Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 May;179(9):3021–3029. doi: 10.1128/jb.179.9.3021-3029.1997

Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A.

T Murray 1, D L Popham 1, P Setlow 1
PMCID: PMC179068  PMID: 9139922

Abstract

Amino acid sequence analysis of tryptic peptides derived from purified penicillin-binding protein PBP2a of Bacillus subtilis identified the coding gene (now termed pbpA) as yqgF, which had been sequenced as part of the B. subtilis genome project; pbpA encodes a 716-residue protein with sequence similarity to class B high-molecular-weight PBPs. Use of a pbpA-lacZ fusion showed that pbpA was expressed predominantly during vegetative growth, and the transcription start site was mapped using primer extension analysis. Insertional mutagenesis of pbpA resulted in no changes in the growth rate or morphology of vegetative cells, in the ability to produce heat-resistant spores, or in the ability to trigger spore germination when compared to the wild type. However, pbpA spores were unable to efficiently elongate into cylindrical cells and were delayed significantly in spore outgrowth. This provides evidence that PBP2a is involved in the synthesis of peptidoglycan associated with cell wall elongation in B. subtilis.

Full Text

The Full Text of this article is available as a PDF (768.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Antoniewski C., Savelli B., Stragier P. The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. J Bacteriol. 1990 Jan;172(1):86–93. doi: 10.1128/jb.172.1.86-93.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atrih A., Zöllner P., Allmaier G., Foster S. J. Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol. 1996 Nov;178(21):6173–6183. doi: 10.1128/jb.178.21.6173-6183.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1513–1515. doi: 10.1128/jb.154.3.1513-1515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghuysen J. M. Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol. 1991;45:37–67. doi: 10.1146/annurev.mi.45.100191.000345. [DOI] [PubMed] [Google Scholar]
  7. Imae Y., Strominger J. L. Relationship between cortex content and properties of Bacillus sphaericus spores. J Bacteriol. 1976 May;126(2):907–913. doi: 10.1128/jb.126.2.907-913.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ishino F., Matsuhashi M. Peptidoglycan synthetic enzyme activities of highly purified penicillin-binding protein 3 in Escherichia coli: a septum-forming reaction sequence. Biochem Biophys Res Commun. 1981 Aug 14;101(3):905–911. doi: 10.1016/0006-291x(81)91835-0. [DOI] [PubMed] [Google Scholar]
  9. Ishino F., Mitsui K., Tamaki S., Matsuhashi M. Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A. Biochem Biophys Res Commun. 1980 Nov 17;97(1):287–293. doi: 10.1016/s0006-291x(80)80166-5. [DOI] [PubMed] [Google Scholar]
  10. Ishino F., Tamaki S., Spratt B. G., Matsuhashi M. A mecillinam-sensitive peptidoglycan crosslinking reaction in Escherichia coli. Biochem Biophys Res Commun. 1982 Dec 15;109(3):689–696. doi: 10.1016/0006-291x(82)91995-7. [DOI] [PubMed] [Google Scholar]
  11. Kleppe G., Strominger J. L. Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis. J Biol Chem. 1979 Jun 10;254(11):4856–4862. [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Murray T., Popham D. L., Setlow P. Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3. J Bacteriol. 1996 Oct;178(20):6001–6005. doi: 10.1128/jb.178.20.6001-6005.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neyman S. L., Buchanan C. E. Restoration of vegetative penicillin-binding proteins during germination and outgrowth of Bacillus subtilis spores: relationship of individual proteins to specific cell cycle events. J Bacteriol. 1985 Jan;161(1):164–168. doi: 10.1128/jb.161.1.164-168.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Patel-King R. S., Benashki S. E., Harrison A., King S. M. Two functional thioredoxins containg redox-senesitive vicinal dithiols from the Chlamydomonas outer dynein arm. J Biol Chem. 1996 Mar 15;271(11):6283–6291. doi: 10.1074/jbc.271.11.6283. [DOI] [PubMed] [Google Scholar]
  17. Piras G., Raze D., el Kharroubi A., Hastir D., Englebert S., Coyette J., Ghuysen J. M. Cloning and sequencing of the low-affinity penicillin-binding protein 3r-encoding gene of Enterococcus hirae S185: modular design and structural organization of the protein. J Bacteriol. 1993 May;175(10):2844–2852. doi: 10.1128/jb.175.10.2844-2852.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Popham D. L., Helin J., Costello C. E., Setlow P. Analysis of the peptidoglycan structure of Bacillus subtilis endospores. J Bacteriol. 1996 Nov;178(22):6451–6458. doi: 10.1128/jb.178.22.6451-6458.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Popham D. L., Helin J., Costello C. E., Setlow P. Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15405–15410. doi: 10.1073/pnas.93.26.15405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Popham D. L., Setlow P. Phenotypes of Bacillus subtilis mutants lacking multiple class A high-molecular-weight penicillin-binding proteins. J Bacteriol. 1996 Apr;178(7):2079–2085. doi: 10.1128/jb.178.7.2079-2085.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Setlow B., Hand A. R., Setlow P. Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA. J Bacteriol. 1991 Mar;173(5):1642–1653. doi: 10.1128/jb.173.5.1642-1653.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Spratt B. G. Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sun D. X., Cabrera-Martinez R. M., Setlow P. Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G. J Bacteriol. 1991 May;173(9):2977–2984. doi: 10.1128/jb.173.9.2977-2984.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Suzuki H., van Heijenoort Y., Tamura T., Mizoguchi J., Hirota Y., van Heijenoort J. In vitro peptidoglycan polymerization catalysed by penicillin binding protein 1b of Escherichia coli K-12. FEBS Lett. 1980 Feb 11;110(2):245–249. doi: 10.1016/0014-5793(80)80083-4. [DOI] [PubMed] [Google Scholar]
  33. Todd J. A., Bone E. J., Ellar D. J. The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro. Biochem J. 1985 Sep 15;230(3):825–828. doi: 10.1042/bj2300825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Yanouri A., Daniel R. A., Errington J., Buchanan C. E. Cloning and sequencing of the cell division gene pbpB, which encodes penicillin-binding protein 2B in Bacillus subtilis. J Bacteriol. 1993 Dec;175(23):7604–7616. doi: 10.1128/jb.175.23.7604-7616.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES