1887

Abstract

The regulatory unit of strain 168 encompassing the structural genes of the -acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons and . Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of . LytR is a 35 kDa protein which acts as an attenuator of the expression of both and operons. Transcription of the operon proceeds from two promoters: P, identified as P (Gilman ., 1984), and an upstream P. The former only is subject to LytR attenuation. Translational initiation of and is directed by UUG start codons, suggesting that and undergo coupled translation. Transcription of is initiated at two start sites, one of which corresponds to a highly intense P promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the operon is discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-9-1949
1992-09-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/9/mic-138-9-1949.html?itemId=/content/journal/micro/10.1099/00221287-138-9-1949&mimeType=html&fmt=ahah

References

  1. Adhin M. R., van Duin J. 1989; Translation regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon. Molecular and General Genetics 218:137–142
    [Google Scholar]
  2. Albertini A. M., Caramori T., Crabb W. D., Scoffone F., Galizzi A. 1991; The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide. Journal of Bacteriology 173:3573–3579
    [Google Scholar]
  3. Bartlett D. H., Frantz B. B., Matsumura P. 1988; Flagellar transcriptional activators FlbB and Flal: gene sequences and 5’ consensus sequences of operons under FlbB and Flal control. Journal of Bacteriology 170:1575–1581
    [Google Scholar]
  4. Beliveau C., Potvin C., Trudel J., Asselin A., Bellemare G. 1991; Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin. Journal of Bacteriology 173:5619–5623
    [Google Scholar]
  5. Birchmeier C., Birnbaum D., Waitches G., Fasano O., Wigler M. 1986; Characterization of an activated human ros gene. Molecular and Cellular Biology 6:3109–3116
    [Google Scholar]
  6. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
    [Google Scholar]
  7. Bohannon D. E., Sonenshein A. L. 1989; Positive regulation of glutamate biosynthesis in Bacillus subtilis. Journal of Bacteriology 171:4718–4727
    [Google Scholar]
  8. Brehm J., Salmond G., Minton N. 1987; Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAM/Jl. Nucleic Acids Research 15:3177
    [Google Scholar]
  9. Chambers S. P., Prior S. E., Barstow D. A., Minton N. P. 1988; The pMTL wc-cloning vectors. I. Improved pUC polylinker region to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149
    [Google Scholar]
  10. Chung C. T., Miller R. H. 1988; A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Research 16:3580
    [Google Scholar]
  11. Del Sal G., Manfioletti G., Schneider C. 1988; A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Research 16:9878
    [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
    [Google Scholar]
  13. Fein J. E., Rogers H. J. 1976; Autolytic enzyme-deficient mutants of Bacillus subtilis 168. Journal of Bacteriology 127:1427–1442
    [Google Scholar]
  14. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. 1983; Construction and properties of an integrable plasmid for Bacillus subtilis. Journal of Bacteriology 154:1513–1515
    [Google Scholar]
  15. Foster S. 1991; Cloning, expression, sequence analysis and biochemical characterisation of an autolytic amidase of Bacillus subtilis 168 trpC2. Journal of General Microbiology 137:1987–1998
    [Google Scholar]
  16. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. 1986; Improved free-energy parameters for predictions of RNA duplex stability. Proceedings of the National Academy of Sciences of the United States of America 83:9373–9377
    [Google Scholar]
  17. Garcia P., Garcia J., L, Garcia E., Sanchez-Puelles J. M., Lopez R. 1990; Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 86:81–88
    [Google Scholar]
  18. Gaur N. K., Oppenheim J., Smith I. 1991; The Bacillus subtilis sin gene, a regulator of alternate developmental process, codes for a DNA-binding protein. Journal of Bacteriology 173:678–686
    [Google Scholar]
  19. Gilman M. Z., Chamberlin M. J. 1983; Developmental and genetic regulation of Bacillus subtilis genes transcribed by σ28-RNA polymerase. Cell 35:285–293
    [Google Scholar]
  20. Gilman M. Z., Wiggs J. L., Chamberlin M. J. 1981; Nucleotide sequences of two Bacillus subtilis promoters used by Bacillus subtilis sigmaσ-28 RNA polymerase. Nucleic Acids Research 9:5991–6000
    [Google Scholar]
  21. Gilman M. Z., Glenn J. S., Singer V. L., Chamberlin M. J. 1983; Isolation of sigma σ-28-specific promoters from Bacillus subtilis DNA. Gene 32:11–20
    [Google Scholar]
  22. Grossberger D. 1987; Minipreps of DNA from bacteriophage lambda. Nucleic Acids Research 15:67–37
    [Google Scholar]
  23. Harrison S. C., Aggarwal A. K. 1990; DNA recognition by proteins with the helix-turn-helix motif. Annual Review of Biochemistry 59:933–969
    [Google Scholar]
  24. Hauser P. M., Crabb W. D., Fiora M. G., Scoffone F., Galizzi A. 1991; Genetic analysis of the JlaA locus of Bacillus subtilis. Journal of Bacteriology 173:3580–3583
    [Google Scholar]
  25. Heery D. M., Gannon F., Powell R. 1990; A simple method for subcloning DNA fragments from gel slices. Trends in Genetics 6:173
    [Google Scholar]
  26. von Heijne G. 1986; A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 12:4683–4690
    [Google Scholar]
  27. Helmann J. D., Chamberlin M. J. 1987; DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative a factor. Proceedings of the National Academy of Sciences of the United States of America 84:6422–6424
    [Google Scholar]
  28. Helmann J. D., Marquez L. M., Chamberlin M. J. 1988; Cloning, sequencing, and disruption of the Bacillus subtilis a2H gene. Journal of Bacteriology 170:1568–1574
    [Google Scholar]
  29. Herbold D. R., Glaser L. 1975; Bacillus subtilis N-acetylmuramic acid L-alanine amidase. Journal of Biological Chemistry 250:1676–1682
    [Google Scholar]
  30. Illing N., Errington J. 1991; Genetic regulation of morphogenesis in Bacillus subtilis: roles of av and σF in prespore engulfment. Journal of Bacteriology 173:3159–3169
    [Google Scholar]
  31. Jones K. A., Yamamoto K. R., Tjian R. 1985; Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42:559–572
    [Google Scholar]
  32. Karamata D., Gross J. D. 1970; Isolation and genetic analysis of temperature-sensitive mutants of Bacillus subtilis 168. Molecular and General Genetics 207:73–81
    [Google Scholar]
  33. Kontinen V. P., Sarvas M. 1988; Mutants of Bacillus subtilis defective in protein export. Journal of General Microbiology 134:2333–2344
    [Google Scholar]
  34. Kontinen V. P., Saris P., Sarvas M. 1991; A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export. Molecular Microbiology 5:1273–1283
    [Google Scholar]
  35. Krupinski J., Coussen F., Bakalyar H. A., Tang W.-J., F einstein P. G., Orth K., Slaughter C., Reed R. R., Gilman A. G. 1989; Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244:1558–1564
    [Google Scholar]
  36. Kuroda A., Sekiguchi J. 1990; Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. Journal of General Microbiology 136:2209–2216
    [Google Scholar]
  37. Kuroda A., Sekiguchi J. 1991; Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. Journal of Bacteriology 173:7304–7312
    [Google Scholar]
  38. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  39. Lopez-Diaz I., Clarke S., Mandelstam J. 1986; spoIID operon of Bacillus subtilis, cloning and sequence. Journal of General Microiology 132:341–354
    [Google Scholar]
  40. Margot P., Karamata D. 1992; Identification of the structural genes for, N-acetylmuramoyl-l-alanine amidase and its modifier in Bacillus subtilis 168: inactivation of these genes by insertional mutagenesis has no effect on growth or cell separation. Molecular and General Genetics 232:359–366
    [Google Scholar]
  41. Margot P., Roten C.-A., Karamata D. 1991; N-Acetyl-muramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain. Analytical Biochemistry 198:15–18
    [Google Scholar]
  42. Marquez L. M., Helmann J. D., Ferrari E., Parker H. M., Ordal G. W., Chamberlin M. J. 1990; Studies of σD-dependent functions in Bacillus subtilis. Journal of Bacteriology 172:3435–3443
    [Google Scholar]
  43. Mauel C., Young M., Margot P., Karamata D. 1989; The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis. Molecular and General Genetics 215:388–394
    [Google Scholar]
  44. Mirel D. B., Chamberlin M. J. 1989; The Bacillus subtilis flagellin gene (hag) is transcribed by the ff28 form of RNA polymerase. Journal of Bacteriology 171:3095–3101
    [Google Scholar]
  45. Ohnishi K., Kutsukake K., Suzuki H., Iino T. 1990; Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. Molecular and General Genetics 221:139–147
    [Google Scholar]
  46. Petersen C. 1989; Long-range translational coupling in the rplJL-rpoBC operon of Escherichia coli. Journal of Molecular Biology 206:323–332
    [Google Scholar]
  47. Pooley H. M. 1976; Turnover and spreading of old wall during surface growth of Bacillus subtilis. Journal of Bacteriology 125:1127–1138
    [Google Scholar]
  48. Pooley H. M., Karamata D. 1983; Correlation ol cell wall turnover and autolytic activity in fla~ and supermotile mutants of Bacillus subtilis. In Target of Penicillin pp. 279–284 Berlin: Walter De Gruyter;
    [Google Scholar]
  49. Pooley H. M., Karamata D. 1984; Genetic analysis of autolysin-deficient and flagellaless mutants of Bacillus subtilis. Journal of Bacteriology 160:1123–1129
    [Google Scholar]
  50. Potvin C., Leclerc D., Tremblay G., Asselin A., Bellemare G. 1988; Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Molecular and General Genetics 214:241–248
    [Google Scholar]
  51. Reed K. C., Mann D. A. 1985; Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Research 13:7207–7221
    [Google Scholar]
  52. Rogers H. J., Perkins H. R., Ward J. B. 1980 Microbial Cell Walls and Membranes London: Chapman & Hall;
    [Google Scholar]
  53. Rogers H. J., Taylor C., Rayter S., Ward J. B. 1984; Purification and properties of autolytic endo-β-N-acetylglucos-aminidase and the N-acetylmuramyl-L-alanine amidase from Bacillus subtilis strain 168. Journal of General Microbiology 130:2395–2402
    [Google Scholar]
  54. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  55. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
    [Google Scholar]
  56. Sekiguchi J., Ohsu H., Kuroda A., Moriyama H., Akamatsu T. 1990; Nucleotide sequence of the Bacillus subtilis flaD locus and a B. licheniformis homologue affecting the autolysin level and flagellation. Journal of General Microbiology 136:1223–1230
    [Google Scholar]
  57. Sharp P. M., Li W.-H. 1987; The codon adaptation index – a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research 15:1281–1295
    [Google Scholar]
  58. Sharp P. M., Higgins D. G., Shields D. C., Devine K. M., Hoch J. A. 1990; Bacillus subtilis gene sequences. In Genetics and Biotechnology of Bacilli vol. 3 pp. 89–98 Edited by Zukowski M. M., Ganesan A. T., Hoch J. A. San Diego: Academic Press;
    [Google Scholar]
  59. Shields D. C., Sharp P. M. 1987; Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Research 15:8023–8040
    [Google Scholar]
  60. Singer V. L. 1987 Characterisation of promoters and genes controlled by Bacillus subtilis sigma 28 RNA polymerase PhD thesis University of California; Berkeley:
    [Google Scholar]
  61. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  62. Studer R. E. 1988 Caracterisation de la paroi native de Bacillus subtilis et etude des proteines qui lui sont associees PhD thesis University of Lausanne;
    [Google Scholar]
  63. Studer R. E., Karamata D. 1988; Cell wall proteins in Bacillus subtilis. In Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function pp. 146–150 Edited by Actor P. Washington: American Society for Microbiology;
    [Google Scholar]
  64. Tinoco I., Borer P. N., Dengler B., Levine M. D., Uhlenbeck O. C., Crothers D. M., Gralla J. 1973; Improved estimation of secondary structure in ribonucleic acids. Nature New Biology 246:40–41
    [Google Scholar]
  65. Tullis R. H., Rubin H. 1980; Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I. Analytical Biochemistry 107:260–264
    [Google Scholar]
  66. Wang X., Wilkinson B. J., Jayaswal R. K. 1991; Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. Gene 102:105–109
    [Google Scholar]
  67. Ward J. B., Curtis C. A. M., Taylor C., Buxton R. S. 1982; Purification and characterisation of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase. Journal of General Microbiology 128:1171–1178
    [Google Scholar]
  68. Yamaguchi K., Yu F., Inouye M. 1988; A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell 53:423–432
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-9-1949
Loading
/content/journal/micro/10.1099/00221287-138-9-1949
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error