No abstract

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N-or C-terminal domain has been deleted (SleB-⌬N or SleB-⌬C) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-⌬N or SleB-⌬C, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N-or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.Spores of the genera Bacillus and Clostridium emerge from dormancy via the process of germination. The germination process comprises a series of sequential biophysical and biochemical reactions that result irreversibly in the spore losing its properties of metabolic dormancy and extreme resistance to various chemical and physical treatments (24, 34). Germination is initiated by the presumed binding of small molecular germinants, commonly amino acids or sugars, to cognate receptors located within the spore inner membrane (25,28). In a process that is poorly understood at the molecular level, this interaction leads to a change in the permeability of the inner membrane, resulting in the release of various solutes from the spore core, including metal ions, calcium dipicolinate (Ca-DPA), and some amino acids (32,33,35). A degree of rehydration of the core is evident at or around the same time, although this is insufficient to permit a significant degree of vegetative metabolism (9, 31). These events, which appear common to all Bacillus species where examined, comprise stage I of germination (31, 32, 34).The major event in stage II of the germination process from a biochemical perspective involves depolymerization of the spore cortex. The spore cortex is a thick layer of peptidoglycan, characterized by the spore-specific muramic acid lactam (MAL) moiety (37, 38), which, together with the thin inner layer of germ cell wall peptidoglycan (36), forms contiguous layers that entirely envelope the spore protoplast. ...

Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/ vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA 2 , indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA ؉ spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.

, respectively. Moreover, thermodynamic (DG, DS, DH) and kinetic constants (K m and V max ) were determined and effects of metal ions were investigated. As a result, these enzymes could be considered as potential candidates for lactose hydrolysis of milk and milk products.