Isolation and characterization of Bacillus subtilis spores that are superdormant for germination with dodecylamine or Ca2+-dipicolinic acid

Authors


Correspondence

Peter Setlow, Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT06030-3305, USA. E-mail: setlow@nso2.uchc.edu

Abstract

Aims

To isolate and characterize spores superdormant (SD) for germination with either Ca2+-dipicolinic acid (CaDPA) or dodecylamine.

Methods and Results

Bacillus subtilis spores were germinated three times with either CaDPA or dodecylamine and germinated spores removed after each germination treatment, yielding 0·9% (CaDPA-SD spores) or 0·4% (dodecylamine-SD spores) of initial dormant spores. Compared to dormant spores, CaDPA-SD spores germinated poorly with CaDPA and better with dodecylamine and nutrient germinants, although release of DPA from individual CaDPA-SD spores was slow during nutrient germination, and this germination was strongly inhibited by TbCl3. The CaDPA-SD spores were sensitive to hypochlorite and had elevated levels of nutrient germinant receptors (GRs) relative to levels in dormant spores. Dodecylamine-SD spores' germination with dodecylamine and nutrients was similar to that of dormant spores, their germination with Ca-DPA was slower than that of dormant spores, and these SD spores' GR levels were lower than in dormant spores. However, dodecylamine-SD spores were not sensitive to hypochlorite, and the nutrient germination of these SD spores was only partially inhibited by TbCl3.

Conclusions

CaDPA-SD spores appear to have a coat defect and accompanying low levels of the cortex-lytic enzyme CwlJ. The defect in dodecylamine-SD spores, however, is not clear.

Significance and Impact of the Study

The results suggest that triggering germination by non-GR-dependent germinants is a potential strategy for efficient spore inactivation.

Introduction

Dormant spores of various Bacillus species are significant causes of much food spoilage and food poisoning and spores of Bacillus anthracis are a potential agent for biological warfare and terrorism (Setlow and Johnson 2012). As a consequence, there is much interest in methods to kill spores, something that is not trivial because of the dormant spores' extreme resistance to many different agents. One attractive strategy for spore killing is to first trigger dormant spores to germinate, because germination results in the loss of most spore resistance properties, and the germinated spores are relatively easy to kill, for example by a relatively mild heat treatment (Setlow 2003; Paredes-Sabja et al. 2011; Setlow and Johnson 2012). While this simple strategy can significantly decrease spore populations, it is not completely effective because of the extreme heterogeneity in germination properties between individuals in spore populations (Setlow and Johnson 2012; Setlow et al. 2012). Thus, during germination of spore populations with nutrient germinants, a small percentage of spores germinates extremely slowly, in some cases not for many h or even d, and these spores will escape mild inactivation treatments following relatively short germination with nutrients. These spores that are slow to germinate are termed superdormant (SD), and recent work has shown that spores SD for germination with particular nutrient germinants have very low levels of the germinant receptors (GRs) that recognize these nutrient germinants (Ghosh and Setlow 2009, 2010; Ghosh et al. 2009, 2012). This heterogeneity in spore germinant receptor (GR) levels that leads to significant heterogeneity in nutrient germination appears to be due in large part to stochastic effects, although it is possible that some factors that regulate expression of genes that encode GRs are actually noise generators (Ramirez-Peralta et al. 2012a,b; Setlow et al. 2012).

While nutrient germinant triggering of GRs is likely the major mechanism triggering spore germination in nature, there are additional agents that can trigger spore germination in processes that do not require GRs (Setlow 2003). Two such agents are as follows: (i) cationic surfactants, most notably dodecylamine, that appear to trigger spore germination by directly activating the SpoVA protein channel that allows the release of spores' large depot (c. 10% of spore dry wt) of the 1 : 1 chelate of Ca2+ and pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] from the spore core (Setlow et al. 2003; Vepachedu and Setlow 2007; Li et al. 2012); and (ii) high concentrations of Ca2+dipicolinic acid (CaDPA) that trigger Bacillus spore germination by activating the cortex-lytic enzyme (CLE) CwlJ that initiates degradation of the spore's peptidoglycan (PG) cortex, with this degradation then triggering subsequent germination events (Paidhungat et al. 2001; Setlow et al. 2009; Heffron et al. 2010).

As CaDPA and dodecylamine trigger spore germination by a process that does not require GRs, it seems likely that these agents would trigger the germination of spores SD for germination with GR-dependent germinants, and this is indeed the case (Ghosh and Setlow 2009, 2010). Consequently, it seems possible that agents that trigger spore germination via pathways initiated by CaDPA or dodecylamine might lead to more complete spore germination prior to a relatively mild decontamination regimen. However, some spores in populations may also be refractory to germination by direct activation of CwlJ or the SpoVA channel and will thus be SD for germination with CaDPA or dodecylamine. To address this possibility, we have germinated spore populations multiple times with either CaDPA or dodecylamine, and isolated spores that appear to be SD for germination with these agents. These novel SD spores have been characterized, and their properties suggest some potential strategies for dealing with spores that are SD for both GR-dependent and GR-independent germinants.

Materials and methods

Bacillus subtilis strain used and spore preparation and purification

The Bacillus subtilis strain used in this work was PS533 (Setlow and Setlow 1996), a prototrophic derivative of strain 168 that carries plasmid pUB110 conferring kanamycin resistance (10 mg l−1). Spores of this strain were prepared at 37°C on 2× Schaeffer's-glucose agar plates without kanamycin as described (Nicholson and Setlow 1990; Paidhungat et al. 2000). After 2–3 days at 37°C, plates were incubated at 23°C for 1–3 days and then spores were scraped from the plates and purified by repeated centrifugations and water washing, with intermittent mild sonication treatment. All spores used in this work were free (>98%) from growing or sporulating cells and germinated spores, as determined by phase contrast microscopy.

Isolation of SD spores

For isolation of spores that are SD for germination with either CaDPA or dodecylamine, termed CaDPA-SD spores and dodecylamine-SD spores, respectively, spores were routinely germinated at an optical density at 600 nm (OD600) of 1 in either 60 mmol l−1 Ca-DPA made to pH 7·5 with Tris base at 23°C, or 1·2 mmol l−1 freshly prepared dodecylamine at 45°C in 25 mmol l−1 K-Hepes buffer pH 7·4, or in a few cases with 25 mmol l−1 KPO4 buffer, pH 7·4. After germination for 3 h, cultures were harvested by centrifugation (15 min; 10 000 g) at 23°C. The dodecylamine-germinated spores were washed with water, while the CaDPA germinated spores were washed with 0·1 mol l−1 EDTA to remove any precipitated CaDPA that interfered with subsequent separation of dormant and germinated spores. Washed spores were suspended at an OD600 of 100 in 1·5 ml of 50% wt/vol Nycodenz (Sigma Chemical Co., St. Louis, MO, USA) and centrifuged for 30 min at 4°C in a microcentrifuge. Under these conditions, dormant spores pellet, while germinated spores float and are discarded. In some cases, brief sonication of the dodecylamine-germinated spores prior to centrifugation with Nycodenz was used to shear DNA released from lysing germinated spores, as this released DNA appeared to trap dormant spores in the floating germinated spores; similar results were obtained by addition of DNAase. The dormant spore pellets were washed several times with water, and the germination and dormant spore isolation described above was repeated twice more. The final SD spore preparations were stored frozen in water at an OD600 of 5–15 in multiple aliquots.

Spore germination

Spore germination with CaDPA as described above was monitored by phase contrast microscopy, with at least 100 spores examined at each time point taken. Dodecylamine germination was also done as described above and at various times 200 μl aliquots were centrifuged in a microcentrifuge, the supernatant fluid made 50 μmol l−1 in TbCl3, and Tb-DPA fluorescence was measured as described (Yi and Setlow 2010). For germination of spores with nutrient germinants, either l-valine or a mixture of l-asparagine, d-glucose, d-fructose and K+ (AGFK), spores were first heat-activated in water at 75°C for 30 min and then cooled on ice. Spore germination with l-valine or AGFK was performed at 37°C in 25 mmol l−1 K-Hepes buffer (pH 7·4) and spores at an OD600 of 0·5, and with either 10 mmol l−1 l-valine or with 10 mmol l−1 l-asparagine, 10 mmol l−1 d-glucose, 10 mmol l−1 d-fructose. In some experiments, germination with l-valine and AGFK was monitored by inclusion of 50 μmol l−1 TbCl3 in the incubation, with samples' fluorescence monitored continuously in a multi-well fluorometer plate reader as described (Yi and Setlow 2010). However, as Tb+3 can strongly inhibit the nutrient germination of spores with coat defects (Yi et al. 2011), germination with l-valine and AGFK was also carried out as described above, but without TbCl3 present continuously, but rather added at various times after the start of germination. Aliquots of germinating cultures were centrifuged in a microcentrifuge, TbCl3 was added to the supernatant fluid to 50 μmol l−1, and Tb-DPA fluorescence was measured as described above.

Analytical procedures

Spore killing by sodium hypochlorite was carried out at 23°C as described with NaOCl (50 mg l−1) at pH 7·5 (Young and Setlow 2003), and spore viability was measured on LB medium plates (Paidhungat et al. 2000) containing kanamycin (10 mg l−1). Isolation of inner membrane fractions from SD spores and initial dormant spores and the analysis of levels of germination proteins in these membrane fractions by Western blot analysis were as described (Ghosh et al. 2012; Ramirez-Peralta et al. 2012a,b). Levels of germination proteins in initial dormant spores or SD spores were compared by running samples with equal amounts of initial dormant or SD spores' inner membrane protein together on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blots. The same Western blot was used for analysis of different germination proteins by stripping the blots following analysis of one protein and then reprobing with a new antiserum as described (Ghosh et al. 2012; Ramirez-Peralta et al. 2012a,b).

Incubations to follow the l-valine or dodecylamine germination of individual dormant and SD spores adhered to glass were done as described above but in the absence of TbCl3, and the germination of multiple individual spores was monitored by differential interference contrast (DIC) microscopy as described (Kong et al. 2011; Zhang et al. 2012). This analysis of the germination of hundreds of individual spores allowed the calculation of a number of kinetic parameters of individual spore's germination including: (i) the time, Tlag, between the mixing of spores with germinant and the initiation of rapid CaDPA release; (ii) the time, Trelease, at which >90% of CaDPA is released; (iii) the time ΔTrelease, which is TreleaseTlag; (iv) the time, Tlys, when hydrolysis of a spore's PG cortex and full spore core hydration are complete, as indicated by the completion of the fall in a spore's DIC image intensity; and (v) the time, ΔTlys, which is TreleaseTlys. ΔTlys is the time for the hydrolysis of the spore's PG cortex and full spore core hydration, since PG cortex lysis takes place only after DPA release (Kong et al. 2011).

Results

Isolation of spores SD for germination with CaDPA or dodecylamine

Germination of Bacillus subtlis spores with either CaDPA or dodecylamine was not complete, even when spores were germinated three times for 3 h, with spores that germinated removed after each germination treatment. When the 3 h germination with either CaDPA or dodecylamine was carried out three times, yields of spores that remained dormant at the end of the third germination were significant (Table 1). These spores that remained dormant were defined as SD, and the CaDPA-SD spores were 0·5–1·6% of the initial dormant spores, and the dodecylamine-SD spores were 0·1–1·1% of initial spores used (Table 1). It was noticeable that the recovery of dormant spores germinated with CaDPA appeared to be levelling off somewhat during the multiple germinations, while recoveries of spores following dodecylamine germination continued to decrease relatively similarly through all three germination treatments.

Table 1. Yields of spores SD for germination with CaDPA or dodecylaminea
Germination withDormant spores recoveredb – % of initial dormant spores
One germinationTwo germinationsThree germinations
  1. a

    Initial dormant spores were subjected to one, two or three germinations with either CaDPA or dodecylamine, and remaining dormant spores were isolated as described in 'Materials and methods'. Values shown are from four independent preparations of spores that were run through the germination procedures with CaDPA or dodecylamine.

  2. b

    Recoveries based on the OD600 of initial and recovered spores.

  3. c

    The highest values for recoveries in these experiments were when the germinated spores were sonicated briefly before applying to density gradients.

CaDPA10 (8–16)2·7 (2·2–3·5)0·9 (0·5–1·6)
Dodecylamine15·5 (14·5–16·5c)4·0 (2·8–5·8c)0·4 (0·1–1·1c)

Germination of CaDPA-SD and dodecylamine-SD spores with various germinants

The CaDPA-SD spores germinated extremely poorly with CaDPA compared with the initial dormant spores as expected, and this was seen with three different preparations of the CaDPA-SD spores (Fig. 1a; and data not shown). In contrast, the germination of these SD spores with dodecylamine was much faster than was the dodecylamine germination of the initial dormant spores; again this was seen with two different preparations of these SD spores (Fig. 1b, and data not shown). Surprisingly, the CaDPA-SD spores exhibited no germination with the GR-dependent germinants l-valine or AGFK when spore germination was monitored by inclusion of Tb+3 in germination incubations and continuous monitoring of the fluorescence due to complex formation between Tb and DPA released during spore germination (Fig. 1c).

Figure 1.

(a–d) Germination of initial dormant spores and CaDPA-SD spores with various germinants. Initial dormant spores (○, ▵) and CaDPA-SD spores (●, ▲) were germinated as described in 'Materials and methods' with: (a) CaDPA; (b) dodecylamine; (c) l-valine (○,●) or AGFK (▵,▲) with TbCl3 present from the start of germination; and (d) l-valine (○,●) or AGFK (▵,▲) with TbCl3 added only at various times after the start of germination. The percentages of spore germination were determined either by phase contrast microscopy (a) or by quantitation of DPA release relative to the total amount of DPA in spores released by boiling (b–d). Results similar to those shown in this experiment were seen with 2–3 independent preparations of CaDPA-SD spores. Note that the initial dormant spore preparation used to generate these CaDPA-SD spores was not the same initial dormant spore preparation used to generate the dodecylamine-SD spores used in Fig. 2.

The apparent minimal GR-dependent germination of the CaDPA-SD spores was surprising, because lack of GR-dependent spore germination had not been selected for in isolation of these SD spores. In addition, spores that are SD for germination with nutrient germinants such as l-valine germinate normally with CaDPA (Ghosh et al. 2009; Ghosh and Setlow 2010). However, a trivial explanation for the absence of GR-dependent germination of the CaDPA-SD spores is that Tb+3 is inhibiting these spores' germination with l-valine or AGFK, as is the case for spores with defective coats (Yi et al. 2011). To test whether the CaDPA-SD spores germinated well with nutrient germinants in the absence of Tb+3, the Tb+3 was added only at various times after initiation of germination and then Tb-DPA fluorescence was measured. When germination was measured in this manner, the CaDPA-SD spores were seen to germinate normally with both l-valine and AGFK, indeed, even notably faster than the initial dormant spores with AGFK (Fig. 1d).

The dodecylamine-SD spores also germinated more poorly than initial dormant spores with CaDPA (Fig. 2a), although not as poorly as did the CaDPA-SD spores (compare Fig. 2a and Fig. 1a). However, dodecylamine germination of the dodecylamine-SD spores was only slightly slower that that of initial dormant spores (Fig. 2b); this small difference was seen with several independent dodecylamine-SD spore preparations (data not shown). The germination of the dodecylamine-SD spores with l-valine and AGFK was also slower that that of the initial dormant spores when TbCl3 was present throughout germination (Fig. 2c), but not nearly as slow as CaDPA-SD spores germinated similarly (compare Fig. 2c and Fig. 1c). However, when TbCl3 was added only at various time points in germination to measure the DPA released at that point, it was again clear that Tb+3 partially inhibited the l-valine and AGFK germination of the dodecylamine-SD spores and that the rates of l-valine and AGFK germination of the dodecylamine-SD spores were actually very similar to those of the initial dormant spores (Fig. 2d).

Figure 2.

(a–d) Germination of initial dormant spores and dodecylamine-SD spores with various germinants. Initial dormant spores (○, ▵) and dodecylamine-SD spores (●, ▲) were germinated as described in 'Materials and methods' with: (a) CaDPA; (b) dodecylamine; (c) l-valine (○,●) or AGFK (▵,▲) with TbCl3 present from the start of germination; and (d) l-valine (○,●) or AGFK (▵,▲) with TbCl3 added only at various times after the start of germination. The percentages of spore germination were determined either by phase contrast microscopy (a) or by quantitation of DPA release relative to the total amount of DPA in spores released by boiling (b–d). Results similar to those shown in this experiment were seen with two independent preparations of dodecylamine-SD spores. Note that the initial dormant spore preparation used to generate these dodecylamine-SD spores was not the same initial dormant spore preparation used to generate the CaDPA-SD spores used in Fig. 1.

Analysis of the germination of individual dormant, CaDPA-SD and dodecylamine-SD spores

The analysis of the germination of the SD spores presented above was carried out with spore populations. However, the germination behaviour of spore populations is quite heterogeneous, and this heterogeneity can often obscure important information about germination of individual spores. Indeed, analysis of the germination of multiple individual spores has given much new information about spore germination (Kong et al. 2011; Zhang et al. 2012). Consequently, we also examined the germination of multiple individual dormant, CaDPA-SD and dodecylamine-SD spores with either l-valine or dodecylamine (Figs 3a–d; 4a–d; Tables 2 and 3). As found previously (Kong et al. 2011; Zhang et al. 2012), germination of individual dormant spores with either l-valine or dodecylamine exhibited a variable lag period, Tlag, following germinant addition and prior to the rapid fall in a spore's DIC image intensity due to rapid release of endogenous CaDPA, with CaDPA release complete at Trelease (Fig. 3a,c). Following Trelease, there was a further slow fall in spores' DIC image intensity due to hydrolysis of the spores' PG cortex and accompanying spore core swelling and water uptake, with this slow decrease ending at Tlys. The additional parameters that can be obtained from these data for individual spores are ΔTrelease = TreleaseTlag, with this value being the time for the release of ≥90% of a spore's CaDPA depot, and ΔTlys, with this value being the time for the hydrolysis of the PG cortex and full spore core hydration (Kong et al. 2011; Zhang et al. 2012).

Table 2. l-valine and dodecylamine germination parameters of initial dormant and CaDPA-SD sporesa
Germinant (spores)Germination parameters (min)
T lag T release T lys ΔTreleaseΔΤlys
  1. DIC, differential interference contrast.

  2. a

    Germination parameters for individual initial dormant spores or CaDPA-SD spores were determined by DIC microscopy as described in 'Materials and methods'. Values shown are averages of data from ≥260 individual spores that germinated. Note that the initial dormant spore preparation used to generate these CaDPA-SD spores was different than the initial dormant spores used in Table 3.

l-valine (initial dormant)26 ± 2030 ± 2044 ± 214·3 ± 1·713 ± 3·7
l-valine (CaDPA-SD)24 ± 2334 ± 2473 ± 369·7 ± 2·839 ± 27
Dodecylamine (initial dormant)190 ± 131198 ± 132247 ± 1427·0 ± 2·649 ± 39
Dodecylamine (CaDPA-SD)26 ± 4030 ± 40617 ± 2904·4 ± 2·0588 ± 293
Table 3. l-valine and dodecylamine germination parameters of initial dormant and dodecylamine-SD sporesa
Germinant (spores)Germination parameters (min)
T lag T release T lys ΔTreleaseΔΤlys
  1. DIC, differential interference contrast.

  2. a

    Germination parameters for individual initial dormant spores or dodecylamine-SD spores were determined by DIC microscopy as described in 'Materials and methods'. Values shown are averages of data from ≥290 individual spores that germinated. Note that the initial dormant spore preparation used to generate these dodecylamine-SD spores was different than the initial dormant spores used in Table 2.

l-valine (initial dormant)22 ± 1525 ± 1535 ± 142·9 ± 0·810 ± 2·5
l-valine (dodecylamine-SD)24 ± 1829 ± 1848 ± 194·7 ± 1·719 ± 6
Dodecylamine (initial dormant)342 ± 267349 ± 268373 ± 2656·3 ± 2·925 ± 10
Dodecylamine (dodecylamine-SD)254 ± 325264 ± 324294 ± 32310 ± 4·131 ± 9
Figure 3.

(a–d) Germination of multiple individual initial dormant and CaDPA-SD spores with l-valine or dodecylamine. The germination of six randomly selected individual initial dormant spores (a, c) and CaDPA-SD spores (b, d) with l-valine (a, b) or dodecylamine (c, d) was followed by differential interference contrast microscopy as described in 'Materials and methods'. In a–d, the times for Tlag, Trelease and Tlys are noted for one spore by the vertical arrows, and in d, the Tlag and Trelease times for this single spore are shown in the inset in the box in panel d with an expanded scale, while the Tlys time for this spore is shown in the full-scale panel. Note that the initial dormant spore preparation used to generate these CaDPA-SD spores was not the same initial dormant spore preparation used to generate the dodecylamine-SD spores used in Fig. 4.

Figure 4.

(a–d) Germination of multiple individual initial dormant and dodecylamine-SD spores with l-valine or dodecylamine. The germination of six randomly selected individual initial dormant spores (a, c) and dodecylamine-SD spores (b, d) with l-valine (a, b) or dodecylamine (c, d) was followed by differential interference contrast microscopy as described in 'Materials and methods'. In a–d, the times for Tlag, Trelease and Tlys are noted for one spore by the vertical arrows. Note that the initial dormant spore preparation used to generate these dodecylamine-SD spores was not the same initial dormant spore preparation used to generate the CaDPA-SD spores used in Fig. 3.

The germination of the individual CaDPA-SD spores was significantly different than that of the initial dormant spores, as individual Ca-DPA spore's germination with both l-valine and dodecylamine exhibited an initial slow fall in DIC image intensity prior to the rapid fall (Fig. 3b,d). However, when average germination parameters from multiple individual initial dormant and CaDPA-SD spores were determined (Table 2), most of these values generally reflected what was seen when compared with germination of spore populations. Thus, the average Tlag values for l-valine germination were almost identical for both the initial dormant spores and CaDPA-SD spores, consistent with the very similar germination of these spore populations with l-valine. In contrast, the average ΔTrelease value was >2-fold longer for the CaDPA-SD spores germinating with l-valine. The CaDPA-SD spores also had much shorter average Tlag times for dodecylamine germination than the initial dormant spores, consistent with the faster dodecylamine germination of the CaDPA-SD spore population. The average ΔTrelease value for dodecylamine germination was also slightly smaller for the CaDPA-SD spores, although this difference may not be significant. The most notable difference between dormant and CaDPA-SD spores' germination with l-valine and dodecylamine was the 3- to 10-fold longer, respectively, average ΔTlys times for the CaDPA-SD spores.

Analysis of the germination of multiple individual dodecylamine-SD spores gave results that were similar in some respects to the results with the individual CaDPA-SD spores (Table 3; compare with Table 2). Thus, average Tlag values in l-valine germination of dodecylamine-SD spores were similar to those for the initial dormant spores, and average ΔTrelease and ΔTlys times for l-valine germination were slightly longer for the dodecylamine-SD spores, although less so than seen with CaDPA-SD spores. However, consistent with the relatively rapid germination of dodecylamine-SD spore populations with dodecylamine, the kinetic parameters of dodecylamine germination of multiple individual dodecylamine-SD and initial dormant spores were quite similar.

Hypochlorite resistance of CaDPA- and dodecylamine-SD spores

The inhibition of the GR-dependent germination of the CaDPA-SD spores by Tb+3 strongly suggested that these spores have a significant coat defect, because Tb+3 strongly inhibits the GR-dependent germination of coat-defective spores (Yi et al. 2011). That these SD spores might have defective coats was also consistent with their more rapid germination with dodecylamine (Setlow et al. 2003). In addition, spores with severely defective coats have extremely low levels of CwlJ, the CLE activated by CaDPA to trigger spore germination (Paidhungat et al. 2001; Ragkousi et al. 2003). To test whether the CaDPA-SD spores do indeed have defective coats, we measured these spores' resistance to hypochlorite, because an intact spore coat is essential for spores' hypochlorite resistance and coat-defective spores are extremely sensitive to this agent (Young and Setlow 2003). Strikingly, the CaDPA-SD spores were killed c. 95% in 15 min by dilute hypochlorite, while the initial dormant spores exhibited no killing by this same reagent through at least 60 min (Fig. 5). In contrast, the dodecylamine-SD spores exhibited no killing with hypochlorite (Fig. 5).

Figure 5.

Hypochlorite resistance of initial dormant spores, dodecylamine-SD spores and CaDPA-SD spores. Initial dormant spores (○), CaDPA-SD spores (●) and dodecylamine-SD spores (▲) were incubated in hypochlorite and spore viability was measured as described in 'Materials and methods'. The viabilities of the starting spores and the SD spores were essentially identical.

Levels of germination proteins in initial dormant, CaDPA-SD and dodecylamine-SD spores

Given some of the differences in the rates of germination of initial dormant, dodecylamine-SD and CaDPA-SD spores, it was of obvious interest to determine whether this was due to differences in levels of various specific germination proteins. Previous work has shown that levels of various GRs can have large effects on rates of spore germination with nutrients, while levels of functional SpoVA proteins can have effects on rates of DPA release during germination (Vepachedu and Setlow 2004, 2007; Wang et al. 2011; Ghosh et al. 2012; Ramirez-Peralta et al. 2012a,b). Notably, levels of the subunits of the GerA, GerB and GerK GR subunits GerAC, GerAA, GerBC and GerKA were 2·5–3·5-fold higher in the CaDPA-SD spores than in the initial dormant spores (Fig. 6; Table 4). In contrast, the level of the SpoVAD protein important in DPA release during spore germination, and the GerD protein important in all GR-dependent spore germination were essentially identical in both initial dormant and CaDPA-SD spores. Figure 6; Table 4). In contrast to the results with CaDPA-SD spores, the dodecylamine-SD spores had 2·5- to 5-fold lower levels of GR subunits, and a comparably lower level of the GerD protein, while these spores' level of SpoVAD was c. 2-fold higher than in the initial dormant spores (Fig. 7; Table 4).

Table 4. Relative levels of germination proteins in initial dormant spores, CaDPA-SD spores and dodecylamine-SD sporesa
ProteinRelative germination protein level
Initial dormantbCaDPA-SDDodecylamine-SD
  1. a

    Levels of various germination proteins in SD spores relative to levels in initial dormant spores were determined by Western blot analyses as described in 'Materials and methods' and the legends to Figs 6 and 7. Protein levels were quantitated by Image J analysis of Western blots.

  2. b

    Levels of all germination proteins in initial dormant spores were set as 1·0.

GerAA1·03·50·4
GerAC1·02·50·4
GerBC1·02·50·3
GerKA1·02·80·2
GerD1·01·10·4
SpoVAD1·01·02·1
Figure 6.

Relative levels of various germination proteins in initial dormant spores and CaDPA-SD spores. Relative levels of various germination proteins in spores were determined by Western blot analysis of equal amounts of spore inner membrane protein as described in 'Materials and methods'. Protein levels in the 1× samples were identical in the initial dormant and CaDPA-SD spore samples. All strips used in the detection of the different antigens were from the same Western blot that was stripped and then reprobed, with both dormant and SD lanes exposed to X-ray film together. All the samples from dormant and SD spores were run adjacent to each other as shown in the figure; the separation between the dormant and SD lanes was put in only to add clarity.

Figure 7.

Relative levels of various germination proteins in initial dormant spores and dodecylamine-SD spores. Relative levels of various germination proteins in spores were determined by Western blot analysis of equal amounts of spore inner membrane protein as described in 'Materials and methods'. Protein levels in the 1× samples are identical in the initial dormant and dodecylamine-SD spore samples. All strips used in the detection of the different antigens were from the same Western blot that was stripped and then reprobed, with both dormant and SD lanes exposed to X-ray film together. All the samples from dormant and SD spores were run adjacent to each other as shown in the figure; the separation between the dormant and SD lanes was put only to add clarity.

Discussion

The work in this communication shows clearly that B. subtilis spores that are SD for CaDPA germination can readily be isolated, and as expected these spores germinate extremely poorly with CaDPA. A variety of evidence indicates that CaDPA-SD spores have a coat defect that results in low levels of the CLE CwlJ whose activation by CaDPA triggers spore germination (Paidhungat et al. 2001). This evidence includes the following: (i) like CaDPA-SD spores, spores with defective coats exhibit little if any CaDPA germination, likely due to their low levels of CwlJ (Paidhungat et al. 2001); (ii) the much more rapid germination with dodecylamine of CaDPA-SD spores, characteristic of coat-defective spores (Setlow et al. 2003); (iii) the longer average ΔTrelease value for individual spore's germination with l-valine, characteristic of the germination of individual spores that are defective in CwlJ (Peng et al. 2009; Setlow et al. 2009), and coat-defective spores can readily lose CwlJ (Paidhungat et al. 2001); and (iv) the CaDPA-SD spore population's greatly decreased resistance to hypochlorite, characteristic of coat-defective spores (Young and Setlow 2003). These results further suggest that in spore populations, spores that are SD for CaDPA germination are those with a significant spore coat defect, because as noted above, coat-defective spores germinate extremely poorly with CaDPA, and would thus be isolated as CaDPA-SD spores. We have no direct measurements of CwlJ levels in CaDPA-SD spores, but all available evidence is consistent with CaDPA-SD spores having low CwlJ levels, and almost certainly because these SD spores have defective coats. Note, however, that the CaDPA-SD spore population is not homogeneous, as a small percentage of the SD spores are resistant to hypochlorite. In addition, the increase in ΔTrelease in the CaDPA-SD spores is only c. 2·3-fold, while complete loss of CwlJ results in much larger increases in average ΔTrelease times in GR-dependent germination (Peng et al. 2009). Thus, CwlJ levels in the CaDPA-SD spores cannot be zero, indeed these SD spores do germinate slowly with CaDPA, but CwlJ levels seem most likely to be low. Interestingly, CaDPA-SD spore populations germinated faster with nutrient germinants than the initial dormant spores, indicating that transit of nutrient germinants through the spore coat is not decreased in the CaDPA-SD spores and may even be faster than in the initial dormant spores. Thus, the CaDPA-SD spores do not have a specific defect in the GerP coat proteins that appear to be essential for rapid nutrient germinant permeation through the spore coats (Butzin et al. 2012).

While it appears likely that a spore coat defect is the reason for B. subtilis spores' dormancy with CaDPA, it was extremely surprising that these spores also had elevated levels of GR subunits, although this latter finding was consistent with the more rapid germination of CaDPA-SD spore populations with nutrient germinants. This finding does, however, suggest that the small percentage of spores' SD for CaDPA may not be due to a small percentage of spores that have been damaged during sporulation or spore purification, as this should not lead to elevated GR levels in these SD spores. Rather it suggests that the small percentage of CaDPA-SD spores in spore populations is due to sporulation heterogeneity that results in simultaneous generation of both a coat defect and elevated GR levels. B. subtilis spore populations are known to be extremely heterogeneous in their GR levels, with low GR levels giving rise to spores' SD for nutrient germination (Ghosh and Setlow 2009, 2010; Setlow et al. 2012). Thus, a small percentage of spores having elevated GR levels would be expected, and perhaps the skewed regulation of operons encoding GRs also results in a spore coat defect, as alterations in gene expression in the developing forespore can have marked effects on expression of genes encoding coat proteins on the mother cell (Setlow et al. 2000).

In contrast to the CaDPA-SD spores that germinated extremely poorly with the germinant used to isolate these SD spores, the dodecylamine-SD spores germinated relatively well with dodecylamine, and when germination of either spore populations or multiple individual spores was measured. This was a very surprising result, and one for which we have no definitive explanation. However, perhaps the dodecylamine germination of spores is to some degree stochastic, and in spore populations, some spores germinate slowly purely by chance. We do not know whether this is the case, but this is at least somewhat consistent with the relatively consistent decreases in yields of spores through multiple germinations with dodecylamine, as compared to the apparent levelling off in yields of spores through multiple germinations with CaDPA.

The dodecylamine-SD spores did, however, have significant differences from the initial dormant spores, even though it is not clear how these differences were selected for in the isolation of the SD spores. Thus, the dodecylamine-SD spores germinated poorly with CaDPA, and their nutrient germination was inhibited somewhat by Tb+3, suggesting that these SD spores also have a coat defect as noted above for the CaDPA-SD spores. However, as the dodecylamine-SD spores germinated normally with dodecylamine and exhibited no sensitivity to hypochlorite, any coat defect must be minor and involve only a small fraction of this SD spore population.

The dodecylamine-SD spores also have some other major differences from the initial dormant spores, in particular in the SD spores' low levels of GR subunits and the auxiliary GR-dependent germination protein GerD. We also have no definitive explanation for the low germination protein levels in the dodecylamine-SD spores, and how these may contribute to the enrichment of these spores in this SD spore population. In addition, why these low GR subunit levels in the dodecylamine-SD spores were not reflected in low rates of germination with GR-dependent germinants is also not clear. Possible explanations are that i) levels of some unknown germination protein do not change in dodecylamine-SD spores and that levels of this protein are normally rate limiting for GR-dependent germination; and ii) the ratio of GerD to GRs is what is crucial for normal rates of GR-dependent spore germination, and this ratio remains relatively constant in dodecylamine-SD spores, while in spores SD for germination with nutrient germinants, the GerD/GR ratio increases significantly (Ghosh et al. 2012). Taking the latter explanation even further, in CaDPA-SD spores, the ratio of GerD/GRs goes down significantly, and this may explain why rates of nutrient germination of these spores do not increase more than was observed.

While the factors leading to some of the properties of CaDPA- and dodecylamine-SD spores are clearly not known, it seems possible that the properties themselves may suggest means to improve spore eradication by germination. A major problem in using spore germination with nutrients to render spores relatively easy to kill is the heterogeneity in spore populations such that a small fraction of spores are SD for germination with nutrients (Setlow et al. 2012). One way to improve upon this strategy would be to use a mixture of germinants, both GR-dependent ones as well as those that trigger germination by activating CwlJ, as does CaDPA, or directly triggering DPA release by activating the SpoVA channel as dodecylamine likely does. However, this would likely require identification of new molecules that trigger germination by these non-GR-dependent pathways. If such a molecule that acts like CaDPA could be identified, then an alternative strategy would be to use this molecule to trigger spore germination, and while SD spores would likely remain, these SD spores would most likely have defective coats, and would thus be relatively easy to kill by mild decontamination regimens. These possibilities certainly seem worth pursuing.

Acknowledgements

This work was supported by a Department of Defense Multi-disciplinary University Research Initiative through the U.S. Army Research Laboratory and the U.S. Army Research Office under contract number W911NF-09-1-0286. We are grateful to L. Kaminsky and A. Ramirez-Peralta for assistance with some experiments.

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