Activity and Regulation of Various Forms of CwlJ, SleB, and YpeB Proteins in Degrading Cortex Peptidoglycan of Spores of Bacillus Species
            <i>In Vitro</i>
            and during Spore Germination

Li, Yunfeng; Butzin, Xuan Y.; Davis, Andrew John; Setlow, Barbara; Korza, George; Üstok, Fatma Işık; Christie, Graham; Setlow, Peter; Hao, Bing

doi:10.1128/jb.00259-13

Cited by 49 publications

(84 citation statements)

References 42 publications

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“…The crystal structure of the catalytic C-terminal domain of SleB has been determined recently (16,17), revealing a protein fold that is reminiscent of those of several bacterium-and phage-lytic transglycosylases, which is consistent with the results of earlier molecular-genetic and biochemical studies aimed at characterizing this CLE hydrolytic-bond specificity (18)(19)(20). Sequence alignments, putative secondary-structure assignments, and site-directed mutagenesis experiments indicate that CwlJ is a lytic transglycosylase also (17,21), although this has yet to be confirmed by biochemical analysis.Knowledge of the structure and function of SleB and, to a lesser extent, of CwlJ is relatively detailed, although several crucial questions remain to be answered. The same cannot be said for another protein, YpeB, whose structural gene is borne by Bacillus subtilis and Bacillus megaterium immediately downstream of sleB in a bicistronic operon and which shares no detectable homology with any protein of known function.…”

supporting

confidence: 67%

“…Furthermore, analysis of peptidoglycan fragments released into germination exudates revealed the presence of anhydromuropeptides, indicating that a lytic transglycosylase other than SleB must be present in these spores. Similar complementation analyses have been conducted recently with B. subtilis sleB cwlJ spores (21). In that case, however, the germination defect was not complemented by sleB N plus ypeB.…”

mentioning

confidence: 48%

“…However, previously conducted immunochemical studies with B. subtilis revealed that YpeB is essential for the presence of SleB in the spore (22), although the physiological basis of this observation was not established. A more recent study revealed that the reciprocal arrangement, i.e., that SleB is required for YpeB's presence in the spore, also holds true (21). Additionally, YpeB, in particular, the putative N-terminal domain of YpeB, was demonstrated to inhibit the lytic activity of SleB against decoated spore substrates, supporting the hypothesis that YpeB may have a role in maintaining SleB in an inactive state during spore dormancy.…”

mentioning

confidence: 48%

“…The construction of the B. megaterium sleB (15), sleB cwlJ and sleB sleL (19), and sleB cwlJ sleL (21) null mutant strains employed in this study has been described previously. Construction of pHT315-derived plasmids with the genes sleB N ypeB and ypeB sleB E208A (sleB with a mutation resulting in an E208A change in the enzyme) has also been described (19).…”

Section: Construction Of B Megaterium Cle Mutant Strainsmentioning

confidence: 99%

“…Additionally, YpeB, in particular, the putative N-terminal domain of YpeB, was demonstrated to inhibit the lytic activity of SleB against decoated spore substrates, supporting the hypothesis that YpeB may have a role in maintaining SleB in an inactive state during spore dormancy. However, despite the apparent codependency for localization and perceived modulation of activity, evidence for direct physical interaction between the proteins was not detected in vitro (21).…”

mentioning

confidence: 99%

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Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Üstok

Packman

Lowe

et al. 2013

Journal of Bacteriology

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Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB N ). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro. Members of the bacterial genera Bacillus and Clostridium form environmentally resistant spores in response to nutrient starvation. Subsequent exposure of spores to conditions that are amenable to vegetative growth and metabolism triggers a series of germination reactions that result ultimately in the emergence of new vegetative cells (1, 2). A major germination event concerns depolymerization of the thick layer of cortical peptidoglycan that surrounds the spore protoplast. The cortex serves to maintain the relatively dehydrated status of the spore core (27 to 55% water by weight versus 70 to 80% water in vegetative cells [3]), and its removal by lytic enzymes is essential for hydration of the core to levels that permit protein mobility and the resumption of metabolic activity (4). The cortex is composed of structurally distinct peptidoglycans comprising linear chains of alternating N-acetylglucosamine and N-acetylmuramic acid (NAM) residues, the latter characterized by enzymatically processed side chains that yield either single L-alanine residues (in ϳ25% of NAM residues) or the spore-specific muramic acid lactam (MAL; prevalent in ϳ50% of NAM residues) (5, 6). Both modifications result in reduced availability of peptide side chains for cross-linking of glycan chains in cortical peptidoglycan, conferring a peptidoglycan sacculus that is relatively loosely cross-linked compared to vegetative cell sacculi (7).The ...

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supporting

confidence: 67%

mentioning

confidence: 48%

mentioning

confidence: 48%

Section: Construction Of B Megaterium Cle Mutant Strainsmentioning

confidence: 99%

mentioning

confidence: 99%

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Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Üstok

Packman

Lowe

et al. 2013

Journal of Bacteriology

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Enzyme‐driven bacillus spore coat degradation leading to spore killing

Mundra

Mehta

et al. 2013

Biotech & Bioengineering

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The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable.

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Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of B acillus spores

2015

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A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi-redundant cortex-lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N-acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/β)8 -barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N-terminal peptidoglycan-binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL.

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Activity and Regulation of Various Forms of CwlJ, SleB, and YpeB Proteins in Degrading Cortex Peptidoglycan of Spores of Bacillus Species In Vitro and during Spore Germination

Cited by 49 publications

References 42 publications

Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Enzyme‐driven bacillus spore coat degradation leading to spore killing

Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of B acillus spores

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