largely activated only at t= 120min (Figures 2 and ​and3;3; Table S1). Interestingly, the assembly of the division machinery correlated with the observed morphological changes. A total of 10 bona fide cell division proteins were identified during the course of spore revival. The two initial division components, FtsZ and FtsA, were detected as early as 60min post-induction of revival, while the others were first identified only at t= 120min (Figure2; Table S1). Observing the dynamics of FtsZ-GFP during revival revealed detectable fluorescence at t= 60min; however, Z-ring formation occurred only at t= 120min (Figure3). Consistently, EzrA, one of the late cell division proteins, was produced and localized to the division site of a reviving spore only at t= 120min (Figure3). Thus, it seems that the temporal production of cell division components contributes to the proper assembly of the division ring. A similar gradual activation pattern was observed for components required for cell wall construction. Notably, the PG synthesizing enzymes were produced at t= 90min, early during outgrowth, while the teichoic and lipoteichoic acids biosynthesis enzymes were produced later on (Figures 2 and ​and3;3; Table S1). This sequential pattern of cell wall production suggests that PG construction precedes teichoic and lipoteichoic acid synthesis.

Protein Synthesis Is Carried Out during Germination and Required for the Process to Occur

The first 30min of revival are composed of two phases: germination, taking place approximately at the first 15min, and early ripening (Figure1C). We next zoomed into this time period to investigate the long-outstanding question of whether protein synthesis takes place during germination. It has been shown previously that Bacilli spores are capable of undergoing germination in the presence of RNA and protein synthesis inhibitors (Steinberg etal., 1965; Vinter, 1970). However, some doubts have been raised concerning these observations, as drugs may not be capable of penetrating the spore protective shells at this early stage (Sussman and Douthit, 1973; Tisdale and DeBusk, 1972). To elucidate this issue, we incubated spores with antibiotics that inhibit translation and are soluble in ethanol, which was shown to increase spore permeability (Tanimoto etal., 1996). Remarkably, when spores were incubated with either lincomycin or tetracycline and then induced to germinate with l-alanine, approximately 80%–90% of the spore population was clearly inhibited from germinating, as manifested by their phase bright appearance (Figure4A; Table S3). Moreover, incubation with both antibiotics blocked germination in more than 99% of the spores (Table S3), indicating that translation is required for the process. Further analysis of the antibiotic treated spores revealed that upon induction of germination, they released DPA at kinetics similar to that of untreated spores and lost their heat resistance (Figure4B; Table S4). Thus, the antibiotic-treated spores initiated germination, but were most likely blocked prior to cortex hydrolysis as they still remained phase bright (Moir and Smith, 1990).

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Figure4

Protein Synthesis Is Carried Out during Germination and Required for the Process to Occur

(A) PY79 (WT) spores were incubated at 37°C for2hr in ethanol with or without lincomycin (250μg/ml). Next, spores were washed with phosphate buffer and incubated with l-alanine for 30min. Shown are phase-contrast images of spores incubated with (right) or without (left) lincomycin, before (t= 0) and after (t= 30min) l-alanine addition. Scale bar represents 1μm.

(B) PY79 (WT) spores were incubated at 37°C for 2hr in ethanol with or without lincomycin (250μg/ml) or tetracycline (250μg/ml). Next, spores were washed with phosphate buffer and incubated with l-alanine. The DPA release to the medium was determined as a measurement of OD₄₄₀. The nongerminating strain RU97 (ΔgerAA) was used as a control.

(C and D) Spores of LS5 (ΔmetE) strain were induced to germinate with l-alanine (C, left), AGFK (C, right) for 30min or with Ca-DPA (D) for 60min in the presence of AHA. Shown are dot blot analyses of protein samples (in duplicates) that were collected before (t= 0, dormant) and after (t= 30, germinated) germination induction. Samples were diluted (1:100 and 1:10) and spotted on a membrane that was subsequently probed with antibiotin antibodies. The obtained signal was compared with known amounts of biotinylated BSA.

(E) Spores of LS5 (ΔmetE) strain were incubated at 37°C for 2hr in ethanol with (+Lin) or without (–Lin) lincomycin (250μg/ml) or in double-distilled water (DDW) (control). Next, spores were washed with phosphate buffer and incubated with l-alanine and AHA for 30min. As a negative control, spores incubated in DDW were also germinated in the presence of methionine instead of AHA (Met). Shown is a dot blot analysis conducted as in (C and D).

We next utilized the BONCAT technique to substantiate that proteins are produced during germination. We took advantage of the observation that dormant spores undergo germination without the ability to execute outgrowth when the germinant factors l-alanine or AGFK are introduced in the absence of additional nutrients (Setlow, 2003). Accordingly, spores were incubated with l-alanine or AGFK in the presence of AHA and the absence of other nutrients. Proteins were extracted, incubated with TAP tag, and dot-blot analysis conducted. Synthesis of labeled proteins was evident for both l-alanine and AGFK germinating spores (Figure4C). Repeating this assay with the non-nutrient germinant Ca-DPA (Setlow, 2003) corroborated that proteins are being synthesized during germination without the need for external nutrients (Figure4D). Furthermore, protein synthesis was almost completely abolished in the presence of lincomycin (Figure4E).

Labeled proteins synthesized during germination were then identified by mass spectrometry. A total of 116 newly synthesized proteins were monitored upon AGFK addition, while 32 and 27 proteins were identified when l-alanine and Ca-DPA were utilized as germinant factors, respectively (Table S5). Analysis of the synthesized proteins revealed that the majority of the proteins detected following l-alanine or Ca-DPA addition were identical (24 of the identified proteins) and included in the list of proteins monitored upon AGFK supplementation (Table S5). The proteins monitored in all assays were part of the revival proteome at t= 30min (Tables S1 and S5). A comparison of the obtained proteomes when categorized into functional groups provided a higher resolution of the earliest events occurring during revival (Table S6). Ca-DPA and l-alanine, which are relatively minimal sources that can trigger spore germination, activated synthesis of proteins categorized into glycolysis, PDH complex, malate utilization, translation elongation, chaperones, and oxidative stress (Table S6). On the other hand, the combination of AGFK triggering molecules supported the synthesis of components belonging to additional pathways, including transcription and translation machineries, electron transport, RNA degradation, and proteolysis (Table S6).

We conclude that protein synthesis is carried out during germination and necessary for its completion. Furthermore, the small group of proteins synthesized in response to Ca-DPA and l-alanine may exclusively represent the germination proteome.

The Translational Factors RpmE and Tig Play a Key Role in Spore Germination

A detailed investigation of the proteins produced in response to l-alanine revealed four newly synthesized translational factors RplU, RplP, RpmE, and Tig (Table S5). RplU, RplP, and RpmE are ribosomal components constituting the 50S subunit (Akanuma etal., 2012), whereas Tig is a ribosome-associated chaperone, which interacts directly with emerging nascent polypeptides (Merz etal., 2008). We reasoned that these factors might be involved in translation of proteins during germination. Since rpmE and tig genes are nonessential for vegetative growth (Akanuma etal., 2012; Reyes and Yoshikawa, 2002), we constructed strains deleted for these genes and followed the ability of mutant spores to revive. Remarkably, both ΔrpmE and Δtig mutant spores exhibited an apparent germination deficiency, as evidenced by their delay in converting from a phase-bright to a phase-dark state in comparison to the WT spores (Figures 5A and 5B). Moreover, spores harboring deletion of both factors displayed a more severe germination defect (Figures 5A and 5B). The ability of these spores to eventually germinate suggests that additional translational factors, such as the essential components RplU and RplP (Akanuma etal., 2012), mediate this developmental transition. Closer investigation of the germinating mutant spores revealed that they released DPA and turned heat sensitive similarly to WT spores (Figure5C; Table S4). Thus, like the antibiotic treated spores, the mutant spores initiated germination but failed to efficiently progress throughout the process. Analysis of the mutant strains during vegetative growth revealed that the Δtig strain grew similarly to WT, while the ΔrpmE strain exhibited only a slight growth perturbation, suggesting that both genes acquired unique germination properties (FigureS2A). Importantly, ΔrpmE and Δtig mutant strains sporulated similarly to WT cells (FigureS2D), and their germination defect was rescued by ectopic insertion of the corresponding WT alleles (Figure5B). Since RpmE and Tig are bona fide translational factors, they could affect the global protein level within spores. It was therefore plausible that the defective germination displayed by the mutant strains was due to decreased amounts of germination receptors in spores. To rule out this possibility, we determined the levels of GerAA, GerAC, GerBC, GerKA, and SpoVAD in mutant and WT spores by western blot analysis (Ramirez-Peralta etal., 2012). The levels of all the investigated proteins were similar in WT and in the mutant spores (Figures S2B and S2C). In addition, the mutant spores showed lysozyme resistance levels similar to WT spores (FigureS2E), indicating that their coat is intact. Taken together, these results imply that the observed defect of the mutant strains is not due to a general deficiency in translation during sporulation but is specific to germination.

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Figure5

The Translational Factors RpmE and Tig Play a Key Role in Spore Germination

(A) Spores of PY79 (WT), LS38 (Δtig), LS26 (ΔrpmE), and LS78 (Δtig ΔrpmE) strains were incubated in LB medium supplemented with l-alanine (4mM) and monitored by time lapse microscopy. Shown are phase contrast images from a representative experiment out of three independent biological repeats. Scale bar represents 1μm.

(B) Spores of PY79 (WT), LS38 (Δtig), LS26 (ΔrpmE), LS78 (Δtig, ΔrpmE), LS101 (Δtig, amyE::tig), and LS100 (ΔrpmE, amyE::rpmE) strains were incubated with AGFK+ l-alanine, and optical density (OD₆₀₀) was measured at the indicated time points. Data are presented as a fraction of the initial OD₆₀₀ of the phase-bright spores. Decreasing OD₆₀₀ signifies spore germination (Moir and Smith, 1990).

(C) Spores of PY79 (WT), LS38 (Δtig), LS26 (ΔrpmE), LS78 (Δtig, ΔrpmE), and RU97 (ΔgerAA) strains were incubated with l-alanine to trigger germination. DPA release to the medium was determined as a measurement of OD₄₄₀.

(D) Spores of LS5 (ΔmetE) (WT), LS82 (ΔmetE, Δtig), and LS83 (ΔmetE, ΔrpmE) strains were induced to germinate with l-alanine in the presence of AHA for 60min. Shown is a dot blot analysis of protein samples (in duplicates) collected 30 and 60min after germination induction. Samples were diluted (1:100 and 1:10) and spotted on a membrane that was subsequently probed with antibiotin antibodies. The obtained signal was compared with known amounts of biotinylated bovine serum albumin.

See also FigureS2.

To substantiate that RpmE and Tig are required for protein production during germination, the BONCAT system was used to follow protein synthesis of ΔrpmE and Δtig mutant spores during this phase. To this end, spores of the different strains were incubated only with l-alanine and AHA, and proteins were extracted from spores at 30 and 60min post-germination induction and subjected to tagging with TAP tag followed by dot blot analysis. No detectable protein synthesis took place in the ΔrpmE and Δtig mutants at t= 30min, whereas protein synthesis was clearly evident in WT spores (Figure5D). After 60min, relatively small amounts of newly synthesized proteins were detected in the ΔrpmE and Δtig mutants, approximately 10-fold less than those found in WT spores (Figure5D).

Finally, we wished to observe protein synthesis during germination in real time. To achieve this goal, we investigated the production of MalS-GFP fusion by time lapse microscopy, as MalS is oneof the earliest proteins produced in germinating spores (Table S5). Remarkably, WT spores harboring MalS-GFP fusion exhibited a dramatic increase in the GFP signal as early as 5min after germination induction by l-alanine. Evidently, under these conditions GFP folding was faster than previous estimates (Tsien, 1998). At this time, spores were clearly residing in their phase-bright state, indicating that germination was still in progress (Figure6A). As a control, we monitored the production of PupG-GFP that was detected in spores (Figure3), but was excluded from the germination proteome (Table S5). Consistent with our data, fluorescence from PupG-GFP did not increase during germination (FigureS3A). Furthermore, the increase in fluorescence from MalS-GFP was considerably delayed in ΔrpmE and Δtig mutant spores, detectable only 40min after germination induction (Figure6B). Of note, mutant spores that exhibited an increase in fluorescence progressed through germination and completed the process at the subsequent time point (Figure6B, circles). These results were further corroborated by western blot analysis (Figures 6C and S3B).

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Figure6

Real-Time Protein Synthesis during Spore Germination

(A and B) Spores of AR71 (WT) (A) and LS80 (Δtig) and LS81 (ΔrpmE) (B) harboring malS-gfp were incubated with l-alanine and followed by time lapse microscopy. Shown are phase contrast (upper) and fluorescence (lower) images taken at the indicated time points. Dashed circles in (B) highlight spores in which increase in MalS-GFP fluorescence is followed by germination. For each strain, images were scaled to the same intensity range. The intensity of a WT (PY79) strain, lacking the gfp gene, was subtracted from the net average fluorescence intensity. A representative experiment out of three independent biological repeats is shown. Scale bars represent 1μm.

(C) Spores of AR71 (WT, malS-gfp), LS81 (Δtig, malS-gfp), LS82 (ΔrpmE, malS-gfp), and AR68 (wild-type, pupG-gfp) strains were induced to germinate with l-alanine, and proteins were extracted before (t= 0) and after (t= 5min) l-alanine addition. Equal amounts of protein extracts were subjected to western blot analysis. Membrane was probed with antibody against GFP. Proteins extracted from PY79 spores lacking gfp (t= 5min) were used as a negative control (Neg).

We surmise that protein synthesis during germination considerably relies on the translational factors RpmE and Tig, which are produced early during the process.

Strains and General Methods

B.subtilis strains are derivatives of PY79 and are listed in Table S7. Plasmids construction is described in Supplemental Experimental Procedures. Sporulation was induced at 37°C by suspending cells in Schaeffer’s liquid medium (Difco Sporulation Medium [DSM]) (Harwood and Cutting, 1990), and mature spores were purified as described in Supplemental Experimental Procedures. Purified spores were heat activated (80°C, 30min) prior to germination and revival experiments. Unless indicated differently, spore revival was induced at 37°C by suspending spores in revival medium composed of S7minimal medium (Vasantha and Freese, 1980) supplemented with AGFK (2.5mM l-aspargine, 5mg/ml d-glucose, 5mg/ml d-fructose, 50mM KCl), 0.01M alanine, and all additional amino acids in concentrations suitable for B.subtilis (Harwood and Cutting, 1990). For BONCAT experiments, AHA (200mg/l; Anaspec) was used instead of methionine. For germination induction by l-alanine and AGFK, heat-activated spores were incubated for 30min, a time in which 99% of the WT spores completed germination. For germination induced byCa²⁺-DPA, heat-activated spores were incubated in 60mM Ca²⁺-DPA (pH 8.3) for 60min at room temperature.

BONCAT Spore Revival Experiments

Cultures of 100ml of reviving spores were centrifuged and washed with PBS ×1. Pellets were resuspended in PBS ×1 supplemented with protease inhibitors (Thermo, 78439), lysed using FastPrep (MP) (6.5, 60 s, ×3), and centrifuged (5min, 14,000 RPM). Under these conditions, less than 0.01% of the spore population remained intact. Supernatants containing a mixed population of AHA-labeled newly synthesized proteins and unlabeled pre-existing proteins were collected. The enrichment for newly translated proteins was performed using BONCAT, as previously described (Dieterich etal., 2007), with the following modifications. For tagging of AHA-labeled proteins, samples were incubated overnight at 4°C with triazole ligand (0.25mM; Sigma), alkyne-bearing biotin-flag tag (0.063mM; Genscript), and CuBr (2mM in DMSO; Sigma). Trypsin-digested samples were analyzed by LC-MS/MS on LTQ-Orbitrap (Thermo). Each presented BONCAT dataset is based on three independent biological repeats. The proteins displayed in Tables S1, S2, and S5 were detected in all repeats. In each experiment, at least 35% of the proteins were identified based on AHA containing peptides, and the majority of the proteins (>95%) were identified based on at least three different peptides. In each experiment, a methionine sample was added as a control. The number of identified proteins in the methionine control samples was always below 1% of the proteins identified in the parallel AHA samples and included mainly abundant spore specific proteins. These proteins were excluded from the analysis.

Additional experimental procedures, including light microscopy, spores purification, l-malate determination, DPA measurements, determination of the levels of spore germination proteins, western blot analysis, and spore resistance to lysozyme treatment, are described in the Supplemental Experimental Procedures.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Document S1. Supplemental Experimental Procedures, Figures S1–S3, and Tables S2–S4, S6, and S7:

Table S1. The Proteomic Timeline of Spore Revival:

The proteomic timeline of reviving LS5 (ΔmetE) spores as determined by BONCAT. The analysis is based on three independent biological repeats. Only proteins detected in all repeats are displayed.

Table S5. The Spore Germination Proteome:

The proteome of germinating LS5 (ΔmetE) spores incubated with l-alanine, AGFK, or Ca-DPA, as determined by BONCAT. The analysis is based on three independent biological repeats. Only proteins detected in all repeats are displayed.

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