The impact of YabG mutations on C. difficile spore germination and processing of spore substrates

Abstract

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabG_C207A (catalytically inactive), C. difficile yabG_A46D, C. difficile yabG_G37E, and C. difficile yabG_P153L strains germinated in response to taurocholic acid alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabG_A46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

Introduction

Susceptibility to Clostridioides difficile infection (CDI) is commonly associated with the use of broad-spectrum antibiotics that disrupt the normally protective colonic microbiota (Theriot et al., 2014, Buffie et al., 2015, Di Bella et al., 2024). Subsequent ingestion of C. difficile spores results in the germination of the spore form to the toxin-producing vegetative form (Paredes-Sabja et al., 2014). C. difficile vegetative cells secrete two toxins that damage the colonic epithelium – resulting in the symptoms associated with CDI (e.g., diarrhea or pseudomembranous colitis) (Smits et al., 2016). Because spores are metabolically dormant and resist antibiotic action / harsh environmental conditions, they are the dispersive and transmissive form of the organism (Deakin et al., 2012). Unfortunately, the primary treatment for CDI is additional broad-spectrum antibiotics (e.g., vancomycin or fidaxomicin) (Zar et al., 2007). Though these antibiotics treat the toxin-producing vegetative cells, but not the dormant spores, they continue to disrupt the colonic microbiota and can leave the patient susceptible to reinfections (Smits et al., 2016). Approximately 15–20% of patients experience recurring disease, and the likelihood of subsequent episodes increases with each recurrence (Di Bella et al., 2024).

C. difficile spores germinate in response to certain host-derived bile acids (e.g., taurocholic acid) and amino acids (e.g., glycine) (Shrestha & Sorg, 2018, Sorg & Sonenshein, 2009, Sorg & Sonenshein, 2008, Howerton et al., 2011, Ramirez et al., 2010, Bhattacharjee et al., 2016b, Shrestha et al., 2017, Wilson, 1983, Wilson et al., 1982). Prior work provided genetic evidence that CspC is the bile acid germinant receptor and CspA is the co-germinant (amino acid) receptor (Francis et al., 2013, Shrestha et al., 2019, Kevorkian & Shen, 2017, Rohlfing et al., 2019). Despite the absence of a catalytic triad in these subtilisin-like proteases, these two pseudoproteases regulate C. difficile spore germination (Kevorkian & Shen, 2017, Kevorkian et al., 2016, Shrestha et al., 2019, Francis et al., 2013). Interestingly, CspA is encoded as a translational fusion to CspB (CspBA) and CspC is encoded downstream in the same operon (Paredes-Sabja et al., 2011, Bhattacharjee et al., 2016b, Adams et al., 2013). CspBA is post-translationally processed to CspB and CspA by the sporulation-specific protease, YabG (Shrestha et al., 2019, Kevorkian et al., 2016, Kevorkian & Shen, 2017). In addition to processing CspBA, YabG also cleaves the pre-sequence from preproSleC (a cortex degrading enzyme) to generate proSleC, the form present in mature spores (Shrestha et al., 2019, Kevorkian et al., 2016). We hypothesize that CspB is associated with CspC and CspA in dormant spores, with CspC and CspA inhibiting the proteolytic activity of CspB. Upon binding of the bile acid and co-germinant, CspC and CspA dissociate from CspB. CspB then cleaves the inhibitory pro-peptide from proSleC, resulting in activation of SleC, cortex degradation, and the subsequent steps in germination (Shrestha et al., 2019, Zhu et al., 2018, Adams et al., 2013, Francis et al., 2015). The absence of yabG has also been shown to influence the extractability/expression of coat and exosporium proteins, SpoIVA, CotA, CotE, and CdeM in a C. difficile yabG mutant, suggesting other targets of YabG (Marini et al., 2023, Zhu et al., 2018).

YabG is a conserved sporulation-specific protease. In Bacillus subtilis, YabG is important for proper assembly of the spore coat and has been shown to process at least six coat proteins (SpoIVA, CotF, CotT, YeeK, YxeA, and SafA) (Takamatsu et al., 2000a, Takamatsu et al., 2000b). Of those proteins, only SpoIVA has an orthologue in C. difficile and may be a YabG substrate (Kevorkian et al., 2016). YabG is produced during sporulation in the mother cell, under the alternative RNA polymerase sigma factors σ^E and σ^K (Fimlaid et al., 2013, Pereira et al., 2013, Saujet et al., 2013, Takamatsu et al., 2000b). C. difficile YabG is required for the expression of two σ^K-dependent genes cotA and cdeM, which encode a coat and an exosporium protein, respectively (Marini et al., 2023).

Using an ethyl methane sulfonate (EMS) screen to identify strains which germinated without a co-germinant, we previously identified mutations in the yabG coding and promoter regions (Figure 1 and Table S3) (Shrestha et al., 2019). Moreover, these yabG mutant strains incorporated preproSleC and CspBA (the unprocessed forms) into spores. One of the yabG mutant alleles, yabG_A46D, (Table S3) appeared to have lower activity than wildtype, but the data was inconclusive (Shrestha et al., 2019).

Figure 1. Mutations in the regulatory region of yabG.

A) The −10 and −35 regions of the yabG promoter, utilized by both σ^E and σ^K in C. difficile, are shown in yellow and the consensus for the two σ factors indicated below. The position of the C-41T and G-8A mutations is shown in red; the C-41T substitution makes the −35 region of the yabG promoter closer to the consensus for σ^A recognition (shown below the sequence). The sequence shown corresponds to the fragment fused to the SNAP^Cd reporter with numbers indicating the position relative to the yabG start codon. B) The protein coding sequence of C. difficile YabG, with mutations, yabG_G37E, yabG_A46D, yabG_P153L, and yabG_C207A shown in red. Numbers indicate the position relative to the fMet. C) Alphafold2 prediction of C. difficile YabG. G37, A46, P153, and C207 are colored black. The CheY-like domain is colored teal, the link is pink, and the SH3-like domain is colored yellow.

Here, we quantified the processing abilities of recombinantly expressed yabG mutant alleles and determined the in vivo effects on C. difficile spore germination. We show that of the previously identified yabG alleles, only YabG_A46D showed any activity in processing preproSleC. Mutations in the yabG locus result in misprocessing of germination proteins and spore coat proteins, whereas mutations in the region upstream of the yabG start codon (promoter and Shine-Dalgarno) affected the timing of expression.

Results

Expression of YabG_A46D leads to reduced processing of preproSleC

YabG cleaves the pre-peptide from preproSleC, with proSleC being incorporated into the mature spore (Shrestha et al., 2019, Kevorkian et al., 2016, Marini et al., 2023). To understand the processing capabilities of the yabG EMS mutant alleles previously identified, we recombinantly expressed and purified C. difficile preproSleC in E. coli. preproSleC was incubated with E. coli lysate expressing YabG, YabG_C207A, YabG_A46D, YabG_P153L, or YabG_G37E from an IPTG-inducible promoter. Due to expression differences of YabG_C207A, YabG_A46D, YabG_P153L, or YabG_G37E in E. coli compared to wild type YabG, various amounts of the E. coli lysate were incubated with the preproSleC to be equal to that of wild type. We then determined the amount of proSleC present in the sample using immunoblotting and quantified by Li-COR imaging. Lysate expressing wild type YabG efficiently processed preproSleC to proSleC to nearly 100% within 1 hour of incubation (Figure 2A and S1A). YabG_C207A, a catalytically inactive mutant, served as a negative control (Figure 2B, S1B) (Marini et al., 2023). Interestingly, of the mutant alleles, only YabG_A46D showed any ability to process preproSleC to proSleC. However, only 65% proSleC was present after 3 hours of incubation, suggesting that YabG_A46D does not process preproSleC as efficiently as the wild type allele (Figure 2C and S1C). YabG_G37E (Figure 2D and S1D) and YabG_P153L (Figure 2E and S1E) did not process preproSleC. During our analyses, we noticed the presence of a proSleC even in the absence of exposure to E. coli lysate (Figure S1, S2A and S2C). To ensure that the presence of proSleC in these assays was a product of preproSleC purification and not due to processing during incubation with E. coli lysate, lysate of E. coli BL21(DE3) containing an empty pET22b vector (pEV) was incubated with the purified preproSleC. Processing was compared to the wild type yabG allele control (Figure S2B and S2D). Because the abundance of the proSleC form does not increase in the E. coli BL21(DE3) pEV control, we conclude that E. coli is not processing preproSleC and that the observed proSleC is a degradation product generated during our preproSleC purification. The presence of this product does not change the outcome or conclusions of these assays.

Figure 2: Processing of preproSleC by mutant YabG variants.

Percent of proSleC present when incubated with the different yabG allele.

The percentage of proSleC processed by: A) Wild type yabG, B) yabG_C207A, C) yabG_A46D, D) yabG_G37E and E) yabG_P153L, were quantified on the LI-COR Odyssey-CLx, (proSleC signal)/(proSleC signal + preproSleC signal) *100. (NL) indicates where no E. coli lysate was added to purified preproSleC. The data represent the averages from three to eight biological replicates and error bars represent the standard error of the mean. Statistical significance was determined using one-way ANOVA with Šídák’s multiple comparisons test (* p < 0.0332; ** p < 0.0021, *** < 0.0001). *Note: the purified SleC used in the assays with yabG_C207A, yabG_G37E, and yabG_P153L had ~15% proSleC present prior to the assay in the sample as seen in the purified preproSleC negative controls (NC).

YabG can shift the processing site of preproSleC to nearby arginine residues

We previously identified the YabG processing site of preproSleC to be immediately after R119 using Edman degradation (Shrestha et al., 2019). Interestingly, the deletion of the identified SRQS sequence resulted in processing of preproSleC after R115 and normal incorporation of proSleC into the spores. This suggested that YabG could shift its processing site to a nearby arginine (Shrestha et al., 2019). To test the limits of YabG to shift its processing site, we first co-incubated preproSleC_R119A with E. coli lysate expressing yabG(referred to as YabG from hereon), as described above. Because the preproSleC_ΔSRQS mutant was processed at R115 instead of R119, we hypothesized that the R119A protein would also be processed at R115 (Shrestha et al., 2019). Indeed, the first 5 amino acids of the resulting proSleC were SFSAQ, suggesting that YabG processed after R115 (Figure 3A). Next, we generated a mutant allele with both arginine amino acids replaced, preproSleC_R119A/R115A, which YabG processed at R112 (Figure 3A). When a triple substitution was tested, preproSleC_{R119A/R115A/R112A}, we observed no processing (Figure 3A). This suggests that YabG processes after arginine residues and that, upon mutation, the processing site shifts within certain limits.

Figure 3: Processing site of recombinant preproSleC by yabG alleles.

A) Recombinantly expressed and purified preproSleC, preproSleC_ΔSRQS, preproSleC_R119A, preproSleC_R119A/R115A, preproSleC_{R119A/R115A/R112A}, preproSleC_R115A/R112A, and preproSleC_R112Awere incubated with E. coli lysate expressing YabG. The N-terminus of the YabG-cleaved preproSleC (proSleC) was determined by Edman sequencing. Arginine after which YabG processes preproSleC are colored at position 119 (red), 115 (green), and 112 (blue). B) C. difficile 630Δerm SleC AlphaFold2 protein structure prediction. Highlighted are R110, R112, R115, and R119 in black. The pre-peptide is colored pink, the pro-domain is colored green, and active SleC is colored orange.

Because Edman degradation only reveals N-terminal amino acids, it was unknown if YabG was processing at these arginine residues in a sequential manner, i.e. at R112, then R115, and lastly R119. To test this hypothesis, preproSleC_R112A and preproSleC_R112A/R115A were recombinantly expressed, purified, and incubated with YabG. Edman degradation revealed that YabG processed both R115A and R115A/R112A alleles at R119 (Figure 3A). There were also no changes in processing efficiency of YabG on the various preproSleC mutants with the exception of preproSleC_{R112A/R115A/R119A,} which was not processed. Using an AlphaFold2 prediction of the preproSleC protein structure, we noticed that the three arginine residues at which YabG processes are in a disordered region (Figure 3B) (Varadi et al., 2024, Jumper et al., 2021). Surprisingly, there is another arginine, R110, within this region that was not cleaved by the YabG in any of our assays. Some AlphaFold2 models predict that the R110 is located within a beta sheet, suggesting that YabG only processes after arginine residues found in the disordered loops (Figure 3B). Alternatively, the presence of a proline at position 109 could be excluding YabG from processing at R110.

Mutations in yabG result in a TA only germination phenotype

YabG processes two proteins involved in C. difficile spore germination: preproSleC and CspBA (Shrestha et al., 2019, Kevorkian et al., 2016). In prior work, we found that a C. difficile yabG::ermB mutant germinated in the presence of taurocholic acid (TA) alone and did not respond to the addition of a co-germinant (Shrestha et al., 2019). To determine if the mutations in yabG affected the ability to respond to co-germinants, isogenic mutants were made of yabG_A46D, yabG_G37E, yabG_P153L, yabG_C207A, yabG_C-41T, and yabG_G-8A in a C. difficile R20291 ΔpyrE strain. Spores derived from the indicated strains in buffer alone did not germinate (Figure 4A and 4D). Wild type C. difficile ΔpyrE does not germinate with the addition of TA alone (Figure 4B and 4E) and requires a co-germinant (glycine) to germinate (Figure 4C and 4F). However, spores derived from the C. difficile ΔpyrE yabG_C207A, C. difficile ΔpyrE yabG_A46D, C. difficile ΔpyrE yabG_P153L, and C. difficile ΔpyrE yabG_G37E strains germinated with the addition of TA but no co-germinant (Figure 4B). Spores derived from the C. difficile ΔpyrE yabG_G-8A and C. difficile ΔpyrE yabG_C-41T mutant strains also germinated in the presence of TA alone (Figure 4E). Germination of all the C. difficile yabG mutant spores was not enhanced by the addition of glycine (Figure 4C and F). Together these results support the previous findings that these mutations in yabG result in spores that no longer respond to co-germinants for germination.

Figure 4: Mutations in YabG allow for germination in the presence of taurocholic acid only.

Spores derived from C. difficile ΔpyrE, C. difficile ΔpyrE; yabG_C207A, C. difficile ΔpyrE; yabG_A46D, C. difficile ΔpyrE; yabG_P153L, C. difficile ΔpyrE; yabG_G37E, C. difficile ΔpyrE; yabG_C-8T, and C. difficile ΔpyrE; yabG_G-41A germinate in the presence of taurocholic acid alone and do not require a co-germinant. 5 μL of OD₆₀₀ = 100 spores derived from C. difficile ΔpyrE (circles), C. difficile ΔpyrE yabG_C207A (triangle), C. difficile ΔpyrE yabG_A46D (square), C. difficile ΔpyrE yabG_P153L (diamond), C. difficile ΔpyrE yabG_G37E (inverted triangle), C. difficile ΔpyrE; yabG_C-8T (inverted open triangle), and C. difficile ΔpyrE; yabG_G-41A (open triangle) were added to 95 μL germination buffer. A and D) Strains were suspended in buffer alone, B and E) in buffer supplemented with 10 mM TA, or C and F) in buffer supplemented with 10 mM TA and 30 mM glycine. Germination was monitored at OD₆₀₀. Data points represent the averages from biological triplicate experiments and error bars represent the standard error of the mean.

Mutations in yabG affect the abundance of YabG processed proteins

YabG is required for the expression of CotA and CdeM, a coat and an exosporium protein, respectively (Marini et al., 2023). In a C. difficile yabG mutant, these proteins have reduced abundance, and the spore has morphological changes (Marini et al., 2023). To investigate if our C. difficile yabG mutant alleles also exhibit changes to cortex/coat/exosporium protein abundance, we performed western blots on spore extracts. YabG was detected in the spore extracts of C. difficile ΔpyrE yabG_G37E, C. difficile ΔpyrE yabG_A46D, C. difficile ΔpyrE yabG_P153L, and C. difficile ΔpyrE yabG_C207A. C. difficile ΔpyrE yabG_C207A spores exhibited the highest amount of YabG, consistent with previous findings that it does not autoprocess (Figure 5A) (Marini et al., 2023). Of the three single amino acid substitution mutants, C. difficile ΔpyrE yabG_P153L showed the highest abundance of YabG, followed by C. difficile ΔpyrE yabG_G37E, and C. difficile ΔpyrE yabG_A46D (Figure 5A). C. difficile ΔpyrE yabG_P153L and C. difficile ΔpyrE yabG_G37E had an abundant processed proSleC, differing from in vitro assays. YabG was not detected in spore extracts derived from the wildtype, C. difficile ΔpyrE yabG_G-8A, and C. difficile ΔpyrE yabG_C-41T mutant strains. This likely results from YabG auto-proteolytic activity combined with reduced expression in the yabG_G-8A and yabG_C-41T. Compared to wildtype, we also detected a greater abundance of the preproSleC form in all mutants tested (Figure 5A).

Figure 5. yabG mutations affect the level and/or extractability of coat and exosporium proteins.

Spores of the WT, yabG_C207A, and the various yabG point mutants, as shown, were purified, fractionated and the coat/exosporium and cortex/core proteins extracted. The proteins were resolved by 15% SDS-PAGE and the gels stained with Coomassie or subjected to immunoblot analysis with anti-YabG, anti-SleC, anti-CdeM (A), anti-CdeC, anti-CotA,anti-CspBA and anti-CotE antibodies (B). The position of the proteins and their main forms, recognized by the different antibodies is indicated by the red arrowheads. The position of molecular weight (MW) markers (in kDa) is indicated on the left side of the panel.

CdeM and CotA were absent from spores derived from the yabG_C207A strain (Figure 5A and 5B). We also observed less CdeM in spores of C. difficile ΔpyrE yabG_C-41T, C. difficile ΔpyrE yabG_G-8A, and C. difficile ΔpyrE yabG_G37E (Figure 5A). There was no effect on the observed CdeC (Figure 5B). The abundance of cleaved CotE was also reduced in all mutant strains. The C. difficile ΔpyrE yabG_A46D strain had increased processing compared to the other mutant strains. This is consistent with this allele retaining some activity against preproSleC (Figure 2C) (Shrestha et al., 2019). Finally, the autoprocessed form of CspB was also detected in all of the mutant spore extracts, consistent with previous findings in spores derived from a C. difficile ΔyabG mutant strain (Figure 5B) (Kevorkian et al., 2016).

Mutations in yabG affect the spore morphology

To understand if spore assembly was affected in the mutant strains, we imaged the spores by transmission electron microscopy (TEM). In spores derived from the C. difficile yabG_C207A mutant, a loose connection between the cortex and coat layers is occasionally seen (Figure 6) (Marini et al., 2023). Also, the spore surface lacks electron density or has a very thin electron dense exosporium which lacks bumps; the appendage, when present, is not electron dense but rather shows a lamellar structure (Figure 6). In C. difficile ΔpyrE yabG_P153L, C. difficile ΔpyrE yabG_A46D and C. difficile ΔpyrE yabG_G37E spores, the exosporium was thin, and, when present, the bumps were much smaller than in the wild type; the appendage region was less electron dense than in the wild type and showed a lamellar pattern (Figure 6). Spores of the C. difficile ΔpyrE yabG_G-8A and C. difficile ΔpyrE yabG_C-41T strains appeared similar. These observations are consistent with the idea that construction of the electron dense outer layer of the spore body and appendage is dependent on CdeM (Marini et al., 2023). In the strains which we observed a reduction in the electron density of the exosporium, we also observed a lower abundance of CdeM in the spore (Figure 5A), consistent with this hypothesis. In all the spores, filamentous projections are seen emanating from the spore surface, which may correspond to the structures formed by the Bcl proteins (Pizarro-Guajardo et al., 2014, Paredes-Sabja et al., 2014, Pizarro-Guajardo et al., 2020).

Figure 6. Morphological alteration in spores of the yabG mutants.

Gradient purified spores of the WT (630Δerm), yabGC207A and the indicated point mutants were analyzed by thin sectioning transmission electron microscopy. The arrowheads point to: the edge of the exosporium, red; the coat, white; the cortex, blue; the spore core, yellow; the electrodense regions within the spore appendage, green. The table refer to the percentage of spores (> 100) in which the indicated feature is present; thick electrodense exosporium, a; thin or absent exosporium, b; electrodense lamellar appendage, c; bumps or robust appendage and all around the spore, d; and small bumpy appendage, e. Scale bars are indicated in the panels.

Characterizing the effects of yabG promoter mutations on yabG expression

To study the timing of yabG expression during sporulation, the fragment derived from the C. difficile yabG regulatory region (Figure 1A) was fused to the SNAP^Cd reporter. In the SNAP^Cd constructs, the C. difficile yabG RBS was fused to the start codon of the reporter so that mutations presumably affecting transcription (C. difficile ΔpyrE yabG_C-41T) or translation (C. difficile ΔpyrE yabG_G-8A) could be assessed (Figure 7A). These experiments were conducted in C. difficile 630Δerm due to better data on the timing of C. difficile sporulation in this strain (Marini et al., 2023). As observed previously, the wild type promoter is active during late stages of sporulation, mainly in sporangia of phase grey/phase bright spores (Figure 7A, white arrows) (Marini et al., 2023). The C. difficile ΔpyrE yabG_C-41T mutation increases expression of the reporter earlier than the wild type (when the forespore is not yet discernible by phase contrast microscopy) (Figure 7A, black/grey arrows) and expression is reduced in sporangia at late stages in sporulation (Figure. 7A, white arrows). In contrast, in the C. difficile ΔpyrE yabG_G-8A mutant, production of the reporter had undetectable levels of expression at all stages of sporulation (Figure 7A). We next quantified the level of expression in sporangia of phase grey/phase bright spores of wild type C. difficile ΔpyrE, C. difficile ΔpyrE yabG_G-8A, and C. difficile ΔpyrE yabG_C-41T strains at various stages (early, phase dark, phase bright) of sporulation (Figure 7B). Consistent with observations in Figure 7A, we observed yabG_C-41T expression earlier than other strains. yabG_C-41T expression was lower in abundance at later stages of sporulation (Figure 7B) compared to wild type, with the yabG_G-8A allele showing the lowest expression in all stages of sporulation (Figure 7B). These results are consistent with the germination data shown in Figure 4E, in which germination occurs in the presence of TA alone as well as the immunoblot in Figure 5A, where yabG is not observed in either of these mutant strains.

Figure 7. Single cell analysis of PyabG expression.

A) C. difficile sporulating cells carrying fusions of the WT yabG promoter or alleles with the G-8A or C-41T mutations fused to the SNAP^Cd reporter in an otherwise WT background (630Δerm) were collected after 20 hours of growth on 70:30 agar plates. The cells were stained with the SNAP substrate TMR-Star and examined by phase contrast (top panels) and fluorescence microscopy (middle panel, SNAP^Cd signal; bottom panels, autofluorescence). The black arrowheads show cells at early stages of sporulation, with no visible signs of a forespore; the grey arrowheads show sporangia of phase dark forespores and the white arrowheads indicate sporangia of phase grey or phase bright forespores. Scale bar, 1 μm. B) Intensity of the fluorescence signal per cell for the P_yabG-SNAP^Cd fusions described in A) in cells at early stages of sporulation or in sporangia of phase dark or phase grey/phase bright forespores. Fluorescence intensity is shown in arbitrary units (AU). The data from three independent experiments was represented using SuperPlots, with each color corresponding to a replicate (Lord et al., 2020). Statistical analysis was carried out using the ordinary one-way ANOVA and Tukey’s test. Asterisks correspond to p-values of p<0.05 (*) or p<0.01 (**).

Discussion

Previous work on YabG has focused mostly on Bacillus subtilis (Takamatsu et al., 2000a, Takamatsu et al., 2000b, Kuwana et al., 2006, Yamazawa et al., 2022). Like in C. difficile, B. subtilis YabG is a sporulation-specific cysteine protease involved in the processing of several spore proteins during sporulation. Most target proteins of B. subtilis YabG (CotF, CotT, YeeK, YxeE, and SafA) are not conserved in C. difficile (Takamatsu et al., 2000a, Takamatsu et al., 2000b, Sebaihia et al., 2006). Additionally, the mechanism of target recognition and any additional targets remain unknown in C. difficile.

In C. difficile, YabG processes CspBA to CspB and CspA and preproSleC to proSleC (Marini et al., 2023, Shrestha et al., 2019, Kevorkian et al., 2016). However, in spores derived from a C. difficile yabG mutant, some CspB is present (Kevorkian et al., 2016). This CspB form is due to either autoprocessing or another protease that is capable of processing CspBA (Kevorkian et al., 2016, Marini et al., 2023, Shrestha et al., 2019). A previous study found a decrease in the abundance of SpoIVA in spores derived from a C. difficile yabG mutant, but the involvement of YabG in the incorporation of SpoIVA into mature spores is poorly understood (Kevorkian et al., 2016). Because C. difficile YabG is the only protease with the ability to cleave preproSleC, and the preproSleC form is the only form incorporated into mature spores in C. difficile yabG mutants, preproSleC was used as our target to quantify the processing capabilities of YabG, YabG_C207A, YabG_A46D, YabG_P153L, and YabG_G37E (Kevorkian et al., 2016, Marini et al., 2023, Shrestha et al., 2019). We found that YabG is very efficient and processed ~50% of preproSleC to proSleC within 10 minutes and ~100% within 1 hour of in vitro incubation (Figure 2A, S1B). However, YabG_A46D only processes 50% of preproSleC within 3 hours of in vitro incubation (Figure 2C, S1C). Interestingly, YabG_P153L and YabG_G37E did not show any activity at the times tested (Figure 2 and S1). Because only one target of YabG was tested, it is possible that these alleles are more active on other unidentified YabG targets. We consider this unlikely because of the defects in processing of numerous spore proteins observed by western blotting (Figure 5). CspBA is another YabG target, but some CspBA is processed and incorporated into mature spores of a C. difficile ΔyabG mutant strain. This suggests that another protease can cleave CspBA to CspB and CspA (Shrestha et al., 2019, Kevorkian et al., 2016). Spores derived from the C. difficile R20291 yabG::ermB mutant strain only contained CspBA and not CspB and CspA (Shrestha et al., 2019) but our isogenic mutants (Figure 5B) contain some processed CspBA suggesting differences between the two C. difficile strains (C. difficile R20291 vs. C. difficile R20291 ΔpyrE) or because yabG is not produced in the C. difficile R20291 yabG::ermB mutant where as it is produced in C. difficile R20291 ΔpyrE yabG_C207A, but not catalytically active.

The processing site of YabG on preproSleC had previously been identified as R119. However, two other arginine residues are near R119, and we wanted to determine if YabG could also process at those sites (Shrestha et al., 2019). Interestingly, we found that upon mutation of preproSleC R119 to alanine, processing occurred at R115. In the preproSleC R119A/R115A mutant, processing occurred at R112. No processing was observed for the combined triple mutant, R119A/R115A/R112A. Interestingly, YabG does not cleave at R110 and in a predicted AlphaFold2 structure of preproSleC shows R119, R115, and R112 all reside within an exposed, unstructured loop, whereas R110 resides within a beta strand, mainly excluding it from processing (Figure 3B). It could also be the presence of P109 that excludes R110 from YabG processing.

All the spores derived from the C. difficile ΔpyrE yabG mutants germinated with TA only and did not require a co-germinant in order to germinate, unlike the parental C. difficile ΔpyrE strain (Figure 4). These results are consistent with previous findings. Interestingly, C. difficile ΔpyrE yabG_C207A appears to germinate more slowly than the other mutants in TA only conditions. However, this may be due to the complete loss of YabG catalytic activity, resulting in a lack of processing of alterative targets (Shrestha et al., 2019).

Spores derived from the C. difficile ΔyabG and C. difficile yabG_C207A strains incorporate less CotA and CdeM (Figure 5A and 5B) (Marini et al., 2023). CotA is a component of the spore coat and is required for assembly of the outer surface layers of the spore (Permpoonpattana et al., 2013, Permpoonpattana et al., 2011). CdeM is a component of the exosporium (Calderón-Romero et al., 2018). Upon investigation of cdeC and cdeM transcript levels of C. difficile ΔyabG sporulating cells, their levels were lower compared to wild type, suggesting YabG was regulating their transcription, though the mechanism by which this occurs remains unknown (Marini et al., 2023). It has also been observed in Clostridium botulinum that a disruption of yabG by a prophage-like DNA insertion (yin element) resulted in early onset sporulation compared to strains lacking that element (Douillard et al., 2023). These findings highlight that YabG in other organisms may be playing a role in sporulation regulationg. To investigate if the C. difficile yabG alleles also affect spore cortex/coat/exosporium proteins, western blots were performed against CdeM, CdeC, CotA, CotE, CspBA, preproSleC, and YabG. CdeM was less abundant or less extractable from the purified spores of C. difficile ΔpyrE yabG_C-41T (Figure 5A). In C. difficile ΔpyrE yabG_C-41T expression of the reporter is even higher than in the wild type (Figure 7B), which could potentially be due to earlier expression of yabG interfering with transcription of cdeM and cotA and/or assembly of the proteins (Figure 5). Interestingly, the levels/extractability of CdeM and CotA, were higher in C. difficile ΔpyrE yabG_A46D and C. difficile ΔpyrE yabG_P153L spores, as compared to C. difficile ΔpyrE yabG_G37E spores (Figure 5A and 5B). One possibility is that the yabG_A46D and yabG_P153L alleles are more efficient than the yabG_G37A allele at promoting cdeM and cotA transcription. Processing of preproSleC, CspBA, and CotE is incomplete (Figure 5A and 5B), and we do not know the location or timing of these events. Because proSleC and CspB and CspA are found in the cortex, we hypothesize that they are processed in the cytoplasm before being transported to the cortex (Baloh et al., 2022).

YabG was not detected in the promoter mutant C. difficile ΔpyrE yabG_C-41T or the RBS mutant C. difficile ΔpyrE yabG_G-8A, similar to wild type. This is most likely due to its auto-proteolytic activity, however the lack of YabG in C. difficile ΔpyrE yabG_G-8A is likely due to loss of expression (Figure 5A and Figure 7B) (Marini et al., 2023). In all yabG mutants with a single amino acid substitution (G37A, A46D, and P153L), YabG was detected in the spore extracts, but at levels lower than the catalytically inactive YabG_C207A allele. This suggests that these variants are less active than the WT protein (Figure 5A). Consistent with the lower activity of YabG_P153L, autoprocessing and processing of preproSleC, CspBA, and CotE was diminished relative to C. difficile yabG_G37E and C. difficile yabG_A46D (Figure 5A and 5B).

C. difficile ΔpyrE yabG_A46D showed the lowest amount of YabG in spore extracts of the three single amino acid substitution mutants. This is consistent with YabG_A46D showing activity against purified preproSleC in vitro (Figure 2C and 5A). Reduced activity of YabG can result from single amino acid substitutions in different parts of the protein (A46D and G37E in the SH3 domain; P153L in the CheY-like, catalytic domain). Together this suggests that the different alleles have various levels of catalytic activity.

The main morphological changes in C. difficile ΔyabG or C. difficile yabG_C207A spores include a loose connection between the cortex and coat layers, a thin outermost electron dense layer (thought to be part of the exosporium), and a lack of electron density in the appendage region, which reveals an underlying pattern of lamellae (Marini et al., 2023). The thinner outer layer and the lack of electron density of the spore appendage is thought to be caused by the absence of CdeM, as it is not expressed in yabG mutants. Accordingly, the electron density of the appendage is restored when cdeM is expressed from the yabG-independent cotE promoter (Marini et al., 2023). C. difficile yabG mutants, were originally studies in C. difficile 630Δerm, however, we used C. difficile R20291 ΔpyrE as a parental strain (Marini et al., 2023). A striking difference between spores of the two strains is that the outermost electron dense spore layer is much thicker in R20291 spores and presents bumps that protrude from the entire surface of the spore (Figure 6, wild type spores). Apart from the C. difficile yabG_C207A mutant, there were no overt signs of an impaired cortex/coat connection in the point mutation strains, suggesting that a lower level of YabG activity is sufficient to prevent this phenotype (Figure 6).

It remains unknown how YabG recognizes its targets in any organism. In B. subtilis, YabG processes coat proteins, however only SpoIVA has an orthologue in C. difficile (Kuwana et al., 2006, Takamatsu et al., 2000a, Takamatsu et al., 2000b, Yamazawa et al., 2022). Moreover, despite universal conservation of the protein in spore forming bacteria, the absence of conserved substrates is intriguing. Future work needs to be done to identify YabG substrates and reveal any conserved features that may help to identify substrates in other non-model, spore forming organisms.

Experimental Procedures

Bacterial growth conditions

All bacterial strains are listed in Table S2. C. difficile strains were derivatives of 630Δerm or R20291 grown in a Coy anaerobic chamber at 37 °C, 3–4% H₂, 5% CO₂, and balanced N₂ on either brain heart infusion (BHI) medium (Difco) supplemented with 0.1% L- cysteine and brain heart infusion supplemented with 5 g / L yeast extract and 0.1% L- cysteine (BHIS) or 70:30 media as indicated. When necessary, media was supplemented with thiamphenicol (10 – 15 μg / mL), kanamycin (50 μg / mL), D-cycloserine (250 μg / mL), tetracycline (5 μg / mL), uracil (2 μg / mL), theophylline (2 g / L), cefoxitin (25 μg / mL), and / or taurocholate (TA) (0.1%). Defined minimal media for C. difficile (CDMM) supplemented with 5-fluoroorotic acid (FOA; 2 mg / mL) and uracil (5 μg / mL) was used for the selection of ΔpyrE mutants. E. coli strains were grown at 37 °C on LB medium supplemented with chloramphenicol (20 μg / mL) and or ampicillin (100 μg / mL) for plasmid maintenance. E. coli BL21 (DE3) was grown in 2x tryptone yeast (2XTY), medium supplemented with chloramphenicol (20 μg / mL) and or ampicillin (100 μg / mL) for plasmid maintenance and used for recombinant protein expression.

Plasmid constructions

The oligonucleotide primers used in this work are listed in Table S1. Plasmids pMS17, pMS43, pMS44, and pMS45 were made from pMTL-YN4 as follows. PMTL-YN4 was digested with MluI / XhoI and purified by gel electrophoresis, extracted, then assembled with their respective regions of homology via Gibson assembly (Gibson et al., 2009). Regions of pMS17 homology were amplified from C. difficile R20291 with primers 5’ yabG C207A pMTL-YN4, 3’ yabG C207A upstream, 5’ yabG C207A downstream, and 3’ yabG C207A pMTL-YN4. Region of pMS43 homology were amplified from C. difficile mutant 20C using primer pairs 5’ yabG A46D_up / 3’ yabG A46D_up, and 5’ yabG A46D_down / and 3’ yabG A46D_down. Regions of pMS44 were amplified from C. difficile mutant 30A using primer pairs 5’ yabG P153L_up / 3’ yabG P153L_up and 5’ yabG P153L_down / 3’ yabG P153L_down, respectively. Regions of pMS45 homology were amplified from C. difficile mutant 30C using primer pairs 5’ yabG G37E_up / 3’ yabG G37D_up and 5’ yabG G37E_down / 3’ yabG G37E_down. Plasmids pJB86 and pJB87 were made from pJB81 as follows. pJB81 was digested by NotI / XhoI and purified by gel electrophoresis, extracted, then assembled with their respective regions of homology via Gibson assembly. Regions of homology for pJB86 were amplified with primers 5’ prom_C-8T_up / 3’ prom_C-8T_up and 5’ prom_C-8T_down / 3’ prom_C-8T_down, while those for pJB87 were amplified with primers 5’ yabG_G-41A_up / 3’ yabG_G-41A_up and 5’ yabG_G-41A_down / 3’ yabG_G-41A_down. After Gibson assembly and transformation into E. coli DH5α (Hanahan, 1983), colonies were re-streaked and tested via PCR for correct assembly, followed by whole plasmid sequencing

Plasmids pMS48, pMS49, pMS50, pMS51, pMS59, and pMS60 were made from pET22b as follows. pET22b was digested with NdeI / XhoI and purified by gel electrophoresis, extracted, then assembled with their respective regions of homology via Gibson assembly. Regions of pMS48 homology were amplified from C. difficile R20291 using primer pairs 5’ pET22b_yabG_up / 3’ yabG A46D_up and 3’ yabG A46D_up / 3’ pET22b_yabG_down. Regions of pMS49 homology were amplified from C. difficile mutant 30A using primer pairs primer pairs 5’ pET22b_yabG_up and 3’ pET22b_yabG_down. Regions of pMS50 were amplified from C. difficile mutant 30C using primer pairs 5’ pET22b_yabG_up and 3’ pET22b_yabG_down. Regions of pMS51 were amplified from C. difficile MRS05 using primer pairs 5’ pET22b_yabG_up and 3’ pET22b_yabG_down. Regions of pMS59 were amplified from C. difficile R20291 using primer pairs 5’ pET22b preproSleC / 3’ pET22b R112A and 5’ pET22b R112A and 3’pET_SleC. Regions of pMS60 were amplified from C. difficile R20291 using primer pairs 5’ pET22b preproSleC / 3’ pET22b R112A/R115A and 5’ pET22b R112A/R115A / 3’pET_SleC. After Gibson assembly and transformation into E. coli DH5α, colonies were re-streaked and tested via PCR for correct assembly, followed by whole plasmid sequencing

Plasmids pAC57, pAC58, and pAC59 were made from pET22b as follows. pET22b was digested with NdeI / BamHI and purified by gel electrophoresis, extracted, then assembled with their respective regions of homology via Gibson assembly. Regions of pAC57 were amplified from C. difficile R20291 using primers SleC 119A_Fp / pet22b_yabg_SleC6his_Rp and pet22b_SleC_Fp / SleC R119A_Rp. Regions of pAC58 were amplified from C. difficile R20291 using primer pairs SleC 119115112A_Fp / pet22b_yabg_SleC6his_Rp and pet22b_SleC_Fp / SleC 119115112A_Rp. Regions of pAC59 were amplified from C. difficile R20291 using primer pairs SleC 119115A_Fp / pet22b_yabg_SleC6his_Rp and pet22b_SleC_Fp / SleC 119115A_Rp. After Gibson assembly and transformation into E. coli DH5α, colonies were re-streaked and tested via PCR for correct assembly, followed by whole plasmid sequencing using Oxford Nanopore Technology by Plasmidsaurus inc. (Eugene, OR).

Conjugations

The plasmids pMS17, pMS43, pMS44, and pMS45 were conjugated separately into C. difficile ΔpyrE for ΔpyrE mediated allele coupled exchange (ACE) (Ng et al., 2013) using E. coli HB101 pRK24 as a conjugal donor. The plasmid was transformed into E. coli HB101 pRK24 and plated on LB-agar supplemented with chloramphenicol and ampicillin and grown overnight. An overnight culture of E. coli HB101 was grown in 5 mL of BHIS supplemented with chloramphenicol and ampicillin, C. difficile ΔpyrE was grown in 5 mL of BHIS. 500 μL of the E. coli overnight culture was pelleted, supernatant discarded, and transferred into the anaerobic chamber. 1 mL of overnight C. difficile ΔpyrE culture was heated for 5 min at 52 °C and let rest for 2 min (Kirk & Fagan, 2016). The E. coli was resuspended with 1 mL of the heat shocked C. difficile ΔpyrE. Four to five spots of 50 μL of mixed culture were plated on BHI and incubated for approximately 24 hrs. Growth was resuspended in 1 mL of BHIS and plated on BHIS supplemented with thiamphenicol, uracil, kanamycin, and D-cycloserine, for selection (BHIS TUCK). The resulting transconjugant colonies were streaked for tetracycline sensitivity and thiamphenicol resistance and confirmed by PCR.

ΔpyrE mediated allelic exchange

C. difficile strains MRS04, MRS05, MRS06, and MRS07 were made as previously described (Ng et al., 2013) from parental strain KNM05 (McAllister et al., 2017) with plasmids, pMS43, pMS17, pMS45, and pMS44 respectively. Briefly, transconjugants were passaged on BHIS supplemented with thiamphenicol, uracil, D-cycloserine, and kanamycin. Integrants were passaged onto CDMM + FOA + uracil. Colonies were passaged onto BHIS and BHIS supplemented with thiamphenicol to screen for plasmid loss. Loss of plasmid was confirmed by PCR and whole genome re-sequencing was performed to confirm genotype.

Theophylline mediated allelic exchange

C. difficile strains JNB25 and JNB26 were made as previously described (Brehm & Sorg, 2024) from parental strain KNM05 with plasmids pJB87 and pJB86, respectively. Briefly, the plasmids were introduced into KNM05 via E. coli HB101 pRK24 conjugation and confirmed by PCR. Transconjugants were plated on BHIS Tm plates, where plasmid integration was screened by colony size. Colonies that contained the integrated plasmid were isolated and plated on BHIS containing 2 g / L theophylline to encourage plasmid excision. Colonies were isolated and tested for their respective mutations by PCR amplification and sequencing, followed by whole genome sequencing of the completed strains. Whole genome sequencing was performed on strains and sent for Illumina sequencing at SeqCoast Genomics LLC (Portsmouth, NH) and raw sequence reads are uploaded to the Sequence Read Archive under BioProject ID: PRJNA1112411.

SNAP^Cd fusions

To construct the PyabG transcriptional fusions to the SNAP^Cd reporter, the promoter regions of yabG were PCR-amplified using as the template genomic DNA of C. difficile R20291 ΔpyrE 31D (strain 4049030, bearing the C-8T mutation), C. difficile R20291 ΔpyrE 27E (strain 4049063, with the G-41A mutation) or C. difficile R20291ΔpyrE (lab strain AHCD774, yabG^WT). The primer pairs used were PyabG-EcoRI-Fw and C8T-SNAP-SOE-Rev for the first strain and PyabG-EcoRI-Fw and YabG-978-SOE-Rev for the last two strains. The PCR reactions produced fragments of 271 bp. The SNAP^Cd gene was PCR amplified from pFT47 (Pereira et al., 2013) using primers SNAP-Fw and SNAPCd-HindIII-Rev to produce a fragment with 558 bp. The two fragments were joined by PCR using primers PyabG-EcoRI-Fw and SNAPCd-HindIII-Rev. This produced fragments of 809 bp which were cleaved using EcoRI and HindIII and inserted between the same sites of pMTL84121. This originated plasmids pCO39 (PyabG-SNAP^Cd), pCO40 (PyabG^C8T-SNAP^Cd) and pCO41 (PyabG^G42A-SNAP^Cd). These plasmids were transformed into E. coli HB101 (RP4) originating strains AHEC1588 (pCO39), AHEC1589(pCO40) and AHEC1590(pCO41) and then transferred to C. difficile 630Δerm pyrE⁺ (AHCD1190) by conjugation to produce strains AHCD1842, AHCD1843 and AHCD1844, respectively. All primers are listed in table S1. All strains and plasmids are listed in Table S2.

Germination assay

Germination was monitored using a Spectramax M3 plate reader (Molecular Devices, Sunnyvale, CA) (Shrestha et al., 2017, Bhattacharjee & Sorg, 2018, Shrestha & Sorg, 2018, Shrestha et al., 2019, Shrestha & Sorg, 2019). OD₆₀₀ = 100 spores were heat activated for 30 min at 65 °C. 5 μL of spores was added to 95 μL of germination buffer containing 50 mM HEPES alone, or HEPES supplemented with 10 mM TA or with 10 mM TA and 30 mM glycine in a 100 μL total volume. The OD₆₀₀ was monitored for 1 hour at 37 °C.

SleC expression and purification

Plasmid pKS08 was transformed into E. coli Rosetta BL21 (DE3) and incubated overnight at 37 °C on LB-agar supplemented with chloramphenicol and ampicillin. The plate was scraped into 1 mL of LB and used to inoculate 1 L of 2XTY supplemented with chloramphenicol and ampicillin in baffled flasks (OD₆₀₀ = 0.01). The culture was incubated at 37 °C at 190 rpm until the OD₆₀₀ was between 0.6 and 0.8, at which point the culture was induced with 250 μM IPTG and incubated for 16 hours at 16 °C and 130 rpm. Cultures were pelleted at 6,370 x g for 15 min at 4 °C. Supernatant was discarded and pellets were stored at −80 °C until use. 1 liter of cells was resuspended in 25 mL of 300 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole (pH 7.5) and 0.03 mM PMSF. Each 25 mL of cells was supplemented with lysozyme and DNase I and was rocked for 30 minutes at 4 °C prior to sonication on ice at 27% amplitude for 20 min. Samples were then centrifuged at 25,900 x g for 30 min at 4 °C and the supernatant was combined with 1 mL of Ni-NTA Agarose beads. Samples were rocked overnight at 4 °C. Beads were washed twice with 300 mM NaCl, 50 mM Tris-HCl, 30 mM imidazole (pH 7.5). Beads were washed once with 300 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole (pH 7.5) and then eluted with the same buffer but supplemented with 500 mM imidazole. Samples were concentrated using a 10 kDA molecular weight cut off centrifugal device. The protein was further purified by size exclusion chromatography using a Cytiva ÄKTA Pure system (Marlborough, MA) on a Superdex 200 increase 10/300 GL column and concentrated again. Protein was diluted to 2.5 mg / mL, aliquoted, flash frozen in a dry ice-ethanol bath, and stored at −80 °C until use.

YabG in vitro lysate preparation

Plasmids containing wild type (pAC28), mutant (pMS48, pMS49, pMS50, and pMS51) yabG, or an empty pET22b vector were transformed into E. coli Rosetta BL21 (DE3), and incubated overnight at 37 °C on LB-agar supplemented with chloramphenicol and ampicillin. The plate was scraped into 1 mL of LB and used to inoculate 50 mL of LB supplemented with chloramphenicol and ampicillin in 250 mL flasks, so the starting culture OD₆₀₀ was at 0.01. The culture was incubated at 37 °C at 170 rpm until the OD₆₀₀ was between 0.6 and 0.7. The culture was induced with 250 μM IPTG and incubated for 1 hour at 37 °C. YabG cultures were pelleted by centrifugation at 4,415 x g, for 15 min at 4 °C. The supernatant was discarded, and cells were resuspended in 4 mL of 300 mM NaCl, 50 mM Tris-HCl (pH 7.5) before sonication on ice at 27% amplitude for 20 min. Bacterial lysate was immediately used in the SleC processing assay.

SleC processing assay

The expression difference between wild type yabG and of each yabG alleles was determined through immunoblotting with an anti-His antibody (Invitrogen). The percent wildtype is as follows; yabG_G37E 19.1% (5.24 x lysate added to assays), yabG_A46D 21.9% (4.57 x lysate added to assays), yabG_P153L 32.1% (3.12 x lysate added to assays), and yabG_C207A 31.2% (3.12 x lysate added to assays).

Recombinantly expressed and purified preproSleC from E. coli was incubated with the indicated recombinantly expressed yabG allele lysate. Incubations were conducted at 37 °C for 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 hour, 2 hours, or 3 hours. The reactions occurred in 25 μL total volume [56 μg preproSleC present in 22.5 μL + 2.5 μL of the indicated yabG allele or buffer 300 mM NaCl, 50 mM Tris-HCl, (pH 7.5)]. As a control, purified preproSleC was added to buffer alone. Samples were denatured at 95 °C for 20 minutes, diluted with 1x Nu-PAGE sample buffer, and stored at 4 °C until use.

42 ng of purified SleC from the SleC processing assays were separated by 12% SDS-PAGE. Protein was then transferred onto low-fluorescence polyvinylidene difluoride membrane (PVDF) using the Hoefer TE 62 tank transfer system at 1.0 A for 1 hour.. The membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) rocking overnight at 4 °C. The membranes were incubated with anti-SleC antibody for 1 hour in 5% skim milk in TBST and washed three times with TBST. For the secondary antibody, LI-COR IRDye 680RD goat anti-rabbit antibody was used to label the membranes for 1 hour in 5% skim milk in TBST in the dark. The membranes were washed three times, in the dark, with TBST. Blots were imaged wet on the Odyssey^® CLx and the Odyssey M imaging systems (Li-COR, Lincoln, NE) using the 700–800 nm channels. The fluorescent bands were visualized and analyzed using ImageStudio software version 5.2 or LI-COR Acquisition software version 1.2 and analyzed with Empiria Studio version 2.3 (LI-COR Biosciences).

Edman sequencing

Plasmids pKS08, pAC57, pAC59, pAC58, pMS60, and pMS59 were induced and protein was purified the same as described above. Purified preproSleC, preproSleC_R119A, preproSleC_R119A/R115A, preproSleC_{R119A/R115A/R112A}, preproSleC_R115A/R112A, and preproSleC_R112A were incubated with E. coli lysate expressing wild type yabG for three hours. The preproSleC processing assays were separated on a 12% SDS-PAGE and transferred to PVDF membrane using the BIO-RAD Trans-Blot Turbo Transfer system at 25 V, 1.0 A for 30 min. The membrane was thoroughly washed under dH₂O for ~ 10 minutes to remove residual glycine. The membrane was stained with Coomassie Brilliant Blue for 20 minutes (50% MeOH, 10% acetic acid, 40% dH₂O, and 0.05% Coomassie). The membrane was destained (50% MeOH, 10% acetic acid, and 40% dH₂O). The proSleC band was excised from the membrane using a fresh razor blade. The band was sent for Edman sequencing of the first five amino acids at The Protein Facility at Iowa State University.

Spore purification

Spores were purified as described previously (Nerber et al., 2023, Aguirre et al., 2022, Baloh et al., 2022, Baloh & Sorg, 2021, Shrestha et al., 2019, Shrestha & Sorg, 2019, Shrestha & Sorg, 2018, Bhattacharjee et al., 2016a). Briefly, cultures were plated on BHIS media (Brain heart infusion with 5% yeast extract) and incubated for 4–5 days. Cells from each plate were scraped into 1 mL of 18 Ω H₂O and incubated overnight at 4 °C. Cells were washed with 18 Ω H₂O approximately 5 times and four tubes were combined into one tube of 1 mL. The washed spores were then layered on top of 9 mL of 50% sucrose and centrifuged at 4,000 x g for 20 minutes at 4 °C. The supernatant was discarded, and the spores were washed approximately five more times with 18 Ω H₂O and stored at 4 °C until use.

Spore production, purification and extraction of spore proteins

Spores were purified from 70:30 agar plates after 20 hours of growth using a step gradient of Gastrografin (Bayer) as described (Marini et al., 2023). The spore titer in the final suspension was estimated by measuring the OD₅₈₀. The sediment was resuspended in 5 ml of BHI centrifuged (4000 x g, for 5 min at 4°C) and the sediment resuspended in 1 ml of extraction buffer [0.125 mM Tris (hydroxymethyl) aminomethane hydrochloric acid (Tris-HCl), 5% β-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 0.025% bromophenol blue, 0.5 mM dithiothreitol (DTT), 5% glycerol, pH6.8)] and the suspension boiled for 8 minutes (Marini et al., 2023).

Transmission electron microscopy (TEM)

Thin sectioning TEM of density gradient-purified C. difficile spores, prepared following 20 hours of growth on 70:30 plates (above) was as described previously (Marini et al., 2023).

SDS-PAGE and immunoblot analysis of spore extracts

Proteins extracted from whole spores were resolved by SDS-PAGE (15% gels) and visualized by Coomassie brilliant blue R-250 staining. Gels run in parallel were subject to immunoblot analysis using the following antibodies at the indicated dilutions: anti-YabG (1:1000), anti-CdeC (1:500), anti-CdeM (1:15000), anti-CotA (1:1000), anti-CspB, anti-CspC, and anti-SleC (1:3000), anti-GPR (1:10000) and anti-SNAP-tag at 1:1000. A rabbit secondary antibody conjugated to horseradish peroxidase (Sigma) was used at a dilution of 1:5000; while anti-mouse IgG (whole molecule)-peroxidase (Sigma), at a dilution of 1:2000). The immunoblots were developed with Super Signal^™ Pico Plus Chemiluminescent Substrate (Thermo Scientific).

SNAP^Cd labelling, phase contrast, fluorescence microscopy and image analysis:

For SNAP labelling, cells were withdrawn from sporulating cultures after 20 hours of growth on 70:30 medium (Putnam et al., 2013); the samples were mixed for 30 min in the dark with the TMR-Star substrate (New England Biolabs) at a final concentration of 250 nM (Pereira et al., 2013). Cells were collected by centrifugation (4,000 x g for 5 min at room temperature), washed four times with 1 mL of phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4), and resuspended in 0.5 mL of PBS. For phase contrast and fluorescence microscopy, cells were mounted on 1.7% agarose coated glass slides and observed on a Leica DM6000B microscope equipped with a phase contrast Uplan F1 100x objective and captured with a CCD Andor Ixon camera (Andor Technologies). Images were acquired and analyzed using the Metamorph software suite (version 5.8; Universal Imaging) and adjusted and cropped using ImageJ.

Statistical analysis:

All processing assays and germination assays were performed in at least biological triplicate and the data represents the averages from the data sets. Error bars represent the standard error of the mean. A one-way ANOVA Šídák’s multiple comparisons test was used to compare the quantified protein amounts.