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.     

Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.Spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of stress factors (31, 32). While spores can remain dormant for long periods, if given the proper stimulus, they can rapidly “return to life” in the process of spore germination followed by outgrowth (30). Since spores are generally present in significant amounts on many foodstuffs and growing cells of a number of Bacillus species are significant agents of food spoilage and food-borne disease (32), there is continued applied interest in spore resistance and germination. While dormant spores can be killed by a treatment such as wet heat, this requires high temperatures that are costly and detrimental to food quality. Consequently, there has long been interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. However, this strategy has been difficult to apply because of the significant heterogeneity in germination rates between individual spores in populations. One reflection of this heterogeneity is the extremely variable lag times following addition of germinants but prior to initiation of germination events; while these lag times can vary from 10 to 30 min for most spores in populations, some spores have lag times of many hours or even many days (2, 12, 13, 15, 25). The spores that are extremely slow to germinate have been termed superdormant spores, and populations of superdormant spores have recently been isolated from three Bacillus species, and their germination properties characterized (9, 10). These superdormant spores germinate extremely poorly with the original germinants used to remove dormant spores from spore populations, thus allowing superdormant spore isolation, and also poorly with a number of other germinants, in particular, germinants that target nutrient germinant receptors different than those activated to isolate the superdormant spores. However, the superdormant spores germinate reasonably well with mixtures of nutrient germinants that target multiple germinant receptors. All reasons for spore superdormancy are not known, but one contributing factor is the number of nutrient germinant receptors in the spore''s inner membrane that trigger spore germination by binding to nutrient germinants (9). The levels of these receptors are most likely in the tens of molecules per spore (24), and thus stochastic variation in receptor numbers might result in some spores with such low receptor numbers that these spores germinate very poorly (23). Indeed, 20- to 200-fold elevated levels of at least one nutrient germinant receptor greatly decreases yields of superdormant spores of Bacillus subtilis (9).Spores of Bacillus species generally exhibit a requirement for an activation step in order to exhibit maximum germination (17). Usually this activation is a sublethal heat treatment that for a spore population exhibits an optimum of 60 to 100°C depending on the species. Spores are also extremely resistant to wet heat, generally requiring temperatures of 80 to 110°C to achieve rapid spore killing, with the major factor influencing the wet-heat resistance of spores of mesophilic strains being the spore core''s water content, which can be as low as 30% of wet weight as water in a fully hydrated spore (8, 19, 27, 28, 31). Invariably, increases in core water content are associated with a decrease in spore wet-heat resistance (8, 19, 22, 25). While spore populations most often exhibit log-linear kinetics of wet-heat killing, the observation of tailing in such killing curves at high levels of killing is not uncommon, suggesting there is significant heterogeneity in the wet-heat resistances of individual spores in populations (27, 28). While there has been no comparable work suggesting that there is also heterogeneity in the temperature optima for heat activation of individual spores in populations, this certainly seems possible and indeed was suggested as one cause of spore superdormancy, as yields of superdormant spores from spore populations that are not heat activated are much higher (9, 10). Consequently, the current work was initiated to test the hypothesis that superdormant spores require heat activation temperatures that are higher than those of the original dormant spores. Once this was found to be the case, the wet-heat resistance and core water content of the superdormant and original dormant spores were compared, and the environment of the spore core''s major small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) was assessed by Raman spectroscopy of individual spores.     

Levels of Germination Proteins in Bacillus subtilis Dormant,Superdormant, and Germinating Spores

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Multigeneration Cross Contamination of Mail with Bacillus Species Spores by Tumbling

In 2001, envelopes loaded with Bacillus anthracis spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a “payload” envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 10³ B. anthracis spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies.In September and October of 2001, letters containing Bacillus anthracis spores were distributed through the U.S. Postal Service (USPS), resulting in contamination of the mail processing and distribution center in Hamilton, NJ, as well as affiliated processing centers in Washington, DC, in New York City, NY, and in Wallingford, CT, as well as postal facilities along the path transited by letters mailed to a targeted media company in Florida. Subsequently, 22 individuals, including postal workers, persons who received or handled the contaminated letters, and persons exposed to environments contaminated by the letters, developed cases of anthrax, including both the inhalation and cutaneous forms of the disease (5, 18-20). Five of these cases of anthrax resulted in death (4, 7). There have been investigations into the relationships of infection and exposure in areas where known exposures occurred (1, 6, 8). However, for two of the individuals who developed inhalational anthrax, an elderly woman in Connecticut and a nurse in New York City, no B. anthracis spores were detected (based on environmental sampling) on their mail or in their homes (2, 17, 19, 20). A third individual, a bookkeeper from New Jersey, survived a cutaneous anthrax infection, and only a single positive environmental sample in her workplace was identified (19).For the three specific cases mentioned above, the authors of the corresponding studies hypothesized that infection may have resulted from exposure to mail cross contaminated by mail that went through the same sorting equipment around the time that the letters to Senators Leahy and Daschle were processed. Without evidence of B. anthracis spores in their homes and other areas they were known to have frequented and the lack of additional cases in these geographic areas, there is no way to confirm the route of their exposure. We hypothesize that these people may have been exposed by inhaling spores released from envelopes that they tore open and then discarded. The delay between exposure and disease would have been sufficient to permit the discarded items to enter into the solid waste or recycling stream, and any residual spores may have been removed by normal housekeeping activities. Alternatively, the true source of exposure may have been undetectable due to a low concentration of spores.Those cases of anthrax raise the question of what, if any, hazards may have been encountered in handling mail with secondary and tertiary contamination. These cases raise particular questions concerning the ability of disease-causing organisms to spread through cross contamination of second- and even third-generation fomites in sufficient numbers to cause infection and possible death.Following the attacks, numerous studies were conducted in the contaminated postal buildings to assess the degree of contamination and to better understand sampling methodologies. Subsequent laboratory studies have been performed to improve B. anthracis sample collection and detection (11, 16, 22, 24, 30). Programs have monitored aerosols within federal buildings, hospitals, and mail facilities (10, 15, 25, 27). Additionally, studies of mail sorting machinery and the potential of this machinery to cross contaminate mail have been done (3, 10). However, to date, no laboratory studies that examined the potential for cross contamination of mail through contact or mixing with contaminated letters have been published.Reaerosolization in general is a poorly studied phenomenon. Characterization of reaerosolization under a variety of circumstances was undertaken following the B. anthracis incidents in 2001 (21, 29). The concept of fomite-to-fomite transference of powdered pathogen residues has been even less well studied.The settling of a primary aerosol comprised of charged particles may be due at least in part to an increase in the mass of these charged particles that occurs when they interact with oppositely charged particles. Once deposited on a surface, several factors may act against reaerosolization. Charged particles that have interacted with oppositely charged particles and have effectively increased in mass may be substantially more difficult to entrain in an aerosol than the initial particles. For charged particles that have not interacted with other particles, there may be a direct electrostatic interaction between the charged particle and the surface on which it has landed which would tend to hold these particles onto the surface. Both of these effects should reduce the potential for reaerosolization.Particulate preparations have a variety of properties, such as hydrophobicity, zeta potential, particle shape, and other characteristics that may also affect the potential for reaerosolization. It would be interesting to characterize a large number of powders, to create a database of the characteristics and their potential for aerosol formation and reaerosolization of these powders, and to use this database of information for comparison of unknown powders. Knowing this information may assist in the public health and risk management decision making processes. Unfortunately, there is no comprehensive database for these characteristics, nor is there any well-accepted unifying theory for deriving the likelihood of reaerosolization from the characteristics of powders that are commonly measured. In addition, there may be unknown variables that have an impact on aerosolization or reaerosolization that become known over time with improvements in understanding the theory of aerosolization and technology for measurement of these variables. A further confounding factor would be the inability to collect this information from the actual material used in any incident. In the case of the 2001 attacks (and likely in future incidents), there was (and will likely be) little material available for such study. The material used in the attacks is inherently hazardous and must be handled in highly controlled settings. The material is therefore difficult and expensive to work with (23). Material used in an attack is also generally sequestered as evidentiary material, and information concerning preparation of a biological weapon used in an attack may be considered too sensitive for public release. This sensitivity may include unwillingness to provide access to information on the efficacy of a specific preparation method to malevolent individuals and the requirement to preserve information for use in successful identification and prosecution of the perpetrator of such an attack. However, it may be possible to collect fomites contaminated with trace amounts of the agent in the course of public health investigations. The current study details a method for dealing with these contaminated fomites to yield information useful for public health protection.A confounding factor in these cases may be the necessity to treat as much of the available bulk material as can be collected as evidence. As evidence, even small amounts of this material may not be available for scientific testing. There may also be restrictions on the handling and treatment of fomites contaminated with residual traces of biological threat agents. For instance, the owners of the fomites may value them highly and may not wish to see them destroyed in the hope that the object may be somehow decontaminated and returned or the owner may wish to prevent public disclosure of the nature or contents of a contaminated object, such as a letter. It is therefore incumbent upon researchers to develop methods that are as minimally invasive and destructive as possible to investigate the potential for fomite-to-fomite transmission.We constructed a device designed to expose uncontaminated fomites to envelopes bearing a powdered preparation of spores or to fomites that had been exposed to other fomites contaminated by the initial powder-bearing envelope. Specifically, fomites used in this study were envelopes containing a piece of paper. This device was designed to conduct the exposure in a consistent, reproducible manner and to allow investigation of the interaction and cross contamination that might be encountered between a “payload” letter (a letter that had been loaded internally with a powdered spore preparation) and other pieces of mail. Uncontaminated envelopes were tumbled with a single envelope containing a payload of milled Bacillus atrophaeus subsp. globigii spores. After tumbling three successive generations of envelopes, CFU counts from the outsides of the envelopes were taken. These estimates of spore loads on the outside of these envelopes may be compared to published human 50% lethal dose (LD₅₀) estimates for aerosolized B. anthracis spores (12, 13). An additional series of envelopes was exposed to envelopes that had been contaminated during this first round of exposures, and those envelopes were found to be externally contaminated as well. We also studied opening an envelope that had been exposed to a payload envelope with either a finger or a letter opener to determine if these activities caused an aerosolization or reaerosolization of a sufficient number of spores to pose a risk of disease through inhalation.It is difficult to balance the concerns of making information public during a public health response and providing sufficient information for information risk management decision making while at the same time preserving the evidence for use by law enforcement agencies for eventual prosecution of individuals accused of committing crimes. We identified a nondestructive procedure by which contaminated mail can be analyzed and biological material collected while still preserving evidence for law enforcement agencies, allowing the payload envelope to be used as evidence while still permitting an assessment of its biological contaminant burden.     

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.     

Factors Influencing Germination of Bacillus subtilis Spores via Activation of Nutrient Receptors by High Pressure

Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl₂.     

Manganese Oxidation by Spores and Spore Coats of a Marine Bacillus Species

Bacillus sp. strain SG-1 is a marine bacterial species isolated from a near-shore manganese sediment sample. Its mature dormant spores promote the oxidation of Mn²⁺ to MnO₂. By quantifying the amounts of immobilized and oxidized manganese, it was established that bound manganese was almost instantaneously oxidized. When the final oxidation of manganese by the spores was partly inhibited by NaN₃ or anaerobiosis, an equivalent decrease in manganese immobilization was observed. After formation of a certain amount of MnO₂ by the spores, the oxidation rate decreased. A maximal encrustment was observed after which no further oxidation occurred. The oxidizing activity could be recovered by reduction of the MnO₂ with hydroxylamine. Once the spores were encrusted, they could bind significant amounts of manganese, even when no oxidation occurred. Purified spore coat preparations oxidized manganese at the same rate as intact spores. During the oxidation of manganese in spore coat preparations, molecular oxygen was consumed and protons were liberated. The data indicate that a spore coat component promoted the oxidation of Mn²⁺ in a biologically catalyzed process, after adsorption of the ion to incipiently formed MnO₂. Eventually, when large amounts of MnO₂ were allowed to accumulate, the active sites were masked and further oxidation was prevented.     

Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure

The effect of hydrostatic pressures as high as 1,700 atm at 25 C on the heat and radiation resistance of Bacillus pumilus spores was studied. Phosphate-buffered spores were more sensitive to compression than spores suspended in distilled water. Measurements of the turbidity of suspensions, the viability, refractility, stainability, dry weight, and respiratory activity of spores, and calcium and dipicolinic acid release were made for different pressures and times. Initiation of germination occurred at pressures exceeding 500 atm and was the prerequisite for inactivation by compression. The rate of initiation increased with increasing pressure at constant temperature. This result is interpreted as a net decrease in the volume of the system during initiation as a result of increased solvation of the spore components.     

Comparison of Pressure Resistances of Spores of Six Bacillus Strains with Their Heat Resistances

The pressure resistances of the spores of six Bacillus strains were examined at 5 to 10(deg)C and were compared with their heat resistances. The pressure treatments (at 981 MPa for 40 min and at 588 MPa for 120 min) did not inactivate the spores of B. stearothermophilus IAM12043, B. subtilis IAM12118, and B. licheniformis IAM13417. However, these spores had large differences in heat resistance. The spores of B. megaterium IAM1166 were 9.3 times more pressure resistant but 246 times less heat resistant than those of B. stearothermophilus IAM11001. The spores of B. coagulans IAM1194 were activated by the pressure treatments. There was no correlation between these pressure and heat resistances.     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

Comparison of UV Inactivation of Spores of Three Encephalitozoon Species with That of Spores of Two DNA Repair-Deficient Bacillus subtilis Biodosimetry Strains

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m², respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.     

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.     

Kinetics of Initiation of Germination of Bacillus pumilus Spores by Hydrostatic Pressure

The kinetics of initiation of germination and inactivation by hydrostatic pressure of phosphate-buffered Bacillus pumilus spores is shown to be a consecutive first-order process at 25 C. The effect of increasing pressure at constant temperature was studied, and rate constants were derived by using the criteria of heat resistance, refractility, and stainability. The calculated volume change of activation (DeltaVdouble dagger) was -139 +/- 6 cm(3)/mole for loss of heat resistance, -158 +/- 8 cm(3)/mole for the loss of refractility, and -153 +/- 4 cm(3)/mole for the change in permeability to dilute stains for the pressure range 800 to 1,010 atm at 25 C. It is suggested that the spore exists as a Donnan phase and that pressure triggers germination by influencing the equilibrium.     

Peptide Ligands That Bind Selectively to Spores of Bacillus subtilis and Closely Related Species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

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?     

High Salinity Alters the Germination Behavior of Bacillus subtilis Spores with Nutrient and Nonnutrient Germinants

The effect of high NaCl concentrations on nutrient and nonnutrient germination of Bacillus subtilis spores was systematically investigated. Under all conditions, increasing NaCl concentrations caused increasing, albeit reversible, inhibition of germination. High salinity delayed and increased the heterogeneity of germination initiation, slowed the germination kinetics of individual spores and the whole spore population, and decreased the overall germination efficiency, as observed by a variety of different analytical techniques. Germination triggered by nutrients which interact with different germinant receptors (GRs) was affected differently by NaCl, suggesting that GRs are targets of NaCl inhibition. However, NaCl also inhibited GR-independent germination, suggesting that there is at least one additional target for NaCl inhibition. Strikingly, a portion of the spore population could initiate germination with l-alanine even at NaCl concentrations near saturation (∼5.4 M), suggesting that spores lack a salt-sensing system preventing them from germinating in a hostile high-salinity environment. Spores that initiated germination at very high NaCl concentrations excreted their large depot of Ca²⁺-pyridine-2,6-dicarboxylic acid and lost their heat resistance, but they remained in a phase-gray state in the phase-contrast microscope, suggesting that there was incomplete germination. However, some metabolic activity could be detected at up to 4.8 M NaCl. Overall, high salinity seems to exert complex effects on spore germination and outgrowth whose detailed elucidation in future investigations could give valuable insights on these processes in general.     

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.