Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation

The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival. During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases. GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination. Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold. The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer. Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore.     

Glucose-triggered germination of Bacillus megaterium spores.

Triggering of germination in Bacillus megaterium QM B1551 spores with D-glucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a non-metabolizable glucose analog, methyl-alpha-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination.     

Glucose catabolism during germination of Bacillus megaterium spores.

When heat-activated spores of Bacillus megaterium germinated in glucose-containing medium, 10 to 30% of the glucose was found to be oxidized to gluconate.     

Biochemistry of L-proline-triggered germination of Bacillus megaterium spores.

The mechanism by which L-proline triggers germination in Bacillus megaterium QM B1551 spores was investigated. First, brief exposure of spores to L-proline, followed by dilution, was sufficient to trigger germination. Once germination was triggered, the spores continued initiation of germination and did not require high concentrations of L-proline. Triggering of germination was pH and temperature dependent. Second, enzymes for L-proline catabolism were absent in spores, and several non-metabolizable analogs of L-proline were effective trigger compounds. Third, triggering of germination occurred in the presence of inhibitors of proton motive force production, oxygen uptake, and metabolism. Fourth, uptake of L-proline occurred after the triggering of germination. These results argue that neither uptake nor metabolism of L-proline was necessary to trigger germination. Instead, L-proline probably causes a biophysical alteration in the spores that triggers the biochemical changes in germination.     

The amino acid sequence specificity of a protease from spores of Bacillus megaterium

Previous work has shown that the degradation of 20% of total protein which occurs early in germination of Bacillus megaterium spores is initiated by an endoprotease. This enzyme is found only in the spore and is active only on the spore proteins degraded during germination. Action of the spore protease in vitro on the three major proteins (Proteins A, B, and C) which are degraded in vivo during germination results in cleavage of one (A and C protein) or two (B protein) peptide bonds. The sequences surrounding the cleavage sites are -Tyr-Glu- Ile-Ala-Ser-Glu-Phe- in the A protein, -Phe-Glu- Ile-Ala-Ser-Glu-Phe- in the C protein, and -Thr-Glu- Phe-Gly-Ser-Glu-Thr-, and -Thr-Glu- Phe-Ala-Ser-Glu-Thr- in the B protein, with cleavage taking place at the glutamyl bond noted by the arrow. The similarity of these four sequences suggests the possibility that the specificity of the spore protease may be due to its requirement for a specific pentapeptide sequence of the type -R-Glu-(Phe or Ile)-(Gly or Ala)-Ser-Glu-R- for recognition and cleavage. However, it is also possible that it is the conformation of the A, B, and C proteins which determines their site of cleavage by the spore protease.     

Collapse of cortex expansion during germination of Bacillus megaterium spores

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast-plus-cortex, C/(P+C), of the CdCl2-germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose-induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.     

Effect of ion channel blockers on germination of Bacillus megaterium spores

Abstract We surveyed 23 drugs that can interact with membrane components, such as ion channels, for their effect on spore germination. The results showed that triggering of spore germination was inhibited by specific calcium (Ca²⁺) potassium (K⁺) and sodium (Na⁺) channel blockers.     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

Lethal germination of spores ofBacillus megaterium 9885

Washed spore suspensions germinated promptly without prior heat shock in a basal germination solution containingl-leucine.Germination was inhibited by dipicolinic acid. The inhibition was reversed by eitherl-leucine or phosphate.Phosphate accelerated the rate and increased the extent of germination, which was accompanied by an uncommonly large fall in the optical density of the suspension, but phosphate also caused a massive lysis after germination. This was accompanied by a sudden shedding of the spore coats. The suspensions consisted of shrivelled, cellular walls and membranes attached to the empty spore coats.Lysis of the germinated cells was prevented by fairly high concentrations of Ca or Mg.During germination, exogenous Ca we used Ca⁴⁵ was absorbed by the cells. Both cells and sonically disrupted cellular particles firmly retained the calcium, and evidence suggested that much of the Ca was bound in the cytoplasmic membranes.The cations contained in plain agar enabled spores which germinated on tryptone soya agar plates to develop into colonies; in the corresponding broth medium these spores lysed upon germination.Hypertonic sucrose delayed but did not prevent lysis.     

Red pigment in Bacillus megaterium spores

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Isolation and properties of membranes from Bacillus megaterium spores.

Membranes from dormant and heat-activated spores of Bacillus megaterium QM B1551 were isolated and purified by gentle lysis procedures followed by differential and sucrose density gradient centrifugations. The purified membranes were enriched for inner membranes and were characterized by their density and content of proteins, phospholipids, enzymes, cytochromes, and carotenoids. These purified spore membranes could be used to investigate their role in the triggering of germination.     

Germinability of coat-lacking spores of Bacillus megaterium

Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus megaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germinability must not exist in the coat.     

An evaluation of respiration chain-associated functions during initiation of germination of Bacillus megaterium spores.

In Bacillus megaterium QM B1551, spore germination could be initiated by glucose in the absence of detectable oxygen consumption, ATP synthesis or a pH decrease in the external media, suggesting that none of those reactions were mandatory. In addition, initiation of germination was insensitive to a variety of inhibitors of energy production or protonmotive force uncouplers. Therefore the respiratory chain-associated functions are not prerequisites for initiation of germination but these functions may be necessary to drive energy-dependent transport systems and other biosynthetic reactions during outgrowth.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.