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

Li, Yunfeng; Jin, Kai; Setlow, Barbara; Setlow, Peter; Hao, Bing

doi:10.1128/jb.00877-12

Cited by 39 publications

(57 citation statements)

References 56 publications

(99 reference statements)

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“…There is some evidence that SleB's partner protein, YpeB, can inhibit SleB's catalytic activity (63), but it is not clear how this inhibition is achieved, since no direct interactions have been seen in vitro between YpeB and SleB. The structure of SleB from several Bacillus species has been solved by X-ray crystallography, and these structures are consistent with SleB being a lytic transglycosylase (64,65). However, the structures alone provide no insight into how SleB activity is regulated.…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores' return to life within minutes in the process of germination. This process requires a number of sporespecific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. T he germination of the dormant and highly resistant spores formed by members of the Firmicutes phylum, in particular bacilli and clostridia, has long been of significant research interest for four major reasons, as follows: (i) fascinating regulatory systems allow such spores to remain in their dormant, resistant state for years and yet return to active growth in minutes; (ii) while spores of most Firmicutes do not cause disease, spores of some bacilli and clostridia cause food spoilage and food-borne disease, as well as human diseases like gas gangrene, tetanus, botulism, anthrax, and pseudomembranous colitis; (iii) spores of Bacillus anthracis are a major bioterrorism threat; and (iv) spores of Clostridium difficile are an emerging public health threat (1-3). Invariably, it is the germination of spores of these organisms that leads to disease or food spoilage, and yet, when spores germinate and lose their dormancy, they lose their extreme resistance properties and become relatively easy to kill. Germination is thus both an essential part of disease pathogenesis or food spoilage and a major weak spot in these organisms' life cycle. Consequently, there has long been applied interest in spore germination, with researchers seeking to understand this process better in order to either prevent spore germination or accelerate it and then kill the newly sensitive germinated spores. This review will concentrate on the germination of spores of bacilli, primarily because of the large amount of detailed knowledge of the germination of spores of these species compared to that of spores of clostridia. However, some of the differences and similarities between the germination of spores of these related genera will also be presented. Most discussion will focus on the germination of the model sporeformer, Bacillus subtilis, although the mechanisms of germination of B. subtilis spores appear to be similar for spores of other bacilli. The properties of the various proteins that are specifically involved in the germination process will not be discussed in great detail, since these have recently be...

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Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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“…Deletion of a predicted destabilizing loop corresponding to residues 124 to 135 was accomplished by overlap PCR (6), giving ⌬spoVAEa. The resulting PCR products, ⌬spoVAEa and the intact spoVAEa gene, were cloned into a modified pET15b vector containing a His 6 tag and a tobacco etch virus (TEV) protease cleavage site (13). The ⌬SpoVAEa protein (residues 2 [⌬124-135] to 203) and intact SpoVAEa were expressed in Escherichia coli BL21 Star(DE3) (Invitrogen, Grand Island, NY) initially by growth at 37°C in Luria broth (LB) (14) plus ampicillin (100 g/ml) and then by induction with 1 mM isopropyl-␤-D-thiogalactopyranoside at an optical density at 600 nm (OD 600 ) of 0.8 and subsequent growth at 21°C for 16 h. Both SpoVAEa proteins were soluble and were purified by Ni 2ϩ -nitrilotriacetic acid affinity chromatography under native conditions, followed by TEV protease cleavage of the His 6 tag and cation-exchange chromatography (SD200; GE Healthcare, Piscataway, NJ) (6,13).…”

Section: Preparation Of Purified Spovaea and Generation And Purificatmentioning

confidence: 99%

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

Pérez-Valdespino

Setlow

et al. 2014

Journal of Bacteriology

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The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.T he spoVA operon of Bacillus subtilis is expressed only in the developing spore during sporulation and encodes seven proteins, with the genes in the order spoVAA, -B, -C, -D, -Eb, -Ea, and -F. At least five of these SpoVA proteins, SpoVAA, -B, -C, -D, and -Eb, are necessary for normal B. subtilis spore formation (1, 2). These proteins are essential for the uptake of dipicolinic acid (DPA) into the dormant spore (3-7); in turn, DPA is essential for spore stability, as DPA-less B. subtilis spores germinate spontaneously after their release from the sporangium and during spore purification (8). At least some SpoVA proteins are also essential for DPA release in spore germination, and one SpoVA protein, SpoVAD, binds DPA specifically, with this binding essential for DPA uptake in sporulation (6). SpoVAD is a soluble protein and has no obvious signal peptide or membrane-spanning or anchor sequences. However, assessment of SpoVAD's location and accessibility in spores indicates that this protein is on the outer surface of the spore's inner membrane (IM) (4, 9, 10).In contrast to SpoVAD, SpoVAA, -B, -C, -Eb, and -F appear to be integral membrane proteins on the basis of predictions from primary sequences and in some cases the localization of proteins expressed in growing bacteria (3,11,12). Although SpoVAF exhibits significant sequence identity to the A subunits of the spore's nutrient germinant receptors (GRs) (2), SpoVAA, -B, -C, -Eb, and -Ea exhibit no significant identity to known proteins. However, SpoVAEa is also predicted to be a soluble protein. Consequently, in this work, we have examined the location an...

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“…This was unexpected, since neither sleB N nor YpeB was thought to be associated directly with lytic activity during spore germination, and indeed, further evidence to support noncatalytic roles for both polypeptides has been obtained since (17,21). Similarly, sleB cwlJ spores complemented with ypeB and with a gene for the C-terminal portion of SleB in which catalytic glutamate 208 was changed to alanine also displayed a considerably enhanced germinative response compared to that of the parental sleB cwlJ spores (19) ( Table 1).…”

Section: Analysis Of B Megaterium Ypeb and Slel In Vivomentioning

confidence: 99%

“…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.…”

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confidence: 99%

“…Where examined, the viability of sleB cwlJ null mutant spores, as determined by their colony-forming ability on rich medium, is diminished by several orders of magnitude compared to that of wild-type (wt) spores of several species of Bacillus (10, 14, 15). 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.…”

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

Cited by 39 publications

References 56 publications

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

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