Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes

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. ...

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

“…The high cortex-lytic activity of SleB C and the lack of activity of SleB N are consistent with the notion that the CTD of SleB comprises the catalytic domain, with the NTD binding to the PG substrate. CwlJ that lacks a PG-binding domain is also active, at least in vivo, and in B. megaterium spores lacking SleB and CwlJ, synthesis of SleB C alone is sufficient for cortex hydrolysis during spore germination and full spore viability (6). Nonetheless, the fact that the PG-binding domain was not essential for SleB C hydrolytic activity on decoated spores was surprising.…”

“…Nonetheless, the fact that the PG-binding domain was not essential for SleB C hydrolytic activity on decoated spores was surprising. In cwlJ sleB B. megaterium spores, synthesis of SleB N or SleB M with the putative catalytic glutamate changed to alanine alone are sufficient for cortex hydrolysis during spore germination as judged by spore viability (6). Clearly, more work is needed to assess the function of SleB's NTD and CTD in cortex degradation during spore germination.…”

“…As we failed to find any structures homologous to SleB C through an automated search, we attempted to superimpose the SleB C structure directly on those of proteins with similar enzymatic activity. SleB has been characterized as a lytic transglycosylase (3,6,15,16) that cleaves the ␤-1,4-glycosidic bond between NAM and NAG residues in PG, as do lysozymes, but unlike lysozyme, SleB makes a new glycosidic bond between the C-6 hydroxyl group and C-1 of the same NAM residue, forming a 1,6-anhydromuramic acid terminal residue. Despite lacking sequence similarity and having disparity in overall tertiary structure, catalytic domains of most bac- , where F H is the structure factor amplitude for anomalous scatterers and ε is the residual lack of closure error.…”

“…The purified CTD of B. anthracis SleB has also been shown to hydrolyze MALcontaining PG in vitro, albeit significantly slower than the fulllength protein (16). Interestingly, work with SleB mutants in Bacillus megaterium spores indicates that SleB's NTD alone is sufficient for complementation of the germination and cortex hydrolysis defects in cwlJ sleB spores and is even better than SleB's CTD (6). Clearly, a number of questions about SleB function and mechanism remain, including the following.…”

“…However, cwlJ sleB double mutant spores have extremely low viability, as the great majority cannot complete germination unless provided with an exogenous lytic enzyme. SleB, the subject of this communication, has been characterized as a lytic transglycosylase, and its action generates large fragments from the spore cortex with these fragments degraded further by other enzymes, although these auxiliary enzymes are not essential for completion of spore germination (3,5,6,16). SleB is synthesized in the developing spore midway in sporulation, and the great majority is translocated across the forespore membrane.…”