Metabolic differentiation and intercellular nurturing underpin bacterial endospore formation – Science Advances

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Experimental methods

Strain construction. All the strains used in this study are derivatives of B. subtilis PY79. A complete list of strains is provided in table S2. The plasmids and oligonucleotides used to construct the different strains are provided in tables S3 and S4, respectively. Detailed descriptions of plasmid construction can be found in Supplementary Text.

Here, we provide a description of the proteins tagged with ssrA* (Fig. 2 and figs. S2 and S3). TCA cycle: CitZ, major citrate synthase (this protein was degraded in a background lacking the minor citrate synthase, CitA); CitB, aconitase; Icd, isocitrate dehydrogenase; OdhB, E2 subunit of 2-oxoglutarate dehydrogenase; SucD, α subunit of succinyl–coenzyme A (CoA) synthetase; FumC; fumarase; Mdh, malate dehydrogenase. The addition of the ssrA* tag to components of the succinate dehydrogenase complex severely affected protein functionality, and they were excluded from this study. Amino acid biosynthetic proteins: ArgH, argininosuccinate lyase, required for arginine synthesis; IlvD, dihydroxy-acid hydratase, required for the synthesis of branched-chain amino acids (leucine, isoleucine, and valine); ThrB, homoserine kinase, required for threonine synthesis. Ribosomal proteins: RpsB, ribosomal protein S2; RplL, ribosomal protein L12.

Culture conditions. B. subtilis strains were routinely grown on LB plates at 30°C. We induced sporulation in two different ways: (i) by resuspension in A + B sporulation medium containing glutamate as the sole carbon source (25), after growing the bacteria in one-fourth diluted LB to OD600 (optical density at 600 nm) ~ 0.5 (26) (sporulation induction was considered to be the moment in which the cells were resuspended in A + B medium), and (ii) by exhaustion in DSM (27), containing beef extract and peptones as carbon and nitrogen sources. In DSM, the cells grow until the nutrients become limiting, and sporulation is induced at the onset of stationary phase, which is characterized by a plateau in the growth curve. Sporulation cultures were grown at 37°C for batch culture experiments and at 30°C for time-lapse microscopy experiments. Arginine auxotrophic strains lacking both ArtPQR and RocC were cultured in LB medium supplemented with a high concentration of l-arginine (10 mM), which allowed enough nonspecific import of l-arginine to support growth.

Fluorescence microscopy. Cells were visualized on an Applied Precision DV Elite optical sectioning microscope equipped with a Photometrics CoolSNAP-HQ2 camera. Images were deconvolved using SoftWoRx v5.5.1 (Applied Precision). The median focal planes are shown.

Batch culture microscopy to follow sporulation progression

We used microscopy to assay for the completion of two developmental milestones: engulfment and forespore maturation (fig. S3F). Engulfment completion was determined using a well-characterized membrane fission assay (28). Briefly, cells are treated with a red membrane-impermeable membrane dye, FM 4-64, and a green membrane-permeable membrane dye, MitoTracker Green. During engulfment, forespore membranes are accessible to both the red and green dyes. However, after engulfment, forespore membranes cannot be stained by the red dye and are clearly visible as green ovals inside the mother cells. Forespore maturation was assessed by phase-contrast microscopy. Mature spores contain low water content, which endows them with a bright appearance under phase-contrast microscopy. This allows the extent to which developing spores become phase bright to be used as a proxy for spore maturation.

For batch culture microscopy, cells contained in 12 μl of culture were transferred to 1.2% agarose pads, prepared using A + B medium or one-fourth diluted DSM, either 3 or 6 hours after sporulation induction. Membranes were stained with FM 4-64 (0.5 μg/ml) (Life Technologies) and MitoTracker Green (1 μg/ml) (Life Technologies). FM 4-64 was added to the agarose pad, whereas MitoTracker Green was mixed with the cells before transferring them to the pad.

Time-lapse microscopy

For time-lapse microscopy, sporulation was induced at 30°C. To visualize the membranes, FM 4-64 (0.5 μg/ml) was added to the culture 1 hour after sporulation induction and incubated for two additional hours under standard culturing conditions. Seven-microliter samples were taken 3 hours after resuspension and transferred to agarose pads prepared as follows: two-third volume of supernatant from the sporulation culture, one-third volume of 3.6% agarose in fresh A + B sporulation medium, and FM 4-64 (0.17 μg/ml). Pads were partially dried, covered with a glass slide, and sealed with petroleum jelly to avoid dehydration during time-lapse imaging (29). Pictures were taken in an environmental chamber at 30°C every 5 min for at least 2 hours. Excitation/emission filters were tetramethyl rhodamine isothiocyanate (TRITC)/CY5 for membrane imaging and fluorescein isothiocyanate (FITC)/FITC for GFP imaging. Excitation light transmission was set to 5% for membrane imaging and 32% for GFP imaging to minimize phototoxicity. Exposure time was 0.1 and 0.3 s for membrane and GFP imaging, respectively. For presentation purposes, sporangia were aligned vertically (with forespore on top) by rotating them using Photoshop.

Spore titer assays. Sporulation was induced in 2 ml of DSM or in 10 ml of A + B resuspension medium and was allowed to proceed at 37°C for 24 hours. Two milliliters of culture was then heated at 80°C for 20 min, serially diluted in 1× T-base, plated on LB, and incubated overnight at 30°C. Spore titers were calculated based on colony counts.

BONCAT. Sporulation was induced in either A + B medium or DSM, as indicated, at 37°C. One hour after sporulation induction, FM 1-43FX (0.5 μg/ml) (Thermo Fisher Scientific) was added to cultures. AHA (100 μM) (Thermo Fisher Scientific) was added to cultures 3.75 hours following sporulation induction and incubated under standard culturing conditions for 15 min.

Cells were fixed and permeabilized as described previously (30). Briefly, 0.5 ml of culture was fixed at room temperature for 20 min in a solution containing 20 mM sodium phosphate (pH 7.4), 2.6% (w/v) paraformaldehyde (EM Sciences), and 0.06% (w/v) glutaraldehyde (EM Sciences). Samples were washed three times with phosphate-buffered saline (PBS) and once with GTE [50 mM glucose, 10 mM EDTA, and 20 mM tris (pH 7.4)] and then resuspended in 200 μl of GTE. Fixed cells were deposited on 15-well glass multitest slides (AP Biomedicals LLC) that were pretreated with 0.1% poly-l-lysine (Sigma-Aldrich) for 5 min and washed twice with sterile water. Fixed cells were permeabilized by adding lysozyme (2 mg/ml) and incubating at room temperature for 4 min. Fixed cells on slides were washed three times with PBS.

Strain-promoted click labeling was performed roughly in accordance with previously described methods (31). All of the following incubations were performed in the dark to preserve the fluorescent signal. Fixed cells on multiwell slides were treated with 100 mM 2-chloroacetamide in tris (pH 7.4) and incubated at 37°C for 1 hour to block free thiol functional groups. Following the 1-hour incubation, Alexa Fluor 647 DIBO Alkyne (Thermo Fisher Scientific) was added directly to the 2-chloroacetamide solution to a final concentration of 1 μM and incubated at 37°C for an additional 30 min to promote click labeling. The 2-chloroacetamide dye mixture was then gently removed from the slide by aspiration. Unbound dye was removed through a series of washes, gently aspirating between each step. First, slides were treated with PBS and incubated at 37°C for 10 min. Next, slides were treated with 50% dimethyl sulfoxide in PBS and incubated at room temperature for 20 min. Then, slides were washed three times with PBS, incubating for 3 min at room temperature for each wash. Last, slides were washed and dehydrated with an increasing ethanol series, incubating for 3 min at room temperature for each 50, 80, and 96% ethanol. Slides were air-dried and equilibrated in equilibration buffer (Life Technologies) for 5 min and then washed again with fresh equilibration buffer. Slides were treated with antifade reagent in glycerol/PBS (Life Technologies) and covered with a glass slide.

All BONCAT experiments were performed in a ∆sigG and ∆spoIVA genetic background. sigG encodes the sigma factor responsible for late forespore gene expression. Because some degradation strains fail to complete engulfment efficiently and because early forespore gene expression continues in the absence of SigG (7), performing these experiments in a ∆sigG background ensured that any observed differences in protein synthesis was not a consequence of the failure to activate late gene expression. To facilitate quantification of mother cell and forespore protein synthesis, all BONCAT experiments were performed in strains lacking SpoIVA to block coat assembly around the forespore (fig. S5). The spore coat consists of more than 70 mother cell–synthesized proteins that encase the forespore and comprise 37% of the total spore protein (32). SpoIVA is a protein that constitutes the basement layer of the spore coat and is required for the recruitment and anchoring of coat proteins to the surface of the forespore, but not for the assembly of other coat layers (33). Therefore, ∆spoIVA mutants form aggregates of coat in the mother cell cytoplasm rather than at the forespore periphery, allowing mother cell protein synthesis to be easily distinguished from forespore protein synthesis.

Mother cell and forespore protein synthesis were quantified in sporangia from three microscopy fields using Fiji software. To determine protein synthesis levels, eight optical sections spanning 1.05 μm from deconvolved images were summed using Fiji’s Z Project tool. Mean Alexa Fluor 647 fluorescence intensities of the forespore and mother cell were determined separately by drawing a polygon encompassing the entire cell and then subtracting the mean background intensity plus 2 SDs. Values for the mother cell and the forespore were made relative to the median fluorescence signal of the respective cell type in an isogenic strain expressing only the –ssrA* tagged protein (for cell-specific inactivation of translation or the TCA cycle) or an isogenic wild-type strain (for Q-A mutants).

Fluorescence recovery after photobleaching. Sporulation was induced in A + B medium. Two hours after resuspension, the medium was supplemented with calcein AM (100 μg/ml) (Thermo Fisher Scientific), and the cells were incubated at 37°C for one extra hour. Then, cells were washed three times with 1 ml of fresh A + B medium without calcein AM and placed onto 1.2% agarose pads, supplemented with FM 4-64 to stain membranes. Before performing FRAP, a static membrane picture was taken (excitation/emission, TRITC/CY5; exposure time, 0.15 s) and was used as reference to determine the position of mother cells and forespores. The FRAP experiment was performed as follows: Before the photobleaching event, intracellular fluorescent calcein was imaged using FITC/FITC filters and 0.4-s exposure time. Subsequently, forespore calcein fluorescence was bleached with a 0.1-s pulse from a 488-nm argon laser set to 30% power, and calcein fluorescence images were collected every 3 s for 90 s, using the same filters and exposure time as the prebleach imaging. Quantification was performed as described previously (34). Briefly, we calculated the corrected fluorescence recovery (cFR) by determining the relative intensity of the bleached cell to the unbleached cell, and defining the prebleaching ratio as cFR = 1 and the ratio immediately following the bleaching event as cFR = 0. Average cFR curves were obtained by averaging fluorescence recovery values of different bleached cells at the same time points for each strain.

Fluorescence loss in photobleaching. Sporulation was induced in A + B medium. Two hours after resuspension, the medium was supplemented with calcein AM (100 μg/ml) (Thermo Fisher Scientific), and the cells were incubated at 37°C for one extra hour. Then, cells were washed three times with 1 ml of fresh A + B medium without calcein AM and placed onto 1.2% agarose pads, supplemented with FM 4-64 to stain membranes. Before performing FRAP, a static membrane picture was taken (excitation/emission, TRITC/CY5; exposure time, 0.15 s) and was used as reference to determine the position of mother cells and forespores. The fluorescence loss in photobleaching (FLIP) experiment was performed as follows: Before the photobleaching event, intracellular fluorescent calcein was imaged using FITC/FITC filters and 0.4-s exposure time. Subsequently, mother cell calcein fluorescence was bleached with a 0.1-s pulse from a 488-nm argon laser set to 70% power, and calcein fluorescence images were collected every 3 s for 90 s, using the same filters and exposure time as the prebleach imaging. A standard curve was generated by measuring the average fluorescence loss of cells distal to the bleaching event (fig. S7D), and this was used to correct for the loss of fluorescence caused by repeated image acquisition rather than intercellular transport (fig. S7E). Fluorescence values were background-subtracted and normalized such that the prebleach values were equal to 1 for both the mother cell and forespore and such that the mother cell values immediately following the bleaching event were equal to zero. Following correction using the standard curve, average normalized fluorescence values for mother cell and forespore were obtained by averaging normalized fluorescence values of different bleached cells at the same time points for each strain.

Forespore–to–mother cell GFP fluorescence ratio quantification. To be able to unambiguously distinguish if tagged proteins were present in the forespore or in the mother cell, we excluded membrane-associated proteins from the analysis. The ratio of GFP intensity between forespore and mother cell was measured semiautomatically using a custom script in MATLAB 2017b. Forespores and mother cell objects were identified and segmented from a single FM 4-64 channel by taking advantage of the increased fluorescence around the forespore. Thresholding high allowed identification of forespores, while thresholding low gave the whole cell outline. Subtracting the forespore image from the whole cell image allowed mother cells to be identified. Forespores were then paired to their corresponding mother cells first by filtering mother cell objects based on Euclidean distance and then by the orientation of the mother cell. These filters were made strict to reduce false matches. GFP fluorescence ratio was calculated by using the paired forespore–mother cell objects to mask the GFP fluorescence image to obtain a mean fluorescence intensity for both.

Quantification of native CitZ-GFP depletion. Mean GFP fluorescence intensity of the median focal plane was quantified separately for the forespore and mother cell by drawing a polygon encompassing the entire area of each cell type using Fiji. The mean background intensity was subtracted from each measurement. The mean fluorescence intensities for each cell type were averaged, and the initial values were normalized to one.

Quantification of phase-bright spores. Measurements of phase-bright spore intensity were obtained in a semiautomated manner from images of sporulating cells at 6 hours after induction using a custom script in MATLAB 2017b, applied to the median focal plane. FM 4-64, which stained the outer cell membranes, provided a fluorescent image to threshold, segment, and count the total number of cells present. The complement of the fluorescent FM 4-64 image provided a mask to segment the background for subsequent subtraction from the phase-bright image. In addition, a second membrane dye, MitoTracker Green, capable of diffusing into the cell, was used to stain fully engulfed forespore membranes. Thus, an image created from the difference in signals of both membrane dyes would only show fully engulfed forespores. Using this new image and filtering out those forespores, which contained objects from concurrent 4′,6-diamidino-2-phenylindole (DAPI) staining (as DAPI is unable to enter fully engulfed spores), ensured that all identified forespores were engulfed. For each forespore identified in this way, the average phase intensity was calculated, with the average background phase intensity removed. A phase intensity threshold of 0.1 arbitrary unit was used to constitute “phase bright.” This threshold was selected because it was the lowest value above which most of the engulfment completed wild-type population was found at 6 hours after sporulation induction. In addition, the populations of known phase-bright mutants failed to reach this threshold.

Stable isotope labeling with amino acids in cell culture. Sporulation was induced in A + B medium at 37°C. Cultures were supplemented with 0.1 mM l-arginine-13C6,15N4 hydrochloride (Sigma-Aldrich) 2 hours after sporulation induction, and sporulation was allowed to proceed for 72 hours. Formation of mature spores was monitored by phase-contrast microscopy. To achieve arginine uptake by the mother cell, artPQR was expressed from spoIIM or spoIVA promoters. To achieve arginine uptake by the forespore, artPQR was expressed from spoIIQ or spoIIR promoters. To maximize the incorporation of exogenous arginine into sporulation proteins, we used strains in which arginine synthesis was blocked in both the mother cell and the forespore by STRP-mediated degradation of ArgH-ssrA*. In addition, wild-type cells were sporulated in the absence of 0.1 mM l-arginine-13C6,15N4 hydrochloride to ensure that labeled peptides were only detected when the medium was supplemented with heavy arginine.

Spore purification. Seventy-two–hour sporulation cultures were pelleted and washed once with 4°C sterile water. To lyse remaining vegetative cells, spore samples were incubated overnight at 4°C in sterile water. The next day, the spores were pelleted by centrifugation, washed once with 4°C sterile water, and incubated again overnight at 4°C in sterile water. Spores were then further purified over a phosphate–polyethylene glycol aqueous biphasic gradient as previously described (35). The spore-containing organic phase (top) was harvested and washed with 50 or more volumes of 4°C sterile water at least three times. The spores were pelleted, resuspended in fresh sterile water, and stored at 4°C. Sample purity was evaluated using phase-contrast microscopy.

Extraction of spore coat proteins. Purified intact spores were diluted to approximately 5 × 109 spores/ml in 0.1 M NaOH and incubated at 4°C for 15 min, vortexing periodically. Samples were pelleted by spinning at 10,000g for 10 min at 4°C. The supernatant was applied to a 2-ml 3.5K molecular weight cutoff (MWCO) dialysis cassette (Thermo Fisher Scientific) and dialyzed against 1 liter of 0.5 M sodium acetate/acetic acid buffer (pH 5.0) and then against four changes of 1 liter of deionized water at 4°C over the course of 48 hours. Dialyzed material was harvested and submitted for MS analysis.

Extraction of small acid soluble proteins. Purified intact spores were diluted to approximately 5 × 109 spores/ml in 4°C 2 M HNO3 and incubated on ice for 30 min. Samples were pelleted by spinning at 10,000g for 10 min at 4°C. The supernatant was applied to a 2-ml 3.5K MWCO dialysis cassette (Thermo Fisher Scientific) and dialyzed against five changes of 1 liter of 1% acetic acid at 4°C over the course of 48 hours. Dialyzed material was harvested and submitted for MS analysis.

Sample preparation for MS. MS samples were prepared and analyzed by the University of California, San Diego Biomolecular and Proteomics MS Facility (http://bpmsf.ucsd.edu/). An equal volume of 6 M guanidine solution was added to 100 μl of protein extract and mixed. The samples were then boiled for 5 min followed by 5-min cooling at room temperature. The boiling and cooling cycle was repeated a total of three cycles. The proteins were precipitated with addition of methanol to a final volume of 90% followed by vortexing and centrifugation at maximum speed on a benchtop microfuge (14,000 rpm) for 10 min. The soluble fraction was removed by flipping the tube onto an absorbent surface and tapping to remove any liquid. The pellet was resuspended in 200 μl of 8 M urea made in 100 mM tris (pH 8.0). To reduce and alkylate samples, TCEP [tris (2-carboxyethyl) phosphine] was added to a final concentration of 10 mM and chloroacetamide was added to a final concentration of 40 mM and vortexed for 5 min. Three volumes of 50 mM tris (pH 8.0) were added to the sample to reduce the final urea concentration to 2 M. Samples were digested with trypsin (trypsin:protein ratio of 1:50) and incubated at 37°C for 12 hours. The solution was then acidified using trifluoroacetic acid (TFA) (0.5% TFA final concentration) and mixed. Samples were desalted using 100 mg of C18-StageTips (Thermo Fisher Scientific) as described by the manufacturer protocol. Samples were resuspended in sample loading buffer, and the peptide concentration was measured using bicinchoninic acid (BCA) (Thermo Fisher Scientific). A total of 1 μg was injected for each label-free quantification run.

LC-MS/MS analysis. Trypsin-digested peptides were analyzed by ultrahigh-pressure liquid chromatography (UPLC) coupled with MS/MS (LC-MS/MS) using nanospray ionization. The nanospray ionization experiments were performed using an Orbitrap Fusion Lumos Hybrid mass spectrometer (Thermo Fisher Scientific) interfaced with nanoscale reversed-phase UPLC (Thermo Dionex UltiMate 3000 RSLC Nano System) using a 25-cm, 75-μm ID (internal dimensions) glass capillary packed with 1.7-μm C18 (130) ethylene bridged hybrid (BEH) beads (Waters Corporation). Peptides were eluted from the C18 column into the mass spectrometer using a linear gradient (5 to 80%) of ACN (acetonitrile) at a flow rate of 375 μl/min for 1 hour. The buffers used to create the ACN gradient were as follows: buffer A (98% H2O, 2% ACN, and 0.1% formic acid) and buffer B (100% ACN and 0.1% formic acid). Mass spectrometer parameters were as follows: An MS1 survey scan using the Orbitrap detector {mass range [mass/charge ratio (m/z)]: 400 to 1500 (using quadrupole isolation), 120,000 resolution setting, spray voltage of 2200 V, ion transfer tube temperature of 275°C, automatic gain control (AGC) target of 400,000, and maximum injection time of 50 ms} was followed by data-dependent scans (top speed for most intense ions, with charge state set only to include +2 to 5 ions, and 5-s exclusion time), while selecting ions with minimal intensities of 50,000 in which the collision event was carried out in the high-energy collision cell [higher-energy C-trap dissociation (HCD) collision energy of 30%], and the fragment masses were analyzed in the ion trap mass analyzer (with ion trap scan rate of turbo, first mass m/z was 100, AGC target 5000, and maximum injection time of 35 ms). Protein identification and SILAC quantification were carried out using Peaks Studio 8.5 (Bioinformatics Solutions Inc.) For modifications, l-arginine-(13C6,15N4) was set as a variable modification and peptides with this modification were used for SILAC quantification. Regulon assignments were made in accordance with those found on the SubtiWiki databases (http://subtiwiki.uni-goettingen.de/) (23). Genes expressed as part of the SigA, SigH, and/or Spo0A regulons were considered vegetative.

Source of this news: https://advances.sciencemag.org/content/7/4/eabd6385.full

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