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. 1995 Oct;177(19):5582–5589. doi: 10.1128/jb.177.19.5582-5589.1995

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

J Sekiguchi 1, K Akeo 1, H Yamamoto 1, F K Khasanov 1, J C Alonso 1, A Kuroda 1
PMCID: PMC177368  PMID: 7559346

Abstract

DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.

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Selected References

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  1. Alonso J. C., Stiege A. C., Lüder G. Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination. Mol Gen Genet. 1993 May;239(1-2):129–136. doi: 10.1007/BF00281611. [DOI] [PubMed] [Google Scholar]
  2. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  4. Foster S. J. Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins. J Gen Microbiol. 1993 Dec;139(12):3177–3184. doi: 10.1099/00221287-139-12-3177. [DOI] [PubMed] [Google Scholar]
  5. Foster S. J. Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis. J Bacteriol. 1992 Jan;174(2):464–470. doi: 10.1128/jb.174.2.464-470.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foster S. J. Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2. J Gen Microbiol. 1991 Aug;137(8):1987–1998. doi: 10.1099/00221287-137-8-1987. [DOI] [PubMed] [Google Scholar]
  7. Foster S. J., Johnstone K. Germination-specific cortex-lytic enzyme is activated during triggering of Bacillus megaterium KM spore germination. Mol Microbiol. 1988 Nov;2(6):727–733. doi: 10.1111/j.1365-2958.1988.tb00083.x. [DOI] [PubMed] [Google Scholar]
  8. Foster S. J., Johnstone K. Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM. Biochem J. 1987 Mar 1;242(2):573–579. doi: 10.1042/bj2420573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gavrilova E. V., Khasanov F. K., Prozorov A. A. Poluchenie Rec-mutantov Bacillus subtilis s pomoshch'iu insertsionnogo mutageneza. Genetika. 1991 Feb;27(2):222–228. [PubMed] [Google Scholar]
  10. Gavrilova E. V., Mekhedov S. L., Prozorov A. A., Khasanov F. K. Gen rec223 Bacillus subtilis: molekuliarnoe klonirovanie i predpolagaemye funktsii ego belkovogo produkta. Genetika. 1992 May;28(5):29–39. [PubMed] [Google Scholar]
  11. Guinand M., Michel G., Balassa G. Lytic enzymes in sporulating Bacillus subtilis. Biochem Biophys Res Commun. 1976 Feb 23;68(4):1287–1293. doi: 10.1016/0006-291x(76)90336-3. [DOI] [PubMed] [Google Scholar]
  12. Herbold D. R., Glaser L. Bacillus subtilis N-acetylmuramic acid L-alanine amidase. J Biol Chem. 1975 Mar 10;250(5):1676–1682. [PubMed] [Google Scholar]
  13. Illades-Aguiar B., Setlow P. Studies of the processing of the protease which initiates degradation of small, acid-soluble proteins during germination of spores of Bacillus species. J Bacteriol. 1994 May;176(10):2788–2795. doi: 10.1128/jb.176.10.2788-2795.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karmazyn-Campelli C., Bonamy C., Savelli B., Stragier P. Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis. Genes Dev. 1989 Feb;3(2):150–157. doi: 10.1101/gad.3.2.150. [DOI] [PubMed] [Google Scholar]
  15. Kuroda A., Asami Y., Sekiguchi J. Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis. J Bacteriol. 1993 Oct;175(19):6260–6268. doi: 10.1128/jb.175.19.6260-6268.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuroda A., Rashid M. H., Sekiguchi J. Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product. J Gen Microbiol. 1992 Jun;138(6):1067–1076. doi: 10.1099/00221287-138-6-1067. [DOI] [PubMed] [Google Scholar]
  17. Kuroda A., Sekiguchi J. Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases. FEMS Microbiol Lett. 1992 Aug 1;74(1):109–113. doi: 10.1016/0378-1097(92)90745-a. [DOI] [PubMed] [Google Scholar]
  18. Kuroda A., Sekiguchi J. Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. J Gen Microbiol. 1990 Nov;136(11):2209–2216. doi: 10.1099/00221287-136-11-2209. [DOI] [PubMed] [Google Scholar]
  19. Kuroda A., Sekiguchi J. High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation. J Bacteriol. 1993 Feb;175(3):795–801. doi: 10.1128/jb.175.3.795-801.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuroda A., Sekiguchi J. Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. J Bacteriol. 1991 Nov;173(22):7304–7312. doi: 10.1128/jb.173.22.7304-7312.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuroda A., Sugimoto Y., Funahashi T., Sekiguchi J. Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase. Mol Gen Genet. 1992 Jul;234(1):129–137. doi: 10.1007/BF00272354. [DOI] [PubMed] [Google Scholar]
  22. Lazarevic V., Margot P., Soldo B., Karamata D. Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. J Gen Microbiol. 1992 Sep;138(9):1949–1961. doi: 10.1099/00221287-138-9-1949. [DOI] [PubMed] [Google Scholar]
  23. Longchamp P. F., Mauël C., Karamata D. Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX. Microbiology. 1994 Aug;140(Pt 8):1855–1867. doi: 10.1099/13500872-140-8-1855. [DOI] [PubMed] [Google Scholar]
  24. Makino S., Ito N., Inoue T., Miyata S., Moriyama R. A spore-lytic enzyme released from Bacillus cereus spores during germination. Microbiology. 1994 Jun;140(Pt 6):1403–1410. doi: 10.1099/00221287-140-6-1403. [DOI] [PubMed] [Google Scholar]
  25. Margot P., Mauël C., Karamata D. The gene of the N-acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis. Mol Microbiol. 1994 May;12(4):535–545. doi: 10.1111/j.1365-2958.1994.tb01040.x. [DOI] [PubMed] [Google Scholar]
  26. Miki T., Yasukochi T., Nagatani H., Furuno M., Orita T., Yamada H., Imoto T., Horiuchi T. Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme. Protein Eng. 1987 Aug-Sep;1(4):327–332. doi: 10.1093/protein/1.4.327. [DOI] [PubMed] [Google Scholar]
  27. Nicholson W. L., Sun D. X., Setlow B., Setlow P. Promoter specificity of sigma G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters. J Bacteriol. 1989 May;171(5):2708–2718. doi: 10.1128/jb.171.5.2708-2718.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oda Y., Nakayama R., Kuroda A., Sekiguchi J. Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis. Mol Gen Genet. 1993 Nov;241(3-4):380–388. doi: 10.1007/BF00284691. [DOI] [PubMed] [Google Scholar]
  29. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  30. Parquet C., Flouret B., Leduc M., Hirota Y., van Heijenoort J. N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12. Possible physiological functions. Eur J Biochem. 1983 Jun 15;133(2):371–377. doi: 10.1111/j.1432-1033.1983.tb07472.x. [DOI] [PubMed] [Google Scholar]
  31. Potvin C., Leclerc D., Tremblay G., Asselin A., Bellemare G. Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Mol Gen Genet. 1988 Oct;214(2):241–248. doi: 10.1007/BF00337717. [DOI] [PubMed] [Google Scholar]
  32. Rashid M. H., Kuroda A., Sekiguchi J. Bacillus subtilis mutant deficient in the major autolytic amidase and glucosaminidase is impaired in motility. FEMS Microbiol Lett. 1993 Sep 1;112(2):135–140. doi: 10.1111/j.1574-6968.1993.tb06438.x. [DOI] [PubMed] [Google Scholar]
  33. Roels S., Driks A., Losick R. Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis. J Bacteriol. 1992 Jan;174(2):575–585. doi: 10.1128/jb.174.2.575-585.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schaeffer P., Millet J., Aubert J. P. Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A. 1965 Sep;54(3):704–711. doi: 10.1073/pnas.54.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thompson J. S., Shockman G. D. A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls. Anal Biochem. 1968 Feb;22(2):260–268. doi: 10.1016/0003-2697(68)90315-1. [DOI] [PubMed] [Google Scholar]
  36. Tomioka S., Nikaido T., Miyakawa T., Matsuhashi M. Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12. J Bacteriol. 1983 Oct;156(1):463–465. doi: 10.1128/jb.156.1.463-465.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Troup B., Jahn M., Hungerer C., Jahn D. Isolation of the hemF operon containing the gene for the Escherichia coli aerobic coproporphyrinogen III oxidase by in vivo complementation of a yeast HEM13 mutant. J Bacteriol. 1994 Feb;176(3):673–680. doi: 10.1128/jb.176.3.673-680.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tsui H. C., Zhao G., Feng G., Leung H. C., Winkler M. E. The mutL repair gene of Escherichia coli K-12 forms a superoperon with a gene encoding a new cell-wall amidase. Mol Microbiol. 1994 Jan;11(1):189–202. doi: 10.1111/j.1365-2958.1994.tb00300.x. [DOI] [PubMed] [Google Scholar]
  39. Xu K., Elliott T. An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium. J Bacteriol. 1993 Aug;175(16):4990–4999. doi: 10.1128/jb.175.16.4990-4999.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yon J. R., Sammons R. L., Smith D. A. Cloning and sequencing of the gerD gene of Bacillus subtilis. J Gen Microbiol. 1989 Dec;135(12):3431–3445. doi: 10.1099/00221287-135-12-3431. [DOI] [PubMed] [Google Scholar]
  41. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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