Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Role of the gerI Operon of Bacillus cereus 569 in the Response of Spores to Germinants

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to l-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to l-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to l-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to l-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in l-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of l-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.Bacillus species have the ability, under certain nutrient stresses, to undergo a complex differentiation process resulting in the formation of a highly resistant dormant endospore (6). These spores can then persist in the environment for prolonged periods until a sensitive response mechanism detects specific environmental conditions, initiating the processes of germination and outgrowth (9, 21, 25). Germination can be initiated by a variety of agents (12), including nutrients, enzymes, or physical factors, such as abrasion or hydrostatic pressure.The molecular genetics of spore germination has been most extensively studied in Bacillus subtilis 168 (21). B. subtilis spores can be triggered to germinate in response to either l-alanine or to a combination (29) of asparagine, glucose, fructose, and potassium ions (AGFK). Mutants of B. subtilis which are defective in germination responses to one or to both types of germinant have been isolated previously (20, 27). Analysis of these mutants suggests that the germinants interact with separate germinant-specific complexes within the spore (21). This in some way leads to activation of components of the germination apparatus common to both responses, such as germination-specific cortex lytic enzymes, leading in turn to complete germination of the spore (10, 22). The mutations within the gerA operon of B. subtilis specifically block germination initiated by l-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded in the operon suggest that these proteins could be membrane associated, and they are the most likely candidates to represent the germinant receptor for alanine (21).The amino acid l-alanine has been identified as a common but not universal germinant in a variety of Bacillus species, often requiring the presence of adjuncts such as electrolytes and sugars. Ribosides, such as inosine, represent another type of common germinant, although many species are unable to germinate rapidly in response to these without the addition of l-alanine (9).The food-borne pathogen Bacillus cereus is a major cause of food poisoning of an emetic and diarrheal type (13, 16). The germination and growth of Bacillus cereus spores during food storage can lead to food spoilage and the potential to cause food poisoning (16). B. cereus has been shown to germinate in response to l-alanine and to ribosides (11, 18, 23). Spore germination can be triggered by l-alanine alone, but at high spore densities this response becomes inhibited by d-alanine, generated by the alanine racemase activity associated with the spores (8, 11). This auto-inhibition of l-alanine germination can be reduced by the inclusion of a racemase inhibitor (O-carbamyl-d-serine) with the germinating spores (11).Inosine is the most effective riboside germinant for B. cereus T, while adenosine and guanosine are less potent (28). The rate of riboside-triggered germination has been reported to be enhanced dramatically by the addition of l-alanine (18). It is unclear whether ribosides can act as a sole germinant, or whether there is an absolute requirement for l-alanine (28).An attempt has been made to analyze genetically the molecular components of the germination apparatus in B. cereus in order to dissect the germination responses of this species and to determine whether riboside-induced germination involves components related to those already described for amino acid and sugar germinants in B. subtilis.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (cwlJ) Which Affects Germination of Bacillus subtilis Spores

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by Eς^E RNA polymerase. cwlJ-deficient spores responded to both l-alanine and AGFK, the A₅₈₀ values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A₅₈₀ and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.During sporulation and germination of Bacillus subtilis, the action of autolysins is assumed to be required for asymmetric septum peptidoglycan hydrolysis, engulfment, cortex maturation, mother cell lysis, and cortex hydrolysis during germination (28, 33). Mother cell lysis depends on the compensatory effect of cell wall hydrolases CwlB (LytC) and CwlC (11, 13, 34). For cortex maturation, a defect in the cwlD gene leads to a lack of germination and blocking of the formation of muramic acid lactam structure in the cortex (2, 26, 31). Recently, Makino and colleagues reported that the B. cereus sleB gene encodes a 24-kDa mature germination-specific N-acetylmuramoyl-l-alanine amidase which degrades decoated spores from various organisms (18, 22). B. subtilis sleB is homologous to B. cereus sleB, and B. subtilis sleB mutant spores are able to germinate and form colonies. However, B. subtilis SleB showed no activity against degraded decoated spores or other substrates (21).Our work on the B. subtilis genome sequencing project has revealed the ycbQ gene, which is homologous with the cortex-hydrolyzing sleB genes (22, 25). In this study, we describe the regulation and function of the cwlJ (ycbQ) gene and the compensatory effect of the CwlJ and B. subtilis SleB proteins on germination.     

Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro⁴²⁷, Gly⁴²⁸, and Gly⁴²⁹ activate the catalytic residue of the enzyme, Thr³⁵², and stabilize an oxyanion hole via main chain amide hydrogen bonds.Spores of Bacillus anthracis are the causative agents of anthrax disease (1). Upon entry into their hosts, spores germinate and replicate as vegetative bacilli (1). The formation of a thick capsule encasing vegetative forms enables bacilli to escape granulocyte0 and macrophage-mediated phagocytosis, and the pathogen eventually disseminates throughout all tissues of an infected host (2, 3). Bacilli secrete lethal and edema toxins, which cause macrophage necrosis and precipitate anthrax death (4–7). The genes providing for toxin and capsule formation are carried on two large virulence plasmids, pXO1 and pXO2, respectively (8, 9). Loss of any one plasmid leads to virulence attenuation, a feature that has been exploited for the generation of vaccine-type strains (10–14).Unlike polysaccharide-based capsules that are commonly found in bacterial pathogens, the capsular material of B. anthracis is composed of poly-γ-d-glutamic acid (PDGA)³ (3). All the genes necessary for capsule biogenesis are located in the capBCADE gene cluster on plasmid pXO2 (15–19). CapD is the only protein of this cluster that is located on the bacterial surface (16). CapD shares sequence similarity with bacterial and mammalian γ-glutamyl transpeptidases (GGTs; EC 2.3.2.2) (17). GGTs belong to the N-terminal nucleophile hydrolases (Ntn) family (Protein Structure Classification (Class (C), Architecture (A), Topology (T) and Homologous superfamily (H)) (CATH) id 3.60.60.10) (20). These enzymes assemble as a single polypeptide chain and acquire activity by undergoing autocatalytic processing to heterodimer.Bacterial GGTs catalyze the first step in glutathione degradation. For example, Helicobacter pylori GGT removes glutamate from glutathione tripeptide via the formation of a γ-glutamyl acyl enzyme. This intermediate is resolved by the nucleophilic attack of a water molecule, causing the release of γ-glutamate (21, 22). Mammalian enzymes transfer the γ-glutamyl intermediate to the amino group of a peptide, thereby completing a transpeptidation reaction (23). The B. anthracis CapD precursor is also programmed for autocatalytic cleavage (17). Similar to mammalian GGTs, CapD also catalyzes a transpeptidation reaction; however, this reaction promotes the covalent linkage of PDGA to the bacterial envelope (16, 24). We have recently demonstrated the cell wall anchor structure of capsule filaments in the envelope of B. anthracis, identifying an amide bond between the terminal carboxyl group of PDGA and the side amino group of m-diaminopimelic acid cross-bridges within muropeptides (24). The CapD-catalyzed transpeptidation reaction could be recapitulated in vitro using purified recombinant CapD, γ-d-Glu_n peptide, and muropeptide substrates (24). In the absence of the physiological nucleophile (muropeptides), CapD acyl intermediates can be resolved by the nucleophilic attack of water to generate hydrolysis products.Here, we report the high resolution crystal structure of CapD in the absence and presence of a glutamate dipeptide and compare it with the known structures of H. pylori and Escherichia coli GGTs. By combining structural, genetic, and biochemical approaches, we identify the unique features of CapD that distinguish the protein from GGTs and detect several residues that are important for CapD autocatalytic cleavage and PDGA processing. This structural information will further the development of small molecule inhibitors that disrupt CapD activity and that may be useful as anti-infective therapies for anthrax.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes

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-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, 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. While the germ cell wall forms the initial cell wall during vegetative outgrowth, the spore cortex serves primarily to maintain the relatively dehydrated status of the spore protoplast during dormancy (13). Dissolution of the cortex permits complete hydration of the spore core and resumption of vegetative metabolism, leading ultimately to shedding of the spore coat and the emergence of a new vegetative cell (34).A number of studies have indicated that spores of various Bacillus species employ two cortex-lytic enzymes (CLEs), SleB and CwlJ, to initiate hydrolysis of the cortex during stage II of the germination process (16, 19, 32). These enzymes are semiredundant; hence, strains bearing null mutations in either structural gene can still degrade the cortex sufficiently to complete germination, whereas double mutant strains do not appear capable of degrading the cortex at all, resulting typically in a decrease of several orders of magnitude in colony-forming ability (15, 19, 32). Other enzymes, including Bacillus cereus/Bacillus anthracis SleL, are also involved in stage II of germination, apparently hydrolyzing peptidoglycan products of SleB and/or CwlJ to smaller peptidoglycan fragments that can more easily permeate through the spore coats to the surrounding germination medium (21).Studies with SleB and SleL purified from dormant and germinating spores indicate that whereas the latter enzyme degrades only cortical fragments of peptidoglycan (7), SleB has a requirement for intact peptidoglycan that has adopted the precise architecture present within the spore (12, 22). These substrate requirements appear to be important in maintenance of the respective autolysins, which are present in the spore in a mature form, in an inactive state during dormancy. Additionally, whereas the molecular mechanism of activation of SleB remains unclear—a change in cortical stress/architecture induced by stage I events has been hypothesized (12)—the efflux of Ca-DPA from the spore core to the cortex/coat boundary where CwlJ is localized (5) appears to be the mechanism by which this CLE is activated. CwlJ can also be activated by high concentrations of exogenous Ca-DPA, presenting an alternative germination pathway that bypasses the germinant receptors (27).The hydrolytic bond specificity of various CLEs has been examined by both direct and indirect biochemical means. Direct assays are typically conducted by incubation of purified or recombinant enzymes with peptidoglycan fragments or suspensions of spores in which the cortex is rendered accessible by first chemically compromising the permeability of the spore coats (7, 12, 22). Subsequent assays for the generation of reducing groups and/or free amino groups can yield information on the probable hydrolytic bond specificity of the respective enzyme(s) being assayed.More recently, the high-performance liquid chromatography/mass spectrometry (HPLC/MS)-based muropeptide analysis technique has been applied to characterize CLE activity during germination of various spore-forming species (2, 4, 10). This methodology has the resolution to reveal fine structural changes that occur to the peptidoglycan in vivo during germination, and when used in combination with CLE null mutant strains, it can be used to indirectly correlate the generation of certain classes of muropeptides, and therefore the hydrolytic bond specificity, with defined CLEs. Muropeptide analysis has revealed, for example, that an intact copy of the sleB gene in B. subtilis and B. anthracis is required for the presence of anhydromuropeptides in the germination exudates of these respective species, indicating that SleB is a lytic transglycosylase or generates substrate for subsequent lytic transglycosylase activity (6, 16). Conversely, B. cereus SleB was characterized as a probable amidase after enzyme purified from germinating spores was found to liberate a large amount of free amino groups when incubated with coat-stripped spores as a substrate (22). The hydrolytic bond specificity of SleB therefore remains ambiguous and perhaps varies between different species.Contrary to these observations, the overall structural architecture of SleB appears to be well conserved between different Bacillus species. Alignment of the primary amino acid sequence from different species indicates that the mature protein comprises an N-terminal domain that is connected to the C-terminal domain by a linker region that is variable in length and amino acid composition (Fig. ​(Fig.1).1). The N-terminal domain is thought to comprise the peptidoglycan binding domain by virtue of two direct sequence repeats that are reminiscent of cell wall-binding motifs observed in other proteins (26). The C-terminal domain shows homology with that of the other major Bacillus CLE, CwlJ, which lacks a corresponding peptidoglycan binding domain and is therefore thought to comprise the catalytic domain (19), although there is as yet no experimental evidence to substantiate this idea.Open in a separate windowFIG. 1.ClustalW alignment of SleB from various Bacillus species. Residues predicted to comprise putative structural domains are denoted. Stars indicate charged residues that were subjected to amino acid substitution in this work. BM, B. megaterium QM B1551; BC, B. cereus W; BCl, B. clausii KSM-K16; BS, B. subtilis 168.In the current study, we have investigated the molecular function of SleB during germination of Bacillus megaterium QM B1551 spores, employing engineered SleB N- and C-terminal deletion strains, site-directed mutagenesis (SDM), and muropeptide analyses. In addition to revealing several cortex-modifying activities during germination of this species, the presented data indicate that while the presence of either the N- or C-terminal domain of SleB is sufficient for the generation of anhydromuropeptides during germination, this may be an indirect effect, and at least a degree of lytic transglycosylase activity may result from the activity of another as yet unidentified enzyme.