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. 1996 Nov;178(22):6451–6458. doi: 10.1128/jb.178.22.6451-6458.1996

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

D L Popham 1, J Helin 1, C E Costello 1, P Setlow 1
PMCID: PMC178530  PMID: 8932300

Abstract

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.

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Selected References

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  1. Beaman T. C., Gerhardt P. Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl Environ Microbiol. 1986 Dec;52(6):1242–1246. doi: 10.1128/aem.52.6.1242-1246.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blumberg P. M., Strominger J. L. Five penicillin-binding components occur in Bacillus subtilis membranes. J Biol Chem. 1972 Dec 25;247(24):8107–8113. [PubMed] [Google Scholar]
  3. Buchanan C. E., Gustafson A. Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis. J Bacteriol. 1992 Aug;174(16):5430–5435. doi: 10.1128/jb.174.16.5430-5435.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buchanan C. E., Ling M. L. Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis. J Bacteriol. 1992 Mar;174(6):1717–1725. doi: 10.1128/jb.174.6.1717-1725.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Glauner B. Separation and quantification of muropeptides with high-performance liquid chromatography. Anal Biochem. 1988 Aug 1;172(2):451–464. doi: 10.1016/0003-2697(88)90468-x. [DOI] [PubMed] [Google Scholar]
  6. Hayashi K. A rapid determination of sodium dodecyl sulfate with methylene blue. Anal Biochem. 1975 Aug;67(2):503–506. doi: 10.1016/0003-2697(75)90324-3. [DOI] [PubMed] [Google Scholar]
  7. Kunst F., Devine K. The project of sequencing the entire Bacillus subtilis genome. Res Microbiol. 1991 Sep-Oct;142(7-8):905–912. doi: 10.1016/0923-2508(91)90072-i. [DOI] [PubMed] [Google Scholar]
  8. Lawrence P. J., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XVI. The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis. J Biol Chem. 1970 Jul 25;245(14):3660–3666. [PubMed] [Google Scholar]
  9. Leighton T. J., Doi R. H. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem. 1971 May 25;246(10):3189–3195. [PubMed] [Google Scholar]
  10. Lewis J. C., Snell N. S., Burr H. K. Water Permeability of Bacterial Spores and the Concept of a Contractile Cortex. Science. 1960 Aug 26;132(3426):544–545. doi: 10.1126/science.132.3426.544. [DOI] [PubMed] [Google Scholar]
  11. Nakashio S., Gerhardt P. Protoplast dehydration correlated with heat resistance of bacterial spores. J Bacteriol. 1985 May;162(2):571–578. doi: 10.1128/jb.162.2.571-578.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ou L. T., Marquis R. E. Electromechanical interactions in cell walls of gram-positive cocci. J Bacteriol. 1970 Jan;101(1):92–101. doi: 10.1128/jb.101.1.92-101.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Popham D. L., Illades-Aguiar B., Setlow P. The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration. J Bacteriol. 1995 Aug;177(16):4721–4729. doi: 10.1128/jb.177.16.4721-4729.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Popham D. L., Sengupta S., Setlow P. Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins. Appl Environ Microbiol. 1995 Oct;61(10):3633–3638. doi: 10.1128/aem.61.10.3633-3638.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Popham D. L., Setlow P. Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor. J Bacteriol. 1995 Jan;177(2):326–335. doi: 10.1128/jb.177.2.326-335.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Popham D. L., Setlow P. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase. J Bacteriol. 1993 May;175(10):2917–2925. doi: 10.1128/jb.175.10.2917-2925.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Popham D. L., Setlow P. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein. J Bacteriol. 1993 Aug;175(15):4870–4876. doi: 10.1128/jb.175.15.4870-4876.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Popham D. L., Setlow P. Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4. J Bacteriol. 1994 Dec;176(23):7197–7205. doi: 10.1128/jb.176.23.7197-7205.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Popham D. L., Setlow P. The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked. J Bacteriol. 1993 May;175(9):2767–2769. doi: 10.1128/jb.175.9.2767-2769.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roten C. A., Pagni M., Margot P., Touri F., Karamata D. Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index. Anal Biochem. 1994 Dec;223(2):208–211. doi: 10.1006/abio.1994.1575. [DOI] [PubMed] [Google Scholar]
  21. Sowell M. O., Buchanan C. E. Changes in penicillin-binding proteins during sporulation of Bacillus subtilis. J Bacteriol. 1983 Mar;153(3):1331–1337. doi: 10.1128/jb.153.3.1331-1337.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tipper D. J., Linnett P. E. Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus. J Bacteriol. 1976 Apr;126(1):213–221. doi: 10.1128/jb.126.1.213-221.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Todd J. A., Roberts A. N., Johnstone K., Piggot P. J., Winter G., Ellar D. J. Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5. J Bacteriol. 1986 Jul;167(1):257–264. doi: 10.1128/jb.167.1.257-264.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Warth A. D., Strominger J. L. Structure of the peptidoglycan from spores of Bacillus subtilis. Biochemistry. 1972 Apr 11;11(8):1389–1396. doi: 10.1021/bi00758a010. [DOI] [PubMed] [Google Scholar]
  25. Warth A. D., Strominger J. L. Structure of the peptidoglycan from vegetative cell walls of Bacillus subtilis. Biochemistry. 1971 Nov 23;10(24):4349–4358. doi: 10.1021/bi00800a001. [DOI] [PubMed] [Google Scholar]
  26. Warth A. D., Strominger J. L. Structure of the peptidoglycan of bacterial spores: occurrence of the lactam of muramic acid. Proc Natl Acad Sci U S A. 1969 Oct;64(2):528–535. doi: 10.1073/pnas.64.2.528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu J. J., Schuch R., Piggot P. J. Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase sigma factor and for a putative DD-carboxypeptidase. J Bacteriol. 1992 Aug;174(15):4885–4892. doi: 10.1128/jb.174.15.4885-4892.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. de Jonge B. L., Chang Y. S., Gage D., Tomasz A. Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A. J Biol Chem. 1992 Jun 5;267(16):11248–11254. [PubMed] [Google Scholar]

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