The wild-type strain of C. sporogenes used in this study is ATCC15579. Isogenic mutants of this wild-type strain included: (i) a quadruple insertional knockout mutant (gerXA^4-) lacking genes for all known GRs (Brunt et al., 2014); and (ii) mutants lacking sleB or cwlJ that were generated using the Clostron system, which inserts an erythromycin resistance cassette into the targeted gene of interest. Target sites were identified [gene; CLOSPO_00754 (sleB) insert site 337s and gene; CLOSPO_02089 (cwlJ) insert site 145s] using the Pertuka method (Perutka et al., 2004) and mutants were generated as previously described (Heap et al., 2010; Brunt et al., 2014).

Vegetative cells of C. sporogenes were grown anaerobically at 37°C in tryptone-yeast medium (TY) broth or on TY agar plates at 37°C. Spores of C. sporogenes strains were prepared in Robertson’s cooked meat broth (Southern Group Laboratories) and spores were cleaned and stored as described elsewhere (Plowman and Peck, 2002). All spores used in this study were >98% free of sporulating cells, germinated spores, and debris, as observed by phase contrast microscopy. Escherichia coli strains used for mutant construction were grown aerobically in Luria-Broth (LB) agar at 37°C. The E. coli strain Top10 (Invitrogen) was used for plasmid maintenance and the E. coli strain CA434 (Purdy et al., 2002) was used as the conjugation donor for plasmid DNA transfer. Where appropriate, growth media were supplemented with antibiotics or indicator reagent at the following final concentrations: chloramphenicol 25 μg mL^-1, cycloserine 250 μg mL^-1, thiamphenicol 15 μg mL^-1, erythromycin 5 μg mL^-1, and the chromogenic substrate 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) 80 μg mL^-1. All bacterial media supplements were purchased from Sigma (Gillingham, United Kingdom).

Clostridium sporogenes spores (10 μL; ∼10⁸ spores mL^-1 in water) were incubated in water for 15 min at high temperatures (70 or 80°C) and then cooled on ice before germination. Unless noted otherwise, spores were routinely heat shocked at 70°C for 15 min since treatment with 70°C gave more spore germination than with the other temperature examined in the study. For monitoring multiple individual spore’s germination using LTRS, spores were incubated at 37°C with 50 or 100 mM L-alanine in Buffer 1 [20 mM Tris-HCl (pH 7.4), 50 mM NaHCO₃, 50 mM L-lactate]. For investigating the effects of loss of SleB, CwlJ, or GR proteins on spore germination, spores were incubated in 100 mM L-alanine in Buffer 1 at 37°C. Spore germination was also carried out with 60 mM CaDPA (made to pH 7.4 with Tris base) at 37°C and 1.0 mM dodecylamine at 45°C in 20 mM Tris-HCl (pH 7.4) and 50 mM NaHCO₃.

In experiments measuring spores’ commitment to germinate, C. sporogenes spores adhered on a coverslip were exposed to 100 mM L-alanine in Buffer 1 at 37°C for 4 or 8 min, followed by germinant removal, 5 rinses with Buffer 1 by vacuum pump suction, and further incubation at 37°C in Buffer 1 for 45 min.

In experiments measuring spores’ memory of germinant stimulation, C. sporogenes spores adhered to a microscope coverslip were given a 4-min pulse of 100 mM L-alanine in Buffer 1 at 37°C, germination solution removed and spores rinsed five times by vacuum pump suction with 37°C Buffer 1 and held in this buffer at 37°C prior to a 2nd 4 min germinant pulse beginning at 19 or 39 min. In this experiment, spores were also germinated with 100 mM L-alanine in Buffer 1 for 8 min, followed by germinant removal and rinsing as described above and further incubation at 37°C. For quantitation of spore memory with two germinant pulses, the percentages of spores that germinated in the 2nd pulse were corrected for the percentages of spores that had germinated due to the 1st pulse.

Spores (1 μL; ∼10⁸ spores mL^-1 in water) were spread on the surface of a microscope coverslip that was then dried in a vacuum desiccator for 10 min. Coverslips were suspended in water and then mounted on and sealed to a microscope sample holder held at 37°C. A phase contrast microscope was used to record the images of 500–1,000 individual spores. Spores that contain DPA appear bright and spores that do not contain DPA appear as dark. The percentages of spores containing DPA were defined as the number of bright spores divided by the total number of spores.

The CaDPA levels of individual spores of various strains in water were determined by LTRS at room temperature. Briefly, an individual spore was captured with laser tweezers and its Raman spectrum was acquired with an integration time of 20 s and a laser power of 20 mW at 780 nm. Spectra of ∼50 individual spores were measured and averaged. The CaDPA level in an individual spore was determined from the peak intensity of the CaDPA-specific Raman band at 1,017 cm^-1, which was calibrated by the peak intensity of the same Raman band from 60 mM CaDPA using the excitation volume of 1 fl to obtain attomoles of CaDPA spore^-1, as described previously (Kong et al., 2011).

The kinetics of CaDPA release during germination of individual C. sporogenes spores optically trapped by laser tweezers in liquid was measured simultaneously by Raman spectroscopy and DIC microscopy as described previously (Zhang et al., 2010b, 2014). The CaDPA level in an individual spore during germination was determined from the intensities of the CaDPA-specific Raman band at 1,017 cm^-1, and the intensity of the DIC image was also recorded. As found previously (Zhang et al., 2010b; Kong et al., 2011), the end of the rapid fall in DIC image intensity during spore germination corresponded to the point at which release of all CaDPA was complete, and this time point was defined as T_release. At this time, the DIC image intensity (I_release) was 25 to 30% of that at T₀, when image intensity at T₀ was set at 1 and the intensity at the end of measurements was set at zero. Consequently, the CaDPA content of wild-type spores at any time relative to T₀ could be estimated from the DIC image intensity, I_t, as 100% × (I_t -I_release)/(1 -I_release), since the DIC intensity was found to be nearly coincident with the CaDPA level prior to T_release (Figure 3B). In addition to T_release, a number of other spore germination parameters have been previously described (Zhang et al., 2010b; Kong et al., 2011; Wang et al., 2015b; Setlow et al., 2017), including: (i) T₁, the time between germinant addition and initiation of a slow release of 10–20% of CaDPA which begins in some spores in populations; (ii) T_lag (termed T_lag1 in the current work; see Results), the time between germinant addition and initiation of fast CaDPA release; T_release, as defined above; T_lag2, when the slow decrease in spores’ DIC image intensity following T_release ends (see T4 in Figure 3B, and in later figures); T_lys, the time when spore refractility and DIC image intensity becomes constant; and ΔT_release and ΔT_lys, which are calculated as T_release-T_lag1 and T_lys-T_release, respectively.

Simultaneous monitoring of the germination of multiple individual spores was by DIC microscopy as described previously (Zhang et al., 2010a,b). In brief, spores were spread on the surface of a coverslip as described above that was dried in a vacuum desiccator for ∼10 min, and coverslips were mounted on and sealed to a microscope sample holder kept at a constant temperature. The DIC images of multiple spores adhered on coverslips were recorded at a rate of 1 frame per 15 s for 30 min to 11 h by a digital charge-coupled-device camera (16 bits; 1,600 by 1,200 pixels) following the addition of preheated germinant solution to the spores on coverslips. The averaged pixel intensity of an area of 40 by 40 pixels that covered each individual spore’s DIC image was calculated, the DIC image intensity of each individual spore was plotted as a function of the incubation time with a resolution of 15 s and with initial image intensity at the first time of measurement, T₀, normalized to 1, and the intensity at the end of the measurement period normally set at zero. Invariably, the DIC image intensity had been constant for ∼10 min at the end of measurements. The degree of germination of spore populations was measured by simultaneously monitoring the germination of ∼300 individual spores by DIC microscopy, and at various times the percentage of these spores that had released their CaDPA was determined as described above.

Laser tweezers Raman spectroscopy showed that the bands from CaDPA dominate the Raman spectra of individual C. sporogenes spores (Figure 1), just as with spores of Bacillus species, C. perfringens and C. difficile, as spores without CaDPA lack significant bands at 1017, 1395, and 1572 cm^-1 (Kong et al., 2011; Wang et al., 2011, 2015c). The average intensities of the major CaDPA-specific 1,017 cm^-1 Raman band from ∼50 individual spores in isogenic wild-type, sleB, cwlJ, and gerXA^4- strains indicated that the CaDPA levels in spores of these four strains, were 447.7 ± 45.6, 460.2 ± 35.0, 462.3 ± 50.2, and 497.4 ± 84.7 amol/spore, respectively, with no significant differences between these values in the spores of the four strains examined. These values for spore CaDPA content are similar to those in other species (Huang et al., 2007; Wang et al., 2011, 2015c).

A short treatment at a high but generally sub-lethal temperature termed a heat shock synchronizes and potentiates the IM GR-dependent germination of spores of Bacillus and at least some Clostridium species (Collado et al., 2006; Zhang et al., 2009; Luu et al., 2015; Setlow et al., 2017). However, the precise mechanism of the heat shock effect is unknown (Zhang et al., 2009). A heat shock also stimulated the L-alanine germination of C. sporogenes spores, and this germination is GR-dependent [(Brunt et al., 2014); and see below]. Heat shock at 70°C for 15 min had a stronger effect on the extent and rate of germination with L-alanine than that at 80°C for 15 min or no heat shock (Figure 2). Consequently, unless noted otherwise, all spores used for L-alanine germination in this work were heat shocked at 70°C for 15 min.

Previous reports showed that L-lactate could stimulate C. sporogenes spore germination with some amino acids, although alone triggered no spore germination (Brunt et al., 2014). We also found that 50 mM L-lactate/20 mM Tris-HCl (pH 7.4)/50 mM NaHCO₃ (Buffer 1) or 20 mM Tris-HCl (pH 7.4) did not trigger C. sporogenes spore germination (<0.3% with 300 spores examined) (Figure 2), consistent with the previous work (Brunt et al., 2014). However, we routinely added L-lactate/NaHCO₃ to Tris-HCl buffer in L-alanine germinations, in case we later had to test spore germination by other amino acids.

Laser tweezers Raman spectroscopy and DIC microscopy were used to analyze the dynamic germination of individual C. sporogenes spores (Figure 3). The results with a single optically trapped C. sporogenes wild-type spore germinating with 100 mM L-alanine indicated that with some spores, beginning at T₁ there was an initial period of slower CaDPA release (Figures 3C,D) preceding the rapid fall in the spore’s DIC image intensity beginning at T_lag1 and ending at T_release. This fast DIC image intensity decrease closely paralleled the fast release of CaDPA seen in the spore’s time lapse Raman spectra (Figures 3A,B). The slower initial CaDPA release (5–10% of all CaDPA) was often difficult to see in spores germinating rapidly, but was more obvious in spores germinating more slowly (Figures 3E,F). Single spores followed by LTRS also exhibited a further decrease in their DIC image intensity (20–40% of the total) between T_release and T_lys (Figures 3C–F), presumably due to cortex hydrolysis and core swelling and water uptake. Indeed, for at least one spore, significant DIC image intensity was retained until until T₅, long after all CaDPA had been released (Figure 3B). These observations were also made upon examination of changes in the CaDPA and DIC image intensity of more individual spores germinating with L-alanine (Figures 3C–F). However, following T_release, some of these spores exhibited a slow decrease in DIC image intensity which was then followed by a faster decrease in ending at T_lys (Figure 3C). With these latter spores, the end of the pause or the slow decline in DIC image intensity between T_release and T_lys in the L-alanine germination of C. sporogenes spore was termed T_lag2, and therefore T_lag was routinely termed T_lag1 (Figure 3C). Given the good correspondence between CaDPA release and loss of DIC image intensity in spore germination, as in previous studies with B. subtilis spores (Zhang et al., 2010b; Kong et al., 2011), DIC microscopy alone was used to monitor the germination of multiple individual C. sporogenes spores in further experiments. The two phases in the decline in DIC image intensity following T_release during L-alanine germination were more accurately quantitated when L-alanine germination of ∼300 individual spores with or without heat shock was followed by DIC microscopy (Figures 3E,F and Table 1). In these experiments, of the spores germinating with or without an optimal heat shock, 35 and 22%, respectively, showed an obvious T_lag2 inflection. Notably, while the average values of times for fast release of CaDPA (ΔT_release) in the L-alanine germination of multiple individual spores were similar with spores with and without a heat shock, optimally heat-shocked spores showed the lowest values of T₁, T_lag1, T_release, and T_lys, (Table 1). These results suggest that the primary effect of an optimal heat shock on C. sporogenes spore germination is to shorten the T₁ values for individual spores, as seen with spores of other species (Yi and Setlow, 2010; Zhang et al., 2010a; Byun et al., 2011; Setlow et al., 2017).

Previous work has found that C. sporogenes spores lacking most GR proteins exhibit minimal germination with L-alanine over many hours (Brunt et al., 2014). This was also found in the current work, as the gerXA^4- spores exhibited ≤1% germination in 2 h, and ≤1% germination even in 12 h (Figure 4A; and data not shown), consistent with the absence of all functional GRs and the tremendously reduced viability of gerXA^4- spores (Brunt et al., 2014). In Bacillus species, two CLEs, SleB, and CwlJ, play redundant roles in spore germination as either protein alone can give full cortex hydrolysis, and CaDPA release is only slightly slowed in cwlJ sleB spores (Setlow et al., 2017). SleB appears to be a lytic transglycosylase that is synthesized in the forespore and is present in spores in a mature form; this is likely also the case for CwlJ, although its bond specificity is not clear (Moriyama et al., 1996, 1999; Setlow et al., 2017). Surprisingly, the absence of either CwlJ or SleB greatly reduced C. sporogenes spore germination with L-alanine, as there was ≤2% germination in 2 h (Figure 4A). However, if the L-alanine germination time was followed for 12 h, both cwlJ and sleB spores exhibited >90% spore germination (Figures 5A,C). Notably, almost all germinating cwlJ spores examined exhibited obvious T_lag2 times, while germinating sleB spores did not (Figures 5B,D and Table 2). Although almost all average times of various events in the L-alanine germination of individual cwlJ and sleB C. sporogenes spores were much larger than with wild-type spores under the same germination conditions, the average value of ΔT_release increased only ∼ 2-fold, and only with sleB spores (Tables 1, 2).

In addition to germination stimulated by the GR-dependent germinant L-alanine, we also tested whether wild-type C. sporogenes spores were responsive to the GR-independent germinants CaDPA and dodecylamine that trigger germination of spores of Bacillales and some Clostridiales spores (Setlow, 2003; Olguin-Araneda et al., 2015; Wang et al., 2015c; Setlow et al., 2017) (Figures 4B,C). Wild-type C. sporogenes spores germinated >90% in 60 mM CaDPA in 2 h, but only ∼50% with 1 mM dodecylamine, and this value had become level at 50 min. Compared to germination with L-alanine, analysis of the germination of multiple individual wild-type C. sporogenes spores with dodecylamine (Figures 6A,B) gave a smaller average ΔT_release time, larger average T₁, T_lag1, T_release, and T_lys times and similar average ΔT_lys times; germination with CaDPA had larger average T_lag1, T_release and T_lys times and smaller average ΔT_release and ΔT_lys values (Table 1). In addition, 16% of wild-type spores germinating with CaDPA showed T_lag2 times following T_release, but minimal if any wild-type spores germinating with dodecylamine exhibited a T_lag2 (Figures 6A,B and Table 1).

CaDPA and dodecylamine also triggered the germination of gerXA^4- and sleB spores, albeit to a lesser extent than with wild-type spores (Figures 4B,C, 6C–F). Minimal if any of these two mutant spores exhibited T_lag2 times in germination with CaDPA and dodecylamine, and average times for various germination events were otherwise relatively similar between wild-type and gerXA^4- spores but higher for sleB spores (Tables 1, 2). Dodecylamine also germinated cwlJ spores, and with larger germination time parameters than with wild-type spores, and a very low percentage of spores with a T_lag2 (Figure 4C and Table 2). However, CaDPA gave minimal germination of cwlJ spores in 2 h (<5%), consistent with CwlJ being activated by CaDPA as is the case in spores of Bacillus species.

A notable feature of Bacillus and C. difficile spores’ germination is their irreversible commitment to germination, followed by CaDPA release after a short GR-dependent exposure (Yi and Setlow, 2010; Luu and Setlow, 2014; Wang et al., 2015b). Thus we examined if C. sporogenes spores also showed commitment after brief exposures to L-alanine, followed by germinant removal, extensive rinsing, and further incubation in buffer. Like Bacillus and C. difficile spores, C. sporogenes spores given a short germinant exposure continued to release CaDPA after germinant removal. Indeed, C. sporogenes spores given 4 or 8 min pulses of L-alanine that gave only 3 and 22% germination, respectively, at the time of germinant removal ultimately released 26 and 50% of their CaDPA, respectively (Figure 7A). These results suggest that C. sporogenes spores also commit to germination shortly after germinant exposure.

A recent novel finding in spore germination is that spores of at least several Bacillus species as well as C. difficile exhibit memory of germinant stimuli such that spores given a 1st germinant pulse exhibit greater germination after a 2nd pulse soon after the 1st one; however, this memory decays quickly when the separation between the two pulses is increased (Wang et al., 2015a). With C. sporogenes spores given a 4 min L-alanine pulse, there was ∼24% germination which ended at ∼10 min (Figure 7B). If a 2nd 4 min L-alanine pulse was then given at 19 min, ∼30% of the remaining dormant spores germinated, but if the 2nd pulse was not given until 39 min, only 23% of the spores remaining dormant after the 1st pulse germinated after the 2nd pulse (Figure 7B). However, this difference of 7% was small, and not as large that in B. subtilis spores germination memory, indicating that C. sporogenes spore memory in GR-dependent germination is low.