Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis.

Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore. The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized. The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores. This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure. Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane. Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B. subtilis chromosome. This is a region where there are no other known spo, ger, or PBP genes. In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis. In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5*. The two PBPs could be distinguished by immunoassays.     

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.     

Restoration of vegetative penicillin-binding proteins during germination and outgrowth of Bacillus subtilis spores: relationship of individual proteins to specific cell cycle events.

The order in which the vegetative penicillin-binding proteins (PBPs) are first synthesized and the rate of their return to normal levels during germination and outgrowth of Bacillus subtilis spores were determined. The rate of synthesis of most of the PBPs was much faster than that of the majority of other membrane proteins, which is consistent with the involvement of PBPs in biosynthesis of the rapidly expanding peptidoglycan. The pattern of PBP changes that occurred during the cell cycle, including sporulation, suggests a likely role for PBP 2A in cell elongation and a unique requirement for PBP 2B during both symmetric and asymmetric septum formation. PBP 3 is the only PBP that appears to be equally necessary for vegetative and cortical peptidoglycan synthesis.     

Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.     

Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.     

A mother cell-specific class B penicillin-binding protein, PBP4b, in Bacillus subtilis

The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing sigma(E). Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.     

Germination-specific cortex-lytic enzyme is activated during triggering of Bacillus megaterium KM spore germination

Antibodies were raised against purified germination-specific cortex-lytic enzyme (GSLE) from spores of Bacillus megaterium KM which neutralized the ability of GSLE to germinate permeabilized spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE demonstrated that GSLE is spore-specific and that greater than 90% of the GSLE is associated with the dormant spore cortex peptidoglycan as a phosphorylated 63kD pro-form, which could only be visualized after lysozyme digestion of the peptidoglycan. During germination, the 63kD pro-form of GSLE is processed to release the active enzyme, which had an apparent molecular weight of 30kD. Inhibitor studies demonstrated that GSLE activation occurs as part of the commitment reaction and thus represents the first-identified enzymatic event to occur during germination triggering. Proteins that cross-react with anti-GSLE sera are present in spore fractions of other species.     

Stability and synthesis of the penicillin-binding proteins during sporulation

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined after incubation of vegetative and sporulating cultures with chloramphenicol, an inhibitor of protein synthesis. The results indicate that the sporulation-specific increases in vegetative PBPs 2B and 3 and the appearance of two new PBPs, 4* and 5*, depend on concurrent protein synthesis, which is most likely to be de novo synthesis of the PBPs rather than synthesis of an activator or processing enzyme. It was also learned that in vivo the PBPs differ in their individual stabilities, which helps to explain some of the quantitative changes that occur in the PBP profile during sporulation. All the membrane-bound PBPs, except possibly PBP 1, were found to be stable in the presence of crude extracts of sporulating cells that contained proteolytic activity.     

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.     

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.     

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function. Received: 18 March 1998 / Accepted: 26 May 1998     

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.     

Ultrastructural studies of sporulation in Bacillus sphaericus.

Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.     

Location of peptidoglycan lytic enzymes in Bacillus sphaericus.

The level of three peptidoglycan hydrolases was determined in the mother cell compartment and forespores of Bacillus sphaericus. Vegetative and sporulating cells contained in LD-carboxypeptidase active only on the vegetative cell wall peptidoglycan, and we have previously shown that sporulation is accompanied by the production of two new enzymes active only on the spore cortex peptidoglycan. These gamma-D-glutamyl-meso-diaminopimelate endopeptidase and a meso-diaminopimelate-D-alanine dipeptidase. The LD-carboxypeptidase activity appeared to be located in the membranes of both the mother cells and forespores. Endopeptidase activity was located in the integument fraction of the forespores, and the dipeptidase activity was only found in the forespore cytoplasm. These different locations comply with the probable different functions of these enzymes.     

Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis.

Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.     

Transpeptidase activity of penicillin‐binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation‐specific class B penicillin‐binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain – one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation‐specific thiol‐disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra‐cytoplasmic protein disulfides in bacterial cells.     

The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.     

Influence of cations on lysozyme-induced germination of coatless spores of Clostridium perfringens 8-6

Bacterial endospore germination is powerfully influenced by inorganic salts, cations having especially important effects. Spores of Clostridium perfringens 8-6 are unusual in lacking a spore coat; these spores germinate only in the presence of lysozyme, which readily digests the exposed cortex. Lysozyme-induced germination showed the same response to ionic strength and valence of cations as does lysozyme hydrolysis of peptidoglycan, and close parallels are evident in the influence of inorganic cations on germination of normal spores. La3+ and transition element cations inhibited lysozyme-induced germination at low concentration, again demonstrating parallels with their action on lysozyme digestion of peptidoglycan and on the germination of normal spores. The poly-cations poly(L-lysine) and Ruthenium Red inhibited at extremely low concentrations. Mn2+ and Co2+, at appropriately low concentrations, stimulated lysozyme germination of 8-6 spores and also lysis of Micrococcus lysodeikticus.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.