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.     

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.     

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.     

The Effects of Heat Activation on Bacillus Spore Germination,with Nutrients or under High Pressure,with or without Various Germination Proteins

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores'' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the l-asparagine–glucose–fructose–K⁺ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores'' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores'' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.     

The Clostridium perfringens Germinant Receptor Protein GerKC Is Located in the Spore Inner Membrane and Is Crucial for Spore Germination

The Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens causes a variety of diseases in both humans and animals, and spore germination is thought to be the first stage of C. perfringens infection. Previous studies have indicated that the germinant receptor (GR) proteins encoded by the bicistronic gerKA-gerKC operon as well as the proteins encoded by the gerKB and gerAA genes are required for normal germination of C. perfringens spores. We now report the individual role of these GR proteins by analyzing the germination of strains carrying mutations in gerKA, gerKC, or both gerKB and gerAA. Western blot analysis was also used to determine the location and numbers of GerKC proteins in spores. Conclusions from this work include the following: (i) gerKC mutant spores germinate extremely poorly with KCl, l-asparagine, a mixture of asparagine and KCl, or NaP_i; (ii) gerKC spores germinate significantly more slowly than wild-type and other GR mutant spores with a 1:1 chelate of Ca²⁺ and dipicolinic acid and very slightly more slowly with dodecylamine; (iii) the germination defects in gerKC spores are largely restored by expressing the wild-type gerKA-gerKC operon in trans; (iv) GerKC is required for the spores'' viability, almost certainly because of the gerKC spores'' poor germination; and (v) GerKC is located in the spores'' inner membrane, with ∼250 molecules/spore. Collectively, these results indicate that GerKC is the main GR protein required for nutrient and nonnutrient germination of spores of C. perfringens food-poisoning isolates.     

Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments

The germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affect Bacillus subtilis spore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores'' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca²⁺-dipicolinic acid, and water fluxes.     

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.     

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.     

Activity and Regulation of Various Forms of CwlJ,SleB, and YpeB Proteins in Degrading Cortex Peptidoglycan of Spores of Bacillus Species In Vitro and during Spore Germination

Germination of Bacillus spores requires degradation of a modified layer of peptidoglycan (PG) termed the spore cortex by two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, plus SleB''s partner protein, YpeB. In this study, in vitro and in vivo analyses have been used to clarify the roles of individual SleB and YpeB domains in PG degradation. Purified mature Bacillus cereus SleB without its signal sequence (SleB^M) and the SleB C-terminal catalytic domain (SleB^C) efficiently triggered germination of decoated Bacillus megaterium and Bacillus subtilis spores lacking endogenous CLEs; previously, SleB''s N-terminal domain (SleB^N) was shown to bind PG but have no enzymatic activity. YpeB lacking its putative membrane anchoring sequence (YpeB^M) or its N- and C-terminal domains (YpeB^N and YpeB^C) alone did not exhibit degradative activity, but YpeB^N inhibited SleB^M and SleB^C activity in vitro. The severe germination defect of B. subtilis cwlJ sleB or cwlJ sleB ypeB spores was complemented by ectopic expression of full-length sleB [sleB(FL)] and ypeB [ypeB(FL)], but normal levels of SleB^FL in spores required normal spore levels of YpeB^FL and vice versa. sleB(FL) or ypeB(FL) alone, sleB(FL) plus ypeB(C) or ypeB(N), and sleB(C) or sleB(N) plus ypeB(FL) did not complement the cortex degradation defect in cwlJ sleB ypeB spores. In addition, ectopic expression of sleB(FL) or cwlJ(FL) with a Glu-to-Gln mutation in a predicted active-site residue failed to restore the germination of cwlJ sleB spores, supporting the role of this invariant glutamate as the key catalytic residue in SleB and CwlJ.     

Role of carbon dioxide in germination of spores of Streptomyces viridochromogenes

CO₂ in required continuously during germination of Streptomyces viridochromogenes spores. Spores incubated in a defined germination medium in the absence of CO₂ remain phase bright and do not release spore carbon. In the presence of CO₂, the spores initiate germination accompanied by loss of refractility and spore carbon. The CO₂ requirement is replaced by oxaloacetate or a mixture of tricarboxylic acid cycle (TCA) intermediates. Labeled CO₂ is taken up by germinating spores, and is incorporated into protein and RNA. TCA cycle intermediates and related amino acids contain most of the acid-soluble label following short term exposures of germinating spores to ¹⁴CO₂. TCA cycle inhibitors repress germination and ¹⁴CO₂ uptake whereas folic acid antagonists do not. The results indicate that CO₂ is incorporated into oxaloacetate which is converted to biosynthetic intermediates required for germination. Operation of the TCA cycle appears to be essential for spore germination. The conclusion is reached that CO₂ is required during germination in order to maintain the cycle by an anaplerotic reaction.Abbreviations SN sucrose-nitrate medium - TX buffer Trisbuffer pH 7.3 containing-Triton X-100 - DGM defined germination medium - TX salts TX buffer plus Mg and Ca ions - TA trichloroacctic acid - TCA tricarboxylic acid     

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores'' return to life within minutes in the process of germination. This process requires a number of spore-specific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores'' IM through which the spore core''s huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. (i) How do nutrient germinants penetrate through spores'' outer layers to access GRs in the IM? (ii) What happens during the highly variable and often long lag period between the exposure of spores to nutrient germinants and the commitment of spores to germinate? (iii) What do GRs and GerD do, and how do these proteins interact? (iv) What is the structure of the SpoVA channel in spores'' IM, and how is this channel gated? (v) What is the precise state of the spore IM, which has a number of novel properties even though its lipid composition is very similar to that of growing cells? (vi) How is CLE activity regulated such that these enzymes act only when germination has been initiated? (vii) And finally, how does the germination of spores of clostridia compare with that of spores of bacilli?     

Isolation and Characterization of Superdormant Spores of Bacillus Species

Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores'' defect was not genetic. The superdormant spores did, however, germinate with Ca²⁺-dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores.Spores of various Bacillus species are formed in sporulation and are metabolically dormant and very resistant to environmental stress factors (21, 37). While such spores can remain in this dormant, resistant state for long periods, they can return to life rapidly through the process of germination, during which the spore''s dormancy and extreme resistance are lost (36). Spore germination has long been of intrinsic interest, and continues to attract applied interest, because (i) spores of a number of Bacillus species are major agents of food spoilage and food-borne disease and (ii) spores of Bacillus anthracis are a major bioterrorism agent. Since spores are much easier to kill after they have germinated, it would be advantageous to trigger germination of spores in foods or the environment and then readily inactivate the much less resistant germinated spores. However, this simple strategy has been largely nullified because germination of spore populations is heterogeneous, with some spores, often called superdormant spores, germinating extremely slowly and potentially coming back to life long after treatments are applied to inactivate germinated spores (8, 9, 16). The concern over superdormant spores in populations also affects decisions such as how long individuals exposed to B. anthracis spores should continue to take antibiotics, since spores could remain dormant in an individual for long periods and then germinate and cause disease (3, 11).In many species, spore germination can be increased by a prior activation step, generally a sublethal heat treatment, although the changes taking place during heat activation are not known (16). Spore germination in Bacillus species is normally triggered by nutrients such as glucose, amino acids, or purine ribosides (27, 36). These agents bind to germinant receptors located in the spore''s inner membrane that are specific for particular nutrients. In Bacillus subtilis, the GerA receptor responds to l-alanine or l-valine, while the GerB and GerK receptors act cooperatively to respond to a mixture of l-asparagine (or l-alanine), d-glucose, d-fructose and K⁺ ions (AGFK [or Ala-GFK]) (1, 27, 36). There are even more functional germinant receptors in Bacillus megaterium spores, and these respond to d-glucose, l-proline, l-leucine, l-valine, or even salts, such as KBr (6). Glucose appears to trigger germination of B. megaterium spores through either of two germinant receptors, GerU or GerVB, while l-proline triggers germination through only the GerVB receptor, and KBr germination is greatly decreased by the loss of either GerU or GerVB (6). Nutrient binding to the germinant receptors triggers the release of small molecules from the spore core, most notably the huge depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) present in spores predominantly as a 1:1 diluted chelate with Ca²⁺ (Ca-DPA) (35, 36). Ca-DPA release then triggers the activation of one of two redundant cortex lytic enzymes (CLEs) that degrade the spore''s peptidoglycan cortex, and cortex degradation completes spore germination and allows progression into outgrowth and then vegetative growth (27, 33, 36).Spore germination can also be triggered by nonnutrient agents, including Ca-DPA and cationic surfactants (27, 33, 36). With B. subtilis spores, Ca-DPA triggers germination by activating one particular CLE, termed CwlJ, and bypasses the spore''s germinant receptors. Germination by the cationic surfactant dodecylamine also bypasses the germinant receptors, and this agent appears to release small molecules including Ca-DPA from the spore core either by opening a normal channel in the spore''s inner membrane for Ca-DPA and other small molecules or by creating such a channel (31, 38, 39).Almost all work on the specifics of the germination of spores of Bacillus species has focused on the majority of spores in populations, and little detailed attention has been paid to that minority of spores that either fail to germinate or germinate extremely slowly. However, it is these latter spores that are most important in unraveling the cause of superdormancy and perhaps suggesting a means to germinate and thus easily inactivate such superdormant spores. Consequently, we have undertaken the task of isolating superdormant spores from spore populations, using buoyant density centrifugation to separate dormant spores from germinated spores. The properties of these purified superdormant spores were then studied, and this information has suggested some reason(s) for 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.     

Dosage effect study of the microsporidian Octosporea muscaedomesticae in the adult black blowfly Phormia regina,with reference to the reproductive cycle of the pathogen

The quantitative pathogenicity of the microsporidian Octosporea muscaedomesticae in adult Phormia regina was studied. Dosage levels ranging from 10² to 10⁶ spores per fly were administered to five and six groups of newly emerged, starved adult flies in two trials. Rates of mortality and infection were determined. A direct relationship between number of spores ingested and subsequent infection rate was found in a 4-day trial while no such relationship was found in an 18-day trial, using the same source of inoculum and host flies from the same colony. The lack of a direct relationship between spore dose and rate of infection in the 18-day trial is explained on the basis of the short spore-to-spore development time of the parasite. New generations of spores formed within the host tissues obscure the results in relation to the spore dose initially administered. An appreciable number of spores in the inoculum is needed to initiate frank infection. The ID₅₀ (median infective dose) was 4.4 × 10⁴ spores per fly after 4 days.     

GerO,a Putative Na+/H+-K+ Antiporter,Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations (K⁺ and/or Na⁺) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, l-asparagine, and a 1:1 chelate of Ca²⁺ and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli Na⁺/H⁺ antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein''s ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in K⁺ uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.Clostridium perfringens is a gram-positive, spore-forming anaerobic pathogen that causes diseases in animals and humans (13). C. perfringens spores are metabolically dormant, are resistant to many environmental insults, and can survive for long periods. Once conditions are favorable, these spores can germinate, outgrow, return to vegetative growth, and then release toxins and cause disease (14).Bacterial spores initiate germination when they sense a variety of compounds termed germinants, which include nutrients, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (Ca-DPA) and cationic surfactants (21, 31). In spores of Bacillus species, nutrient germinants are sensed by specific germinant receptors located in the spore''s inner membrane, each generally encoded by tricistronic operons of the gerA family. In Bacillus megaterium spores, the interaction of nutrient germinants with their cognate receptors leads to an energy independent efflux of ∼80% of the spore''s depot of Na⁺ and K⁺, as well as much H⁺ efflux causing a rise of the spore core''s pH, all within the first 5 min of germination; this efflux is followed by reuptake of K⁺ by an energy-dependent system (33). The spores'' large depot of Ca-DPA is also released shortly after monovalent cation release. The mechanism of release of monovalent cations during spore germination is not known, but monovalent cation antiporters could be involved somehow in this event. Indeed, a member of the CPA-2 monovalent cation-proton antiporter family of membrane transport proteins (27), GrmA, is essential for germination of B. megaterium ATCC 12872 spores (34), since grmA inactivation makes spores unable to release their DPA and complete germination with a variety of germinants. Similarly, in Bacillus cereus ATCC 10876, a GrmA-type homologue, GerN, is essential for spore germination with inosine but not l-alanine (35), and studies with everted vesicles have shown that GerN possesses electrogenic Na⁺/H⁺-K⁺ antiporter activity (32). The GerN homolog, GerT, also plays a minor role in B. cereus spore germination with inosine, as well as a major role in spore outgrowth under some conditions (29). However, in contrast to these latter results, GrmA-like antiporters appear to have no role in the germination of spores of B. megaterium QM B1551 and Bacillus subtilis (3).In C. perfringens, there is no intact tricistronic gerA-like operon, and the only locus that encodes the three proteins (A, B, and C) of a likely germinant receptor is the gerK locus, comprising a bicistronic gerKA-gerKC operon, and a gerKB gene located just upstream of gerKA-gerKC but in the opposite orientation (16). However, GerKA and GerKC appear able to function in spore germination in the absence of GerKB (23). The lack of a classical GerA-type germinant receptor and the fact that C. perfringens spores germinate with K⁺ ions alone (21), raises the possibility that GrmA-like antiporters might also play some role in C. perfringens spore germination. The genome of C. perfringens strain SM101 has two genes encoding putative GrmA-like antiporters (see Fig. S1 in the supplemental material) that we have termed gerO (CPR0227) and gerQ (CPR1038). Orthologs of the gerO and gerQ genes are also present in the genomes of nine additional C. perfringens strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). In present study we have constructed gerO, gerQ, and gerO gerQ strains of C. perfringens and have examined the roles of GerO and GerQ in spore germination. The results show that GerO is essential for normal germination of C. perfringens spores, whereas GerQ plays at most only a minor role.     

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).     

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.     

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.