Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (T_lag and T_release times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT_release times (T_release?T_lag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔT_release times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in T_lag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T_lag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.     

Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, T_lag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T₁; (ii) the period of slow DPA leakage (ΔT_leakage = T_lag − T₁) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T₁ times but increased values of ΔT_leakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T₁ times of less than 10 min, suggesting that T₁ is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T₁ and T_lag times but not the amount of DPA released in ΔT_leakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔT_leakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.     

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores'' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores'' germination kinetics were heterogeneous; (ii) individual spores'' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T_lag) prior to a period of the rapid release (ΔT_release) of the spores'' dipicolinic acid in a 1:1 chelate with Ca²⁺ (CaDPA); (iii) spore germination at 50 MPa had longer average T_lag values than that at ∼150 MPa, but the ΔT_release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔT_release values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T_lag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores'' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.     

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore''s DPA once rapid DPA release had begun (ΔT_release) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ΔT_release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination ≥15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T_lag, between mixing spores with nutrient germinants and the beginning of ΔT_release. (iv) A number of factors decrease spores'' T_lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25, 29, 30). The germinants that most likely trigger spore germination in the environment are low-molecular-weight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore''s inner membrane that recognize their cognate germinants in a stereospecific manner (17, 24, 25, 29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6, 29, 30). Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B. subtilis spore germination by binding of l-alanine or l-valine to the GerA GR. However, many GRs cooperate such that binding of germinants by ≥2 different GRs is needed to trigger germination (2, 29): for example, the triggering of B. subtilis spore germination by the binding of components of a mixture of l-asparagine, d-glucose, d-fructose, and K⁺ ions (AGFK) to the GerB and GerK GRs. The binding of nutrient germinants to GRs triggers subsequent events in germination, although how this is accomplished is not known.The first readily measured biochemical event after addition of nutrient germinants to Bacillus spores is the rapid release of the spore''s large depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus its chelated divalent cations, predominantly Ca²⁺ (Ca-DPA), from the spore core (25, 29). Ca-DPA release then results in the activation of two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, which hydrolyze the spore''s peptidoglycan cortex layer (16, 22, 27, 29). CwlJ is activated by Ca-DPA as it is released from the spore while SleB is activated only after most DPA is released (17, 20, 22, 26, 27). Cortex hydrolysis ultimately allows the spore core to expand and take up more water, raising the core water content from the 35 to 45% of wet weight in the dormant spore to the 80% of wet weight characteristic of growing cells. Full hydration of the spore core then allows enzyme action, metabolism, and macromolecular synthesis to resume in the now fully germinated spore.Germination of spores in populations is very heterogeneous, with some spores germinating rapidly and some extremely slowly (4, 5, 9, 11, 13-15, 19, 26, 31, 32). Where it has been studied, the reason for this heterogeneity has been suggested to be due to a variable lag period (T_lag) between the time of mixing spores with a germinant and the time at which rapid DPA release begins, since once rapid DPA release begins, the time required for release of almost all DPA as well as for subsequent cortex hydrolysis is generally rather short compared to T_lag values in individual spores (5, 11, 13-15, 19, 26, 31, 32). The times required for DPA release and cortex hydrolysis are also similar in wild-type spores with both very short and long T_lag values (5, 15, 19, 27). The reasons for the variability in T_lag times between individual spores in populations are not known, although there are reports that both activation of spores for germination by a sublethal heat treatment (heat activation) as well as increasing concentrations of nutrient germinants can shorten T_lag values (12, 14, 15, 18, 32). However, there has been no detailed study of the causes of the variability in T_lag values between very large numbers of individual spores in populations.In order to study the heterogeneity in spore germination thoroughly, methods are needed to follow the germination of hundreds of individual spores over several hours. Initial studies of the germination of individual spores examined a single spore in a phase-contrast microscope and followed the germination of this spore by changes in the core''s refractive index due to DPA release and core swelling (14, 15, 32, 34). However, this method is labor-intensive for gathering data with hundreds of individual spores. More recently, confocal microscopy and then surface adsorption and optical tweezers have been used to capture single spores, and germination events have been followed by methods such as Raman spectroscopy to directly measure DPA release, as well as phase-contrast microscopy and elastic light scattering (3, 5, 9, 10, 19, 26). While the latter recent advances have allowed accumulation of much information about germination, collection of this type of data for large numbers of individual spores is still labor-intensive, although use of dual optical traps (35) and perhaps multiple traps in the future may alleviate this problem. However, phase-contrast microscopy plus appropriate computer software has recently allowed the monitoring of many hundreds of individual spores for several hours, with automated assessment of various changes in the cells during the period of observation (19). In the present work, we have used both phase-contrast and differential interference contrast (DIC) microscopy to monitor the germination of many hundreds of individual spores of three Bacillus species adhered on either an agarose pad or a glass coverslip for 1 to 2 h. This work, as well as examination of times needed for release of most DPA once rapid DPA release has begun during germination of individual spores under a variety of conditions, has allowed detailed examination of the effects of heat activation, nutrient germinant concentration, GR numbers per spore, and individual CLEs on spore germination heterogeneity and on values of T_lag for individual spores.     

Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores'' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores'' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca²⁺ divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP⁺ spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 μM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores'' inner membrane.     

High-Precision Fitting Measurements of the Kinetics of Size Changes during Germination of Individual Bacillus Spores

High-precision measurements of size changes of individual bacterial spores based on ellipse fitting to bright-field images recorded with a digital camera were employed to monitor the germination of Bacillus spores with a precision of ∼5 nm. To characterize the germination of individual spores, we recorded bright-field and phase-contrast images and found that the timing of changes in their normalized intensities coincided, so the bright-field images can be used to characterize spore size and refractility changes during germination. The major conclusions from this work were as follows. (i) The sizes of germinating B. cereus spores were nearly unchanged until T_release, the time of the completion of CaDPA (a 1:1 chelate of Ca²⁺ and dipicolinic acid [DPA]) release after addition of nutrient germinants. (ii) The minor axis of germinating B. cereus spores rapidly increased by ∼50 nm in a few seconds right after T_release, while the major axis was slightly decreased or unchanged. Both the minor and major axes remained unchanged for a further 30 to 45 s and then increased by 100 to 200 nm by T_lys, the time of completion of cortex lysis. (iii) Individual spores in a population showed significant heterogeneity in the timing of germination events, such as T_release and T_lys, but also variation in size changes during germination. (iv) Bacillus subtilis wild-type spores, B. subtilis spores lacking the cortex-lytic enzyme CwlJ, and wild-type Bacillus megaterium spores showed similar kinetics of size changes during nutrient germination. The size increases in germinating spores probably result from uptake of water and cortex lysis after completion of CaDPA release.     

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T_lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT_release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T_lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T_lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T_lag and ΔT_release values in dodecylamine germination that does not utilize GRs. Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

Germination of dormant Bacillus subtilis spores with specific nutrient germinants is dependent on a number of inner membrane (IM) proteins, including (i) the GerA, GerB, and GerK germinant receptors (GRs) that respond to nutrient germinants; (ii) the GerD protein, essential for optimal GR function; and (iii) SpoVA proteins, essential for the release of the spore-specific molecule dipicolinic acid (DPA) during spore germination. Levels of GR A and C subunit proteins, GerD, and SpoVAD in wild-type spores were determined by Western blot analysis of spore fractions or total disrupted spores by comparison with known amounts of purified proteins. Surprisingly, after disruption of decoated B. subtilis spores with lysozyme and fractionation, ∼90% of IM fatty acids and GR subunits remained with the spores'' insoluble integument fraction, indicating that yields of purified IM are low. The total lysate from disrupted wild-type spores contained ∼2,500 total GRs/spore: GerAA and GerAC subunits each at ∼1,100 molecules/spore and GerBC and GerKA subunits each at ∼700 molecules/spore. Levels of the GerBA subunit determined previously were also predicted to be ∼700 molecules/spore. These results indicate that the A/C subunit stoichiometry in GRs is most likely 1:1, with GerA being the most abundant GR. GerD and SpoVAD levels were ∼3,500 and ∼6,500 molecules/spore, respectively. These values will be helpful in formulating mathematic models of spore germination kinetics as well as setting lower limits on the size of the GR-GerD complex in the spores'' IM, termed the germinosome.     

How moist heat kills spores of Bacillus subtilis

Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but ≥98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.     

Role of the gerP Operon in Germination and Outgrowth of Bacillus anthracis Spores

Germination of Bacillus anthracis spores occurs when nutrients such as amino acids or purine nucleosides stimulate specific germinant receptors located in the spore inner membrane. The gerP_ABCDEF operon has been suggested to play a role in facilitating the interaction between germinants and their receptors in spores of Bacillus subtilis and Bacillus cereus. B. anthracis mutants containing deletions in each of the six genes belonging to the orthologue of the gerP_ABCDEF operon, or deletion of the entire operon, were tested for their ability to germinate. Deletion of the entire gerP operon resulted in a significant delay in germination in response to nutrient germinants. These spores eventually germinated to levels equivalent to wild-type, suggesting that an additional entry point for nutrient germinants may exist. Deletions of each individual gene resulted in a similar phenotype, with the exception of ΔgerPF, which showed no obvious defect. The removal of two additional gerPF-like orthologues was necessary to achieve the germination defect observed for the other mutants. Upon physical removal of the spore coat, the mutant lacking the full gerP operon no longer exhibited a germination defect, suggesting that the GerP proteins play a role in spore coat permeability. Additionally, each of the gerP mutants exhibited a severe defect in calcium-dipicolinic acid (Ca-DPA)–dependent germination, suggesting a role for the GerP proteins in this process. Collectively, these data implicate all GerP proteins in the early stages of spore germination.     

Studies of the Commitment Step in the Germination of Spores of Bacillus Species

Spores of Bacillus species are said to be committed when they continue through nutrient germination even when germinants are removed or their binding to spores'' nutrient germinant receptors (GRs) is both reversed and inhibited. Measurement of commitment and the subsequent release of dipicolinic acid (DPA) during nutrient germination of spores of Bacillus cereus and Bacillus subtilis showed that heat activation, increased nutrient germinant concentrations, and higher average levels of GRs/spore significantly decreased the times needed for commitment, as well as lag times between commitment and DPA release. These lag times were also decreased dramatically by the action of one of the spores'' two redundant cortex lytic enzymes (CLEs), CwlJ, but not by the other CLE, SleB, and CwlJ action did not affect the timing of commitment. The timing of commitment and the lag time between commitment and DPA release were also dependent on the specific GR activated to cause spore germination. For spore populations, the lag times between commitment and DPA release were increased significantly in spores that germinated late compared to those that germinated early, and individual spores that germinated late may have had lower appropriate GR levels/spore than spores that germinated early. These findings together provide new insight into the commitment step in spore germination and suggest several factors that may contribute to the large heterogeneity among the timings of various events in the germination of individual spores in spore populations.Spores of Bacillus species can remain dormant for long times and are extremely resistant to a variety of environmental stresses (26). However, under appropriate conditions, normally upon the binding of specific nutrients to spores'' nutrient germinant receptors (GRs), spores can come back to active growth through a process called germination followed by outgrowth (19, 20, 25, 26). Germination of Bacillus subtilis spores can be triggered by l-alanine or l-valine or a combination of l-asparagine, d-glucose, d-fructose, and K⁺ (AGFK). These nutrient germinants trigger germination by binding to and interacting with GRs that have been localized to the spore''s inner membrane (12, 20). l-Alanine and l-valine bind to the GerA GR, while the AGFK mixture triggers germination by interacting with both the GerB and GerK GRs (25). Normally, l-asparagine alone does not trigger B. subtilis spore germination. However, a mutant form of the GerB GR, termed GerB*, displays altered germinant specificity such that l-asparagine alone will trigger the germination of gerB* mutant spores (1, 18).A number of events occur in a defined sequence during spore germination. Initially, exposure of spores to nutrient germinants causes a reaction that commits spores to germinate, even if the germinant is removed or displaced from its cognate GR (7, 10, 21, 27, 28). This commitment step is followed by release of monovalent cations, as well as the spore core''s large pool of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) along with divalent cations, predominantly Ca²⁺, that are chelated with DPA (Ca-DPA). In Bacillus spores, the release of Ca-DPA triggers the hydrolysis of spores'' peptidoglycan cortex by either of two cortex lytic enzymes (CLEs), CwlJ and SleB (11, 16, 23). CwlJ is activated during germination by Ca-DPA as it is being released from individual spores, while SleB activation requires that most Ca-DPA be released (14, 16, 17). Cortex hydrolysis, in turn, allows the spore core to expand and fully hydrate, which leads to activation of enzymes and initiation of metabolism in the spore core (21, 25).As noted above, commitment is the first event that can be assessed during spore germination, although the precise mechanism of commitment is not known. Since much has been learned about proteins important in spore germination in the many years since commitment was last studied (25, 26), it seemed worth reexamining commitment, with the goal of determining those factors that influence this step in the germination process. Knowledge of factors important in determining kinetics of commitment could then lead to an understanding of what is involved in this reaction.Kinetic analysis of spore germination, as well as commitment, has mostly been based on the decrease in optical density at 600 nm (OD₆₀₀) of spore suspensions, which monitors a combination of events that occur well after commitment, including DPA release, cortex hydrolysis, and core swelling (25-27). In the current work, we have used a germination assay that measures DPA release, an early event in spore germination, and have automated this assay to allow routine measurement of commitment, as well as DPA release from large numbers of spore samples simultaneously. This assay has allowed comparison of the kinetics of DPA release and commitment during germination and study of the effects of heat activation, germinant concentration, GR levels, and CLEs on commitment.     

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.     

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore''s nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore''s inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa''s amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.     

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.     

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105°C, 120°C, and 131°C, respectively. The estimated Z values were 6.3°C, 6.1°C, and 9.7°C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (T_c), the temperature at which half the DPA content has been released within a fixed incubation time. We found T_c values for spores from Bacillus strains 168, A163, and IC4 of 108°C, 121°C, and 131°C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Superdormant Spores of Bacillus Species Germinate Normally with High Pressure,Peptidoglycan Fragments,and Bryostatin

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.Spores of Bacillus species are formed in sporulation, a process that is generally triggered by starvation for one or more nutrients (13, 19). These spores are metabolically dormant and extremely resistant to a large variety of environmental stresses, including heat, radiation, and toxic chemicals, and as a consequence of these properties, these spores can remain viable in their dormant state for many years (13, 18, 19). However, spores are constantly sensing their environment, and if nutrients return, the spores can rapidly return to growth through the process of spore germination (17). Spore germination is generally triggered by specific nutrients that bind to nutrient germinant receptors, with this binding alone somehow triggering germination. However, spore germination can also be triggered by many non-nutrient agents, including cationic surfactants such as dodecylamine, a 1:1 complex of Ca²⁺ with pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA], a major spore small molecule), very high pressures, specific peptidoglycan fragments, and bryostatin, an activator of Ser/Thr protein kinases (17, 19, 20). For nutrient germinants in particular, spore germination is also potentiated by a prior sublethal heat treatment termed heat activation (17).While normally the great majority of spores in populations germinate relatively rapidly in response to nutrient germinants, a small percentage of spores germinate extremely slowly. These spores that are refractory to nutrient germination have been termed superdormant spores and are a major concern for the food industry (8). Recently superdormant spores of three Bacillus species have been isolated by repeated germination of spore populations with specific nutrient germinants and isolation of remaining dormant spores (5, 6). These superdormant spores germinate extremely poorly with the nutrient germinants used in superdormant spore isolation, as well as with other nutrient germinants. All of the specific defects leading to spore superdormancy are not known, although an increased level of receptors for specific nutrient germinants decreases levels of superdormant spores obtained with the nutrients that are ligands for these receptors (5). Superdormant spores also have significantly higher temperature optima for heat activation of nutrient germination than the spore population as a whole (7).In contrast to the poor germination of superdormant spores with nutrient germinants, superdormant spores germinate normally with dodecylamine and Ca-DPA (5, 6). This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11, 15, 17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2, 3, 11, 15, 20, 22, 23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.The spores used in this work were from Bacillus subtilis PS533 (16), a derivative of strain 168 that also carries plasmid pUB110, providing resistance to kanamycin (10 μg/ml), and Bacillus cereus T (originally obtained from H. O. Halvorson). Spores of these strains were prepared and purified as described previously (6, 10, 12). Superdormant spores of B. subtilis were prepared by germination following heat activation at 75°C for 30 min by two germination treatments at 37°C with 10 mM l-valine for 2 h, followed by isolation of remaining dormant spores, all as described previously (5, 10, 12). These superdormant spores germinated extremely poorly with 10 mM valine at 37°C, giving ≤10% germination in 2 h at 37°C, while the initial spore population exhibited >95% germination under the same conditions (data not shown). Superdormant B. cereus spores were isolated similarly, although heat activation was at 65°C for 30 min and the germinant was 5 mM inosine as described previously (6). These superdormant B. cereus spores exhibited <5% germination with inosine in 2 h at 37°C compared to the >95% germination of the initial dormant spores under the same conditions (data not shown).     

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D_120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.