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.     

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.     

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.     

Specificity of cleavage by restriction nuclease from Bacillus subtilis.

The restriction nuclease from B. subtilis (Bsu) which cleaves in the middle of the tetra-nucleotide sequence 5'-GGCC-3' 3'-CCGG-5' has been found to decrease its substrate specificity at high nuclease concentrations. There are special conditions, high pH, low ionic strength, and high glycerol content, which strongly enhance splitting with decreased specificity and also lead to splitting of single-stranded DNA. By sequence analyses it is shown that the reduction in specificity of Bsu corresponds to cleavage predominantly at 5'-GC-3' 3'-CG-5' sequences. No comparable change in specificity has been observed in a restriction nuclease from Haemophilus aegyptius (HaeIII), and isoschizomer of Bsu.     

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.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Autolysis of Bacillus subtilis induced by monovalent cations

Summary Resting cell suspensions of seven Nocardia species catalyzed the production of 10-hydroxystearic acid from oleic acid. Nocardia cholesterolicum NRRL 5767 gave a good yield with optimum conditions at pH 6.5 and 40°C. Yields exceeding 90% can be obtained within 6 h with 0.1 g cells (dry weight) and 178 mg oleic acid in 10 ml of 0.05 M sodium phosphate buffer (pH 6.5). In addition, minor amounts of 10-ketostearic acid were formed as a by-product. The reaction proceeded via hydration of the double bond as shown by labeling experiments with deuterium oxide and ¹⁸O-labeled water. The system was specific for fatty acids with cis unsaturation at the 9 position.A part of this paper was presented at a poster session at the World Conference on Biotechnology for the Fats and Oils Industry, Hamburg, Federal Republic of Germany, September 1987, and at the 194th National American Chemical Society Meeting, New Orleans, September 1987RetiredThe mention of firm names or trade products does not imply that they are endorsed or recommended by USDA over other firms or similar products not mentioned     

Bacillus subtilis Lyt+ and Lyt- strains secrete peptidoglycan hydrolases

The phenotypes characteristic of the pleiotropic lytic-deficient Bacillus subtilis mutants have been attributed to reductions in N-acetyl-muramoyl-L-alanine amidase (EC 3.5.1.28) and endo-beta-N-acetyl-glucosaminidase (EC 3.2.1.30). It is reported here that these peptidoglycan hydrolases are secreted. The FJ3 (lyt-1), FJ6 (lyt-2), and Ni15 (lyt-15) mutants secreted the enzymes in amounts comparable to a Lyt+ strain. Thus, the Lyt- mutants appear not be as deficient in the enzymes' synthesis as their cell-bound activities indicated. Based on the levels of cell-bound and extracellular activities measured during growth, it is suggested that the Lyt- phenotype may be due to a deficiency of the enzymes' acceptor(s) in cell walls.     

Effect of decoyinine on peptidoglycan synthesis and turnover in Bacillus subtilis.

The sporulation of Bacillus subtilis can be induced in the presence of amino acids and glucose by partially depriving the cells of guanine nucleotides. This can be achieved, e.g., by the addition of decoyinine, a specific inhibitor of GMP synthetase. To determine the effect of this and other inhibitors on cell wall synthesis, we measured in their presence the incorporation of acetylglucosamine into acid-precipitable material. The rate of wall synthesis decreased by 50% within 5 min after decoyinine addition; this decrease was prevented by the presence of guanosine. A comparison with the effects of other inhibitors of cell wall synthesis indicated that decoyinine inhibited the final portion of the cell wall biosynthetic pathway, i.e., after the steps inhibited by bacitracin or vancomycin. Decoyinine addition also prevented cellular autolysis and cell wall turnover. It is not known whether these two effects of decoyinine on cell wall synthesis are causally related.     

Rates of peptidoglycan turnover and cell growth of Bacillus subtilis are correlated

Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium.     

Changes of lipid domains in Bacillus subtilis cells with disrupted cell wall peptidoglycan

The cell wall is responsible for cell integrity and the maintenance of cell shape in bacteria. The Gram-positive bacterial cell wall consists of a thick peptidoglycan layer located on the outside of the cytoplasmic membrane. Bacterial cell membranes, like eukaryotic cell membranes, are known to contain domains of specific lipid and protein composition. Recently, using the membrane-binding fluorescent dye FM4-64, helix-like lipid structures extending along the long axis of the cell and consisting of negatively charged phospholipids were detected in the rod-shaped bacterium Bacillus subtilis. It was also shown that the cardiolipin-specific dye, nonyl acridine orange (NAO), is preferentially distributed at the cell poles and in the septal regions in both Escherichia coli and B. subtilis. These results suggest that phosphatidylglycerol is the principal component of the observed spiral domains in B. subtilis. Here, using the fluorescent dyes FM4-64 and NAO, we examined whether these lipid domains are linked to the presence of cell wall peptidoglycan. We show that in protoplasted cells, devoid of the peptidoglycan layer, helix-like lipid structures are not preserved. Specific lipid domains are also missing in cells depleted of MurG, an enzyme involved in peptidoglycan synthesis, indicating a link between lipid domain formation and peptidoglycan synthesis.     

The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked.

Determination by amino acid analyses of the percentage of diaminopimelic acid in the spore cortex of Bacillus megaterium and Bacillus subtilis which is involved in interpeptide cross-links gave values of 31 to 37%. This finding supports the idea that the cortex volume could undergo significant changes in response to changes in pH or ionic strength and could thus play an active role in reducing the water content of the spore protoplast during sporulation.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.