Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Nov;176(21):6518–6527. doi: 10.1128/jb.176.21.6518-6527.1994

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

S Riethdorf 1, U Völker 1, U Gerth 1, A Winkler 1, S Engelmann 1, M Hecker 1
PMCID: PMC197005  PMID: 7961402

Abstract

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.

Full text

PDF
6518

Images in this article

Selected References

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

  1. Amerik AYu, Antonov V. K., Gorbalenya A. E., Kotova S. A., Rotanova T. V., Shimbarevich E. V. Site-directed mutagenesis of La protease. A catalytically active serine residue. FEBS Lett. 1991 Aug 5;287(1-2):211–214. doi: 10.1016/0014-5793(91)80053-6. [DOI] [PubMed] [Google Scholar]
  2. Amerik AYu, Antonov V. K., Gorbalenya A. E., Kotova S. A., Rotanova T. V., Shimbarevich E. V. Site-directed mutagenesis of La protease. A catalytically active serine residue. FEBS Lett. 1991 Aug 5;287(1-2):211–214. doi: 10.1016/0014-5793(91)80053-6. [DOI] [PubMed] [Google Scholar]
  3. Amerik A. Iu, Chistiakov L. G., Ostroumova N. I., Gurevich A. I., Antonov V. K. Klonirovanie, ékspressiia i struktura funktsional'no aktivnogo ukorochennogo gena lon Escherichia coli. Bioorg Khim. 1988 Mar;14(3):408–411. [PubMed] [Google Scholar]
  4. Arnosti D. N., Singer V. L., Chamberlin M. J. Characterization of heat shock in Bacillus subtilis. J Bacteriol. 1986 Dec;168(3):1243–1249. doi: 10.1128/jb.168.3.1243-1249.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benson A. K., Haldenwang W. G. The sigma B-dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock. J Bacteriol. 1993 Apr;175(7):1929–1935. doi: 10.1128/jb.175.7.1929-1935.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boylan S. A., Redfield A. R., Brody M. S., Price C. W. Stress-induced activation of the sigma B transcription factor of Bacillus subtilis. J Bacteriol. 1993 Dec;175(24):7931–7937. doi: 10.1128/jb.175.24.7931-7937.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boylan S. A., Redfield A. R., Price C. W. Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon. J Bacteriol. 1993 Jul;175(13):3957–3963. doi: 10.1128/jb.175.13.3957-3963.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang B. Y., Chen K. Y., Wen Y. D., Liao C. T. The response of a Bacillus subtilis temperature-sensitive sigA mutant to heat stress. J Bacteriol. 1994 Jun;176(11):3102–3110. doi: 10.1128/jb.176.11.3102-3110.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Chuang S. E., Daniels D. L., Blattner F. R. Global regulation of gene expression in Escherichia coli. J Bacteriol. 1993 Apr;175(7):2026–2036. doi: 10.1128/jb.175.7.2026-2036.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Dubnau D., Cirigliano C. Genetic characterization of recombination-deficient mutants of Bacillus subtilis. J Bacteriol. 1974 Feb;117(2):488–493. doi: 10.1128/jb.117.2.488-493.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duvall E. J., Williams D. M., Lovett P. S., Rudolph C., Vasantha N., Guyer M. Chloramphenicol-inducible gene expression in Bacillus subtilis. Gene. 1983 Oct;24(2-3):171–177. doi: 10.1016/0378-1119(83)90077-x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Fischer H., Glockshuber R. ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli. J Biol Chem. 1993 Oct 25;268(30):22502–22507. [PubMed] [Google Scholar]
  19. Gill R. E., Karlok M., Benton D. Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling. J Bacteriol. 1993 Jul;175(14):4538–4544. doi: 10.1128/jb.175.14.4538-4544.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Goff S. A., Goldberg A. L. An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli. J Biol Chem. 1987 Apr 5;262(10):4508–4515. [PubMed] [Google Scholar]
  22. Goff S. A., Goldberg A. L. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985 Jun;41(2):587–595. doi: 10.1016/s0092-8674(85)80031-3. [DOI] [PubMed] [Google Scholar]
  23. Goldberg A. L. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur J Biochem. 1992 Jan 15;203(1-2):9–23. doi: 10.1111/j.1432-1033.1992.tb19822.x. [DOI] [PubMed] [Google Scholar]
  24. Gottesman S., Maurizi M. R. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56(4):592–621. doi: 10.1128/mr.56.4.592-621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  26. Hansson M., Rutberg L., Schröder I., Hederstedt L. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J Bacteriol. 1991 Apr;173(8):2590–2599. doi: 10.1128/jb.173.8.2590-2599.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hoch J. A. Genetic analysis in Bacillus subtilis. Methods Enzymol. 1991;204:305–320. doi: 10.1016/0076-6879(91)04015-g. [DOI] [PubMed] [Google Scholar]
  28. Huisman O., D'Ari R., Gottesman S. Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4490–4494. doi: 10.1073/pnas.81.14.4490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hwang B. J., Park W. J., Chung C. H., Goldberg A. L. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5550–5554. doi: 10.1073/pnas.84.16.5550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Igo M., Lampe M., Ray C., Schafer W., Moran C. P., Jr, Losick R. Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis. J Bacteriol. 1987 Aug;169(8):3464–3469. doi: 10.1128/jb.169.8.3464-3469.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ito K., Udaka S., Yamagata H. Cloning, characterization, and inactivation of the Bacillus brevis lon gene. J Bacteriol. 1992 Apr;174(7):2281–2287. doi: 10.1128/jb.174.7.2281-2287.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Joyce C. M., Grindley N. D. Identification of two genes immediately downstream from the polA gene of Escherichia coli. J Bacteriol. 1982 Dec;152(3):1211–1219. doi: 10.1128/jb.152.3.1211-1219.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Katayama Y., Gottesman S., Pumphrey J., Rudikoff S., Clark W. P., Maurizi M. R. The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J Biol Chem. 1988 Oct 15;263(29):15226–15236. [PubMed] [Google Scholar]
  34. Kroh H. E., Simon L. D. The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5. J Bacteriol. 1990 Oct;172(10):6026–6034. doi: 10.1128/jb.172.10.6026-6034.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Krüger E., Völker U., Hecker M. Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance. J Bacteriol. 1994 Jun;176(11):3360–3367. doi: 10.1128/jb.176.11.3360-3367.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lindner C., Stülke J., Hecker M. Regulation of xylanolytic enzymes in Bacillus subtilis. Microbiology. 1994 Apr;140(Pt 4):753–757. doi: 10.1099/00221287-140-4-753. [DOI] [PubMed] [Google Scholar]
  37. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mitchell J. J., Lucas-Lenard J. M. The effect of alcohols on guanosine 5'-diphosphate-3'-diphosphate metabolism in stringent and relaxed Escherichia coli. J Biol Chem. 1980 Jul 10;255(13):6307–6313. [PubMed] [Google Scholar]
  39. 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]
  40. Phillips T. A., VanBogelen R. A., Neidhardt F. C. lon gene product of Escherichia coli is a heat-shock protein. J Bacteriol. 1984 Jul;159(1):283–287. doi: 10.1128/jb.159.1.283-287.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Polayes D. A., Rice P. W., Garner M. M., Dahlberg J. E. Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. J Bacteriol. 1988 Jul;170(7):3110–3114. doi: 10.1128/jb.170.7.3110-3114.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Schmidt A., Schiesswohl M., Völker U., Hecker M., Schumann W. Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3993–3999. doi: 10.1128/jb.174.12.3993-3999.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schmidt R., Decatur A. L., Rather P. N., Moran C. P., Jr, Losick R. Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G. J Bacteriol. 1994 Nov;176(21):6528–6537. doi: 10.1128/jb.176.21.6528-6537.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schoemaker J. M., Gayda R. C., Markovitz A. Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death. J Bacteriol. 1984 May;158(2):551–561. doi: 10.1128/jb.158.2.551-561.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stout V., Torres-Cabassa A., Maurizi M. R., Gutnick D., Gottesman S. RcsA, an unstable positive regulator of capsular polysaccharide synthesis. J Bacteriol. 1991 Mar;173(5):1738–1747. doi: 10.1128/jb.173.5.1738-1747.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  48. Stülke J., Hanschke R., Hecker M. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol. 1993 Sep;139(9):2041–2045. doi: 10.1099/00221287-139-9-2041. [DOI] [PubMed] [Google Scholar]
  49. Thomas C. D., Modha J., Razzaq T. M., Cullis P. M., Rivett A. J. Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease. Gene. 1993 Dec 22;136(1-2):237–242. doi: 10.1016/0378-1119(93)90471-e. [DOI] [PubMed] [Google Scholar]
  50. Tojo N., Inouye S., Komano T. Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth. J Bacteriol. 1993 Apr;175(8):2271–2277. doi: 10.1128/jb.175.8.2271-2277.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tojo N., Inouye S., Komano T. The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus. J Bacteriol. 1993 Jul;175(14):4545–4549. doi: 10.1128/jb.175.14.4545-4549.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Van Dyck L., Pearce D. A., Sherman F. PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae. J Biol Chem. 1994 Jan 7;269(1):238–242. [PubMed] [Google Scholar]
  54. Varón D., Boylan S. A., Okamoto K., Price C. W. Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B. J Bacteriol. 1993 Jul;175(13):3964–3971. doi: 10.1128/jb.175.13.3964-3971.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Völker U., Engelmann S., Maul B., Riethdorf S., Völker A., Schmid R., Mach H., Hecker M. Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology. 1994 Apr;140(Pt 4):741–752. doi: 10.1099/00221287-140-4-741. [DOI] [PubMed] [Google Scholar]
  56. Völker U., Mach H., Schmid R., Hecker M. Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis. J Gen Microbiol. 1992 Oct;138(10):2125–2135. doi: 10.1099/00221287-138-10-2125. [DOI] [PubMed] [Google Scholar]
  57. Völker U., Riethdorf S., Winkler A., Weigend B., Fortnagel P., Hecker M. Cloning and characterization of heat-inducible promoters of Bacillus subtilis. FEMS Microbiol Lett. 1993 Feb 1;106(3):287–293. doi: 10.1111/j.1574-6968.1993.tb05978.x. [DOI] [PubMed] [Google Scholar]
  58. Wang N., Gottesman S., Willingham M. C., Gottesman M. M., Maurizi M. R. A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11247–11251. doi: 10.1073/pnas.90.23.11247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Watabe S., Kimura T. Adrenal cortex mitochondrial enzyme with ATP-dependent protease and protein-dependent ATPase activities. Purification and properties. J Biol Chem. 1985 Nov 25;260(27):14498–14504. [PubMed] [Google Scholar]
  60. Wetzstein M., Völker U., Dedio J., Löbau S., Zuber U., Schiesswohl M., Herget C., Hecker M., Schumann W. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bacteriol. 1992 May;174(10):3300–3310. doi: 10.1128/jb.174.10.3300-3310.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. Zuber U., Schumann W. CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J Bacteriol. 1994 Mar;176(5):1359–1363. doi: 10.1128/jb.176.5.1359-1363.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES