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.     

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.     

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.     

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.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

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.     

Effect of Monosodium n-Alkylsalicylates on Spores of Bacillus subtilis

The effects of several monosodium n-alkylsalicylates on washed spores of Bacillus subtilis have been examined. None of the compounds was sporicidal, but in general they prevented dormancy induced by incubating the spores in water. This effect was related to the position in the ring and size of the alkyl groups substituted on the salicylic acid nucleus.     

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.     

Initiation of Spore Germination in Glycolytic Mutants of Bacillus subtilis

Enzyme activities of glycolysis and glyconeogenesis are present in spores of Bacillus subtilis, the rate-limiting step of glucose (GLC) metabolism being its phosphorylation. GLC allows initiation of germination in the presence of fructose (FRU) and asparagine (ASN), not because it is used via the Embden-Meyerhof path, but because it is oxidized in the nonphosphorylated form via the spore-specific GLC dehydrogenase. Spores of mutants lacking GLC-phosphoenolpyruvate transferase, FRU-6-P-kinase, or phosphoglucoisomerase activity can still be initiated by the above substrate combination. Furthermore, GLC can be replaced by 2-deoxy-GLC, which is also oxidized by GLC-dehydrogenase, but not by α- or β-methylglucoside, which are not substrates of this enzyme. GLC probably acts by reducing nicotinamide adenine dinucleotide (or nicotinamide adenine dinucleotide phosphate), which is used for some metabolic reaction other than the cytochrome-linked electron transport system, since inhibitors of this system do not inhibit initiation. Spores of a mutant lacking FRU-1-P-kinase activity can no longer be initiated by GLC+FRU+ASN, but they do respond to the combination of GLC+mannose+ASN. Since spores of a FRU-6-P-kinase (or phosphoglucoisomerase) mutant can still respond to either FRU or mannose, FRU-6-P (or some derivative) apparently is needed for initiation (in addition to reduced nicotinamide adenine dinucleotide and an amino donor). Alanine can initiate germination in spores of all of the above mutants, indicating that it can form all required compounds. However, in a mutant lacking P-glycerate kinase activity, alanine initiates only after a long lag and at a slow rate, indicating that some compound in the upper metabolic subdivision is required for initiation, in agreement with the above findings. All initiating agents of B. subtilis probably produce the same required compound(s) by different metabolic routes.     

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.     

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.     

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.     

Effect of Phenol on Bacillus subtilis Spores at Elevated Temperatures

The nature of the recovery medium is shown to influence the number of Bacillus subtilis spores which, after exposure to 2.5 or 5% phenol at high temperatures, can produce a visible colony. Higher survivor counts were obtained in nutrient agar containing L-alanine and D-glucose than in plain nutrient agar.     

Effect of the Method of Presentation of the Medium on the Germination and Growth of Single Spores of Bacillus subtilis

S ummary . Micromanipulation of single spores on an agar surface allowed the observation of normal and heat damaged spores in microculture. In tests for viability on different media, spores recorded as 'dead' in some media were fully viable when nutrients were supplied by diffusion from agar cylinders. In microculture of heat-damaged spores, only those phase-bright spores which exhibited considerable delay in becoming phase-dark were eventually capable of forming visible colonies. Rapidly germinating spores were incapable of any outgrowth, except for a small minority which developed to a maximum of 4–8 cells and then lysed.     

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.     

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.     

Effect of Carbohydrates in Phosphate Buffer on Germination of Bacillus stearothermophilus Spores

Among strains, and among spore suspensions of the same strain, different spore-germination responses were observed when spores were heated in monosaccharides, disaccharides, and polysaccharides in 0.0083 m phosphate buffer (pH 7.1). It was hypothesized that these differences were due to rough and smooth variants in the spore population and to variation in the osmosensitivity of spores of variants within the population when subjected to a heat shock of 110 C.