Characterization of LytH,a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation

The cortex peptidoglycan from endospores of Bacillus subtilis is responsible for the maintenance of dormancy. LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination. The lytH gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor sigma(K). Spores of a lytH mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy. Analysis of the peptidoglycan structure of lytH mutant spores shows the loss of muramic acid residues substituted with L-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain. In a lytH cwlD mutant, the lack of muramic acid residues substituted with L-alanine and delta-lactam leaves 97% of residues substituted with tetrapeptide. These results suggest that lytH encodes an L-Ala-D-Glu peptidase involved in production of single L-alanine side chains from tetrapeptides in the spore cortex. The lack of di- or tripeptides in a lytH mutant reveals the enzyme is an endopeptidase.     

Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan

Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic delta-lactam. Production of muramic delta-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic delta-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic delta-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic delta-lactam. CwlD and PdaA are necessary and sufficient for muramic delta-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.     

The role of peptidoglycan structure and structural dynamics during endospore dormancy and germination

Dormant, bacterial endospores are the most resistant living structures known. The spore cell wall (cortex) maintains dormancy, core dehydration, and heat resistance. The cortex peptidoglycan has a unique, spore specific structure that enables it to fulfill its role. The cross-linking index of spore cortex peptidoglycan is very low, occurring at only 2.9% of the muramic acid residues compared to 33% in vegetative cells. The level of cross-linking of the cortex may be important in maintaining spore dormancy and heat resistance. Approximately 50% of the muramic acid residues in spore cortex are substituted with muramic -lactam. This modification is spore specific and is the major characteristic feature of the cortex. The muramic -lactam has no apparent role in establishing core dehydration, maintaining dormancy or heat resistance. However, the muramic -lactam residues are necessary for spore cortex hydrolysis during germination. They constitute part of the substrate recognition profile of the germination specific lytic enzymes (GSLEs) which are responsible for cortex hydrolysis.Germination results in loss of dormant spore properties and hydrolysis of the cortex is essential for later germination events and outgrowth. Application of muropeptide analysis to determine peptidoglycan structural dynamics during germination has revealed an unexpected degree of complexity in peptidoglycan hydrolysis. At least three hydrolytic activities, an N-acetyl glucosaminidase, a lytic transglycosylase and a possible amidase, are involved. A non-hydrolytic acitivity, likely to be an epimerase of muramic acid also occurs early during germination.The lytic transglycosylase generates anhydro-muropeptides which are released during germination and may be recycled during outgrowth to form part of the new vegetative cell wall.     

Subcellular localization of a germiantion-specific cortex-lytic enzyme, SleB, of Bacilli during sporulation

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.     

Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria.

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.     

Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM.

Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM. Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores. Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species. The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5. It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss. The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed.     

Two class A high-molecular-weight penicillin-binding proteins of Bacillus subtilis play redundant roles in sporulation.

The four class A penicillin-binding proteins (PBPs) of Bacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamed pbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic delta-lactam and L-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.     

Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.

The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.     

A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.     

Envelope structure in three different L-forms ofProteus mirabilis

One A-type, stable and two different B-type, unstable L-forms were obtained from a strain ofProteus mirabilis and studied by electron microscopy and by chemical analysis for the presence of peptidoglycan. The wall of the parent bacterium is characterized by a profile of three superimposed dense lines and a content of 11.07 nmoles of muramic acid (MUR) and of 7.85 nmoles of diaminopimelic acid (DAP) per mg of dry weight. The stable, A-type L-form has completely lost the cell wall of the bacterium and is enveloped only by the plasma membrane to which very small quantities of peptidoglycan components are associated (MUR: 0.041 nmoles/mg; DAP: 0.075 nmoles/mg). The two B-type, unstable L-forms have the same wall structure in only two dense lines, but they differ in their peptidoglycan content. The first one does not contain more peptidoglycan components than the A-type, L-form (MUR: 0.022 nmoles/mg; DAP: 0.016 nmoles/mg), whereas the peptidoglycan content of the second one (MUR: 2.6 nmoles/mg; DAP: 1.65 nmoles/mg) is about one fifth of the content of muramic acid and diaminopimelic acid of the bacterial cell wall.     

Separation and characterization of an autolytic endo-beta-glucosaminidase from Bacillus cereus

1. An autolytic endo-beta-glucosaminidase, capable of cleaving the glycoside linkages of N-unsubstituted glucosamine in the glycan moiety of cell wall peptidoglycan, was purified 470-fold from a salt extract of the 2,000 x g precipitate fraction obtained after sonication of a lysozyme-resistant strain of Bacillus cereus. The properties of this enzyme were studied. 2. The purified enzyme preparation was also active towards the glycan chain of fully N-acetylated cell wall peptidoglycan. 3. The endo-beta-glucosaminidase was inactive towards the cell wall peptidoglycan unless the peptide portion of this polymer was removed either by the action of N-acetylmuramyl-L-alanine amidase or by the treatment with alkali in aqueous dimethyl sulfoxide. 4. Studies on the action of this enzyme towards chemically modified glycans revealed that the carboxyl groups of muramic acid residues are indispensable to a substrate for this enzyme.     

A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL

The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330-5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.     

Effects of cortex peptidoglycan structure and cortex hydrolysis on the kinetics of Ca(2+)-dipicolinic acid release during Bacillus subtilis spore germination

The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-δ-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs.     

Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species

AIMS: The aim of this work was to compare the chemical structure of the spore cortex of a range of species, and to determine any correlation between cortex structure and spore resistance properties. METHODS AND RESULTS: The fine chemical structure of the cortex of Bacillus subtilis, Bacillus megaterium, Bacillus cereus and Clostridium botulinum was examined by muropeptide analysis using reverse phase HPLC. There is a conserved basic structure between peptidoglycan of these species, with the only difference being the level of de-N-acetylation of an amino sugar. In order to determine if an alteration in cortex structure correlates with heat resistance properties, the peptidoglycan structure and properties of B. subtilis spores prepared under different conditions were compared. Peptidoglycan from spores prepared in Nutrient Broth (NB) showed reduction in single L-alanine substituted muramic acid to only 13.9% compared with 20.6% in CCY-grown spores. NB-prepared spores are also unstable, with 161-fold less heat resistance (60 min, 85 degrees C) and 43 times less Mn(2+) content than CCY-grown spores. Addition of MnCl(2) to NB led to a peptidoglycan profile similar to CCY-grown spores, sevenfold more heat resistance (60 min, 85 degrees C) and an 86-fold increase in Mn(2+) content. Addition of CCY salts to NB led all parameters to be comparable with CCY-grown spore levels. CONCLUSION: It has been shown that peptidoglycan structure is conserved in four spore-forming bacteria. Also, spore heat resistance is multifactorial and cannot be accounted for by any single parameter. SIGNIFICANCE AND IMPACT OF THE STUDY: Endospores made by diverse species most likely have common mechanisms of heat resistance. However, the molecular basis for their resistance remains elusive.     

Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars.

A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.     

Chemical structure of the peptidoglycan of Vibrio parahaemolyticus A55 with special reference to the extent of interpeptide cross-linking.

The chemical structure of the cell wall peptidoglycan of Vibrio parahaemolyticus A55 was studied. Estimation of cross linkages between peptide subunits in the peptidoglycan by dinitrophenylation showed that about 30% of the total 2,6-diaminopimelic acid (A2pm) residues were involved in cross linkages. The presence of interpeptide bridges was also demonstrated by isolating bisdisaccharide peptide subunit dimers from Chalaropsis muramidase digests of the cell wall peptidoglycan by gel filtration followed by ion-exchange column chromatography, although most of the building blocks obtained were uncross-linked disaccharide peptide monomers. The chain length of a glycan moiety of the peptidoglycan obtained by treatment with the L-11 enzyme and gel filtration of the digest was also studied. The chain length varied from 7 to 44, but 30% of the glycan fragments had muramic acid at the reducing end and a chain length of 28 to 44. In conformity with the above structural study it was demonstrated that a particulate enzyme fraction obtained by differential centrifugation of a sonicated preparation of V. parahaemolyticus catalyzed a penicillin-sensitive transpeptidation reaction, using UDP-MurNAc-14C-pentapeptide and UDP-GlcNAc as substrates.     

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.     

Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.