Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Apr;174(7):2281–2287. doi: 10.1128/jb.174.7.2281-2287.1992

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

K Ito 1, S Udaka 1, H Yamagata 1
PMCID: PMC205849  PMID: 1551846

Abstract

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.

Full text

PDF
2281

Images in this article

Selected References

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

  1. ADLER H. I., HARDIGREE A. A. ANALYSIS OF A GENE CONTROLLING CELL DIVISION AND SENSITIVITY TO RADIATION IN ESCHERICHIA COLI. J Bacteriol. 1964 Mar;87:720–726. doi: 10.1128/jb.87.3.720-726.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adachi T., Yamagata H., Tsukagoshi N., Udaka S. Multiple and tandemly arranged promoters of the cell wall protein gene operon in Bacillus brevis 47. J Bacteriol. 1989 Feb;171(2):1010–1016. doi: 10.1128/jb.171.2.1010-1016.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adachi T., Yamagata H., Tsukagoshi N., Udaka S. Use of both translation initiation sites of the middle wall protein gene in Bacillus brevis 47. J Bacteriol. 1990 Jan;172(1):511–513. doi: 10.1128/jb.172.1.511-513.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  5. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  6. Buell G., Schulz M. F., Selzer G., Chollet A., Movva N. R., Semon D., Escanez S., Kawashima E. Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I). Nucleic Acids Res. 1985 Mar 25;13(6):1923–1938. doi: 10.1093/nar/13.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charette M. F., Henderson G. W., Markovitz A. ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4728–4732. doi: 10.1073/pnas.78.8.4728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chin D. T., Goff S. A., Webster T., Smith T., Goldberg A. L. Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J Biol Chem. 1988 Aug 25;263(24):11718–11728. [PubMed] [Google Scholar]
  10. Chung C. H., Goldberg A. L. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4931–4935. doi: 10.1073/pnas.78.8.4931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Downs D., Waxman L., Goldberg A. L., Roth J. Isolation and characterization of lon mutants in Salmonella typhimurium. J Bacteriol. 1986 Jan;165(1):193–197. doi: 10.1128/jb.165.1.193-197.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goff S. A., Casson L. P., Goldberg A. L. Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6647–6651. doi: 10.1073/pnas.81.21.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldberg A. L. Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc Natl Acad Sci U S A. 1972 Feb;69(2):422–426. doi: 10.1073/pnas.69.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gottesman S., Squires C., Pichersky E., Carrington M., Hobbs M., Mattick J. S., Dalrymple B., Kuramitsu H., Shiroza T., Foster T. Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc Natl Acad Sci U S A. 1990 May;87(9):3513–3517. doi: 10.1073/pnas.87.9.3513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gottesman S., Zipser D. Deg phenotype of Escherichia coli lon mutants. J Bacteriol. 1978 Feb;133(2):844–851. doi: 10.1128/jb.133.2.844-851.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karn J., Matthes H. W., Gait M. J., Brenner S. A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI. Gene. 1984 Dec;32(1-2):217–224. doi: 10.1016/0378-1119(84)90049-0. [DOI] [PubMed] [Google Scholar]
  18. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  19. Konishi H., Sato T., Yamagata H., Udaka S. Efficient production of human alpha-amylase by a Bacillus brevis mutant. Appl Microbiol Biotechnol. 1990 Dec;34(3):297–302. doi: 10.1007/BF00170046. [DOI] [PubMed] [Google Scholar]
  20. Kop J., Kopylov A. M., Magrum L., Siegel R., Gupta R., Woese C. R., Noller H. F. Probing the structure of 16 S ribosomal RNA from Bacillus brevis. J Biol Chem. 1984 Dec 25;259(24):15287–15293. [PubMed] [Google Scholar]
  21. MARKOVITZ A. REGULATORY MECHANISMS FOR SYNTHESIS OF CAPSULAR POLYSACCHARIDE IN MUCOID MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1964 Feb;51:239–246. doi: 10.1073/pnas.51.2.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matsuyama S., Mizushima S. Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli. J Bacteriol. 1985 Jun;162(3):1196–1202. doi: 10.1128/jb.162.3.1196-1202.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maurizi M. R., Clark W. P., Kim S. H., Gottesman S. Clp P represents a unique family of serine proteases. J Biol Chem. 1990 Jul 25;265(21):12546–12552. [PubMed] [Google Scholar]
  24. McKenzie T., Hoshino T., Tanaka T., Sueoka N. The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid. 1986 Mar;15(2):93–103. doi: 10.1016/0147-619x(86)90046-6. [DOI] [PubMed] [Google Scholar]
  25. Menon A. S., Goldberg A. L. Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J Biol Chem. 1987 Nov 5;262(31):14929–14934. [PubMed] [Google Scholar]
  26. Mizusawa S., Gottesman S. Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci U S A. 1983 Jan;80(2):358–362. doi: 10.1073/pnas.80.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  28. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  29. 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]
  30. Takahashi W., Yamagata H., Yamaguchi K., Tsukagoshi N., Udaka S. Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA. J Bacteriol. 1983 Dec;156(3):1130–1134. doi: 10.1128/jb.156.3.1130-1134.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Takano T. Bacterial mutants defective in plasmid formation: requirement for the lon + allele. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1469–1473. doi: 10.1073/pnas.68.7.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Torres-Cabassa A. S., Gottesman S. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J Bacteriol. 1987 Mar;169(3):981–989. doi: 10.1128/jb.169.3.981-989.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Udaka S., Tsukagoshi N., Yamagata H. Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol Genet Eng Rev. 1989;7:113–146. doi: 10.1080/02648725.1989.10647857. [DOI] [PubMed] [Google Scholar]
  34. Uretz R. B., Markovitz A. Dominance of ultraviolet radiation resistance in partial diploids of Escherichia coli K-12. J Bacteriol. 1969 Nov;100(2):1118–1120. doi: 10.1128/jb.100.2.1118-1120.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yamagata H., Nakahama K., Suzuki Y., Kakinuma A., Tsukagoshi N., Udaka S. Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci U S A. 1989 May;86(10):3589–3593. doi: 10.1073/pnas.86.10.3589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES