Macromolecular Syntheses During Germination and Outgrowth of Bacillus subtilis Spores

Alanine and glucose used jointly are known to be necessary and sufficient for spore germination in Bacillus subtilis 168. By testing them separately, we have verified that alanine provokes optimal phase-darkening of the spores but inhibits macromolecular syntheses, while glucose is specifically needed for initiating those syntheses. By using them in succession we obtained evidence suggesting that: (i) sporal modifications which lead to phase-darkening must occur before macromolecular synthesis can start; (ii) the amino acid pool, on which the early protein synthesis is solely dependent, expands during incubation in alanine which allows degradative but prevents synthetic activities; and (iii) progression of degradations in alanine not promptly followed by syntheses in glucose produce a metabolic imbalance in the germinating spore. A sharp transition in the origin of building blocks was shown by using a tryptophan-defective mutant. At first the synthesis of proteins depended on pre-existing amino acids from turnover of sporal material since it occurred in the absence of any exogenous amino acid and its rate remained unaltered by supplying either all amino acids except tryptophan or tryptophan alone. Eventually, protein synthesis became dependent strictly on exogenous tryptophan and strongly on the supply of several other amino acids, not required later during vegetative growth. Clearly, by the start of outgrowth, all building blocks must be provided either by endogenous de novo synthesis or by exogenous supply.     

Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.     

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

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

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Chromosomes in Bacillus subtilis Spores and Their Segregation During Germination

Spores of a thymine-requiring mutant of Bacillus subtilis 168 leucine(-), indole(-), thymine(-)) were uniformly labeled with (3)H-thymidine. These were seeded on thinlayer agar plates where they germinated into long-chained microcolonies. Autoradiograms were used to measure the distribution of labeled deoxyribonucleic acid in the chains of cells, which ranged in length from 2 to 32 cells. Four major grain clusters appeared in most chains. These clusters were homogeneous in size; their grain numbers were distributed symmetrically from 9 to 15 with an average of 12.0. When three or fewer major clusters appeared in short chains, some of them were composed of two subclusters. However, there were always four clusters per chain when these subclusters were counted as individuals. Groupings containing two to eight grains appeared, as well as the four major clusters in longer chains. These minor groups were fragments of the major clusters. In contrast to the symmetrical distribution of major clusters, fragmented clusters were distributed at random, indicating random fragmentation. The total number of major and minor clusters increased at a constant exponential rate when measured against total cell number per chain, i.e., number of generations. It was calculated from the rate that a detectable fragmentation, at least 16% of a conserved unit (defined as a single strand of the complete chromosome), occurred every 6.0 generations. These results led us to conclude that each B. subtilis spore contained four conserved units or two completed chromosomes. Segregation of the four units into progeny cells was almost random. The one notable exception was a conserved unit which frequently appeared in a terminal cell to which an empty spore coat was attached. The presence of two chromosomes in the spore is consistent with our proposed structure of the completed chromosome, in which two sister chromosomes are covalently linked at the initiation region. This double chromosome may be incorporated into the spore without further structural change.     

Biosynthesis of 50S Ribosomal Proteins During the Outgrowth of Bacillus subtilis Spores

The biosynthesis of 14 protein bands from 50S ribosomes was studied during the outgrowth of Bacillus subtilis W23 spores. The time at which the synthesis of each protein band begins and the patterns of synthesis that each displays were determined by comparing the incorporation of (3)H-phenylalanine during five intervals throughout the first hour of outgrowth with the incorporation of (14)C-phenylalanine between 145 and 150 min. The majority of the proteins in the 14 bands analyzed were synthesized as early as 5 to 10 min. The bands could be grouped on the basis of their labeling patterns as well as the time required for them to achieve a high rate of synthesis. Three bands did not achieve a high rate of synthesis until after 30 min of outgrowth, and at least one of the proteins from these bands appeared to be located on the exterior of the 50S subunit. The significance of the proteins from these three bands is discussed in terms of the late formation of new, mature 50S subunits during outgrowth.     

Effects of Temperature on Activation, Germination, and Outgrowth of Bacillus megaterium Spores

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.     

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn²⁺ or Ca²⁺ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.     

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.     

Comparative Study of Pressure-Induced Germination of Bacillus subtilis Spores at Low and High Pressures

We have studied pressure-induced germination of Bacillus subtilis spores at moderate (100 MPa) and high (500 to 600 MPa) pressures. Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used. Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa. Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination. DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination. These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa. The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to α/β-type SASPs, since mutants deficient in α/β-type SASPs were more sensitive to inactivation at 600 MPa. Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa. These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case. The implications for the use of high pressure as a preservation treatment are discussed.     

Role of GerKB in Germination and Outgrowth of Clostridium perfringens Spores

Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.Spores of Bacillus and Clostridium species can break dormancy upon sensing a variety of compounds (termed germinants), including amino acids, nutrient mixtures, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), and cationic surfactants such as dodecylamine (20). Nutrient germinants are sensed by their cognate receptors, located in the spore''s inner membrane (6), which are composed of proteins belonging to the GerA family (10, 11). In Bacillus subtilis, three tricistronic operons (gerA, gerB, and gerK) expressed uniquely during sporulation in the developing forespore each encode the three major germinant receptors, with different receptors responding to a different spectrum of nutrient germinants (5, 9, 20). Null mutations in any cistron in a gerA family operon inactivate the function of the respective receptor (9, 11). In contrast, Clostridium perfringens, a gram-positive, spore-forming, anaerobic pathogenic bacterium, has no tricistronic gerA-like operon but only a monocistronic gerAA that is far from a gerK locus. This locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream of and in the opposite orientation to gerKA-gerKC (Fig. ​(Fig.1A)1A) (16). GerAA has an auxiliary role in the germination of C. perfringens spores at low germinant concentrations, while GerKA and/or GerKC are required for l-asparagine germination and have partial roles in germination with KCl and a mixture of KCl and l-asparagine (AK) (16). In contrast to the situation with B. subtilis, where germinant receptors play no role in Ca-DPA germination (12, 13), GerKA and/or GerKC is required for Ca-DPA germination (16). The partial requirement for GerKA and/or GerKC in C. perfringens spore germination by KCl and AK suggests that the upstream gene product, GerKB, might also have some role in KCl and AK germination of C. perfringens spores. Therefore, in this study we have investigated the role of GerKB in the germination and outgrowth of C. perfringens spores.Open in a separate windowFIG. 1.Arrangement and expression of gerKB in C. perfringens SM101. (A) The arrangement of the gerK locus in C. perfringens SM101 is shown, and the locations of the primers used to amplify the upstream regions of the gerKB gene and the putative promoters of gerKB and gerKA are indicated. The gerKB promoter was predicted to be within the intergenic regions between gerKB and the gerK operon. (B) GUS specific activities from the gerKB-gusA fusion in strain SM101(pDP84) grown in TGY vegetative (filled squares) and DS sporulation (open squares) media were determined as described in the text. Data represent averages from three independent experiments with the error bars representing standard deviations, and time zero denotes the time of inoculation of cells into either TGY or DS medium.To determine if gerKB is expressed during sporulation, 485 bp upstream of the gerKB coding sequence, including DNA between gerKB and gerKA, was PCR amplified with primer pair CPP389/CPP391, which had SalI and PstI cleavage sites, respectively (see Table S2 in the supplemental material). The PCR fragment was cloned between SalI and PstI cleavage sites in plasmid pMRS127 (17) to create a gerKB-gusA fusion in plasmid pDP84 (see Table S1 in the supplemental material). This plasmid was introduced into C. perfringens SM101 by electroporation (3), and Em^r transformants were selected. The SM101 transformant carrying plasmid pDP84 was grown in TGY vegetative growth medium (3% Trypticase soy, 2% glucose, 1% yeast extract, 0.1% l-cysteine) (7) and in Duncan-Strong (DS) (4) sporulation medium and assayed for β-glucuronidase (GUS) activity as described previously (23). Vegetative cultures of strain SM101 carrying plasmid pMRS127 (empty vector) or pDP84 (gerKB-gusA) exhibited no significant GUS activity, and strain SM101 grown in DS medium also exhibited no significant GUS activity (Fig. ​(Fig.1B1B and data not shown). However, GUS activity was observed in sporulating cultures of SM101(pDP84) (Fig. ​(Fig.1B),1B), indicating that a sporulation-specific promoter is located upstream of gerKB. The expression of the gerKB-gusA fusion began ∼3 h after induction of sporulation and reached a maximum after ∼6 h of sporulation (Fig. ​(Fig.1B).1B). The decrease in GUS activity observed after ∼6 h is consistent with the GerKB-GusA protein being packaged into the dormant spore where it cannot be easily assayed and thus with gerKB being expressed in the forespore compartment of the sporulating cell (8). These results confirm that, as with the gerKA-gerKC operon (16), gerKB is also expressed only during sporulation.To investigate the role of GerKB in C. perfringens spore germination, we constructed a gerKB mutant strain (DPS108) as described previously (14-16). A 2,203-bp DNA fragment carrying 2,080 bp upstream of and 123 bp from the N-terminal coding region of gerKB was PCR amplified using primers CPP369 and CPP367, which had XhoI and BamHI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). A 1,329-bp fragment carrying 134 bp from the C-terminal and 1,195 bp downstream of the coding region of gerKB was PCR amplified using primers CPP371 and CPP370, which had BamHI and KpnI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). These PCR fragments were cloned into plasmid pCR-XL-TOPO, giving plasmids pDP67 and pDP69, respectively (see Table S1 in the supplemental material). An ∼2.2-kb BamHI-XhoI fragment from pDP67 was cloned into pDP1 (pCR-XL-TOPO carrying an internal fragment of gerAA), giving plasmid pDP68, and an ∼1.4-kb KpnI-BamHI fragment from pDP69 was cloned in pDP68, giving pDP73 (see Table S1 in the supplemental material). The latter plasmid was digested with BamHI, the ends were filled, and an ∼1.3-kb NaeI-SmaI fragment carrying catP from pJIR418 (1) was inserted, giving plasmid pDP74. Finally, an ∼4.8-kb KpnI-XhoI fragment from pDP74 (see Table S1 in the supplemental material) was cloned between the KpnI and SalI sites of pMRS104, giving pDP75, which cannot replicate in C. perfringens. Plasmid pDP75 was introduced into C. perfringens SM101 by electroporation (3), and the gerKB deletion strain DPS108 was isolated as described previously (18). The presence of the gerKB deletion in strain DPS108 was confirmed by PCR and Southern blot analyses (data not shown). Strain DPS108 gave ∼70% sporulating cells in DS sporulation medium, similar to results with the wild-type strain, SM101 (data not shown).Having obtained evidence for successful construction of the gerKB mutant, we compared the germinations of heat-activated (80°C; 10 min) gerKB and wild-type spores as previously described (16). Both the gerKB and wild-type spores germinated identically and nearly completely in 60 min at 40°C in brain heart infusion (BHI) broth as determined by the fall in optical density at 600 nm (OD₆₀₀) of germinating cultures and phase-contrast microscopy (data not shown). This result suggests that GerKB plays no essential role in spore germination in rich medium. The role of GerKB in C. perfringens spore germination was also assessed with individual germinants identified previously (16). Heat-activated wild-type and gerKB spores germinated similarly with high (100 mM) concentrations of KCl, l-asparagine, and AK, all in 25 mM sodium phosphate (pH 7.0), and in 50 mM Ca-DPA adjusted to pH 8.0 with Tris base (Fig. 2A to D). These results were also confirmed by phase-contrast microscopy (data not shown). However, with lower (10 to 20 mM) concentrations of KCl, l-asparagine, and AK, gerKB spore germination was very slightly (Fig. ​(Fig.2A)2A) to significantly (Fig. 2B and C) slower than that of wild-type spores. These results suggest that while GerKB is not essential for germination with high concentrations of KCl, l-asparagine, or AK, it plays a significant role in germination with low l-asparagine and AK concentrations and, further, that GerKB is not required for Ca-DPA germination. This latter finding is similar to the situation with B. subtilis spores where germinant receptors play no role in Ca-DPA germination (19, 20). However, in C. perfringens spores, GerKA and/or GerKC do play a significant role in Ca-DPA germination (16).Open in a separate windowFIG. 2.Germination of spores of C. perfringens strains with various germinants. Heat-activated spores of strains SM101 (wild type) (filled symbols) and DPS108 (gerKB) (open symbols) were incubated at an OD₆₀₀ of 1 at 40°C with high (squares) and low (triangles) germinant concentrations of 100 and 10 mM KCl (A), 100 and 20 mM l-asparagine (B), 100 and 10 mM AK (C), and 50 mM Ca-DPA (D) as described in the text, and at various times the OD₆₀₀ was measured. No significant germination was observed when heat-activated spores of SM101 and DPS108 were incubated for 60 min at 40°C in 25 mM sodium phosphate buffer (pH 7.0) (data not shown). The data shown are averages from duplicate determinations with two different spore preparations, and error bars represent standard deviations.Bacterial spores can also germinate with dodecylamine, a cationic surfactant (19). In B. subtilis spores, dodecylamine induces germination most likely by opening channels composed, at least in part, of SpoVA proteins (22), allowing release of the spores'' Ca-DPA (19). Spores of B. subtilis lacking all three functional germinant receptors release DPA, as do wild-type spores, upon incubation with dodecylamine (19), while C. perfringens spores lacking GerKA-GerKC incubated with dodecylamine release DPA slower than wild-type spores (16). However, when C. perfringens gerKB spores at an OD₆₀₀ of 1.5 were incubated with 1 mM dodecylamine in Tris-HCl (pH 7.4) at 60°C (2, 16), gerKB spores released their DPA slightly faster than wild-type spores (Fig. ​(Fig.3)3) when DPA release was measured as described previously (16). These results suggest that GerKB has no role in dodecylamine germination.Open in a separate windowFIG. 3.Germination of spores of C. perfringens strains with dodecylamine. Spores of strains SM101 (wild type) (filled squares) and DPS108 (gerKB) (open squares) were germinated with dodecylamine, and germination was monitored by measuring DPA release as described in the text. There was no significant DPA release in 60 min by spores incubated similarly but without dodecylamine (data not shown). Error bars represent standard deviations.Previous work (16) found that C. perfringens spores lacking GerKA-GerKC had lower viability than wild-type spores on rich medium plates, and it was thus of interest to determine gerKB spore viability, which was measured as previously described (14, 16). Strikingly, the colony-forming ability of gerKB spores was ∼7-fold lower (P < 0.01) than that of wild-type spores after 24 h on BHI plates (Table ​(Table1),1), and no additional colonies appeared when plates were incubated for up to 3 days (data not shown). The colony-forming ability of spores lacking GerKA and GerKC determined in parallel was ∼12-fold lower than that of wild-type spores (Table ​(Table1).1). Phase-contrast microscopy of C. perfringens spores incubated in BHI broth for 24 h under aerobic conditions to prevent vegetative cell growth indicated that >90% of wild-type spores not only had germinated but had also released the nascent vegetative cell, while >85% of gerKA gerKC and gerKB spores remained as only phase-dark germinated spores with no evidence of nascent cell release (data not shown), as found previously with gerKA gerKC spores (16). The fact that >85% of gerKB spores germinated in BHI medium in 24 h but most of these germinated spores did not progress further in development strongly suggests that GerKB is needed for normal spore outgrowth (and see below) as well as for normal spore germination.

TABLE 1.

Colony formation by spores of C. perfringens strains^a

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.     

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

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

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Cold Shock of Germinating Bacillus subtilis Spores

Susceptibility of germinating spores of Bacillus subtilis to a rapid chilling was examined by viable countings. Dormant spores were quite resistant to the cold shock but the spores, immediately upon germination, lose viability almost completely by the same treatment. The presence of divalent cation, magnesium, calcium or manganese, in a buffer to which the germinating spores were suspended, markedly protected the cells from the death by the cold shock effect. When the shocked cells were incubated in the buffer containing casein acid hydrolyzate, glucose and magnesium ion for short period of time, a remarkable increase in viable counts was observed. The existence of two critical temperature zones, which were determined by the initial temperature of cell suspension, was confirmed in the cold shock of germinating spores.     

Monitoring of Commitment,Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores'' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T_release) greater than the time of germinant removal (T_b) increased, while the time T_lag − T_b, where T_lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔT_release = T_release − T_lag) were increased; Factors affecting average T_release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.     

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.