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. 1992 May;174(10):3300–3310. doi: 10.1128/jb.174.10.3300-3310.1992

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis.

M Wetzstein 1, U Völker 1, J Dedio 1, S Löbau 1, U Zuber 1, M Schiesswohl 1, C Herget 1, M Hecker 1, W Schumann 1
PMCID: PMC205999  PMID: 1339421

Abstract

By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.

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Selected References

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  1. Ambulos N. P., Jr, Duvall E. J., Lovett P. S. Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis. Gene. 1987;51(2-3):281–286. doi: 10.1016/0378-1119(87)90317-9. [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. 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]
  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. Baird P. N., Hall L. M., Coates A. R. Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. J Gen Microbiol. 1989 Apr;135(4):931–939. doi: 10.1099/00221287-135-4-931. [DOI] [PubMed] [Google Scholar]
  6. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cegielska A., Georgopoulos C. Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. J Biol Chem. 1989 Dec 15;264(35):21122–21130. [PubMed] [Google Scholar]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chitnis P. R., Nelson N. Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 1991 Jan 5;266(1):58–65. [PubMed] [Google Scholar]
  10. Cozens A. L., Walker J. E. The organization and sequence of the genes for ATP synthase subunits in the cyanobacterium Synechococcus 6301. Support for an endosymbiotic origin of chloroplasts. J Mol Biol. 1987 Apr 5;194(3):359–383. doi: 10.1016/0022-2836(87)90667-x. [DOI] [PubMed] [Google Scholar]
  11. Craig E. A. The heat shock response. CRC Crit Rev Biochem. 1985;18(3):239–280. doi: 10.3109/10409238509085135. [DOI] [PubMed] [Google Scholar]
  12. Dedonder R. A., Lepesant J. A., Lepesant-Kejzlarová J., Billault A., Steinmetz M., Kunst F. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol. 1977 Apr;33(4):989–993. doi: 10.1128/aem.33.4.989-993.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
  14. Engel J. N., Pollack J., Perara E., Ganem D. Heat shock response of murine Chlamydia trachomatis. J Bacteriol. 1990 Dec;172(12):6959–6972. doi: 10.1128/jb.172.12.6959-6972.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  18. Garsia R. J., Hellqvist L., Booth R. J., Radford A. J., Britton W. J., Astbury L., Trent R. J., Basten A. Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes. Infect Immun. 1989 Jan;57(1):204–212. doi: 10.1128/iai.57.1.204-212.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Georgopoulos C. P. A new bacterial gene (groPC) which affects lambda DNA replication. Mol Gen Genet. 1977 Feb 28;151(1):35–39. doi: 10.1007/BF00446910. [DOI] [PubMed] [Google Scholar]
  20. Gomes S. L., Gober J. W., Shapiro L. Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures. J Bacteriol. 1990 Jun;172(6):3051–3059. doi: 10.1128/jb.172.6.3051-3059.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  22. Hearne C. M., Ellar D. J. Nucleotide sequence of a Bacillus subtilis gene homologous to the dnaK gene of Escherichia coli. Nucleic Acids Res. 1989 Oct 25;17(20):8373–8373. doi: 10.1093/nar/17.20.8373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Henner D. J., Hoch J. A. The Bacillus subtilis chromosome. Microbiol Rev. 1980 Mar;44(1):57–82. doi: 10.1128/mr.44.1.57-82.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hoch J. A., Barat M., Anagnostopoulos C. Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol. 1967 Jun;93(6):1925–1937. doi: 10.1128/jb.93.6.1925-1937.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Itikawa H., Ryu J. Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. J Bacteriol. 1979 May;138(2):339–344. doi: 10.1128/jb.138.2.339-344.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson C., Chandrasekhar G. N., Georgopoulos C. Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro. J Bacteriol. 1989 Mar;171(3):1590–1596. doi: 10.1128/jb.171.3.1590-1596.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Karamata D., Gross J. D. Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol Gen Genet. 1970;108(3):277–287. doi: 10.1007/BF00283358. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Landick R., Vaughn V., Lau E. T., VanBogelen R. A., Erickson J. W., Neidhardt F. C. Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor. Cell. 1984 Aug;38(1):175–182. doi: 10.1016/0092-8674(84)90538-5. [DOI] [PubMed] [Google Scholar]
  30. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  31. Lipinska B., King J., Ang D., Georgopoulos C. Sequence analysis and transcriptional regulation of the Escherichia coli grpE gene, encoding a heat shock protein. Nucleic Acids Res. 1988 Aug 11;16(15):7545–7562. doi: 10.1093/nar/16.15.7545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Margolin W., Howe M. M. Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription. Nucleic Acids Res. 1986 Jun 25;14(12):4881–4897. doi: 10.1093/nar/14.12.4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mehra V., Sweetser D., Young R. A. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013–7017. doi: 10.1073/pnas.83.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Morrison R. P., Belland R. J., Lyng K., Caldwell H. D. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. J Exp Med. 1989 Oct 1;170(4):1271–1283. doi: 10.1084/jem.170.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Narberhaus F., Giebeler K., Bahl H. Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J Bacteriol. 1992 May;174(10):3290–3299. doi: 10.1128/jb.174.10.3290-3299.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Neidhardt F. C., VanBogelen R. A., Vaughn V. The genetics and regulation of heat-shock proteins. Annu Rev Genet. 1984;18:295–329. doi: 10.1146/annurev.ge.18.120184.001455. [DOI] [PubMed] [Google Scholar]
  39. O'Kane C., Stephens M. A., McConnell D. Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis. J Bacteriol. 1986 Nov;168(2):973–981. doi: 10.1128/jb.168.2.973-981.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Paek K. H., Walker G. C. Escherichia coli dnaK null mutants are inviable at high temperature. J Bacteriol. 1987 Jan;169(1):283–290. doi: 10.1128/jb.169.1.283-290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Parker J. Errors and alternatives in reading the universal genetic code. Microbiol Rev. 1989 Sep;53(3):273–298. doi: 10.1128/mr.53.3.273-298.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saito H., Nakamura Y., Uchida H. A transducing lambda phage carrying grpE, a bacterial gene necessary for lambda DNA replication, and two ribosomal protein genes, rpsP (S16) and rplS (L19). Mol Gen Genet. 1978 Oct 24;165(3):247–256. doi: 10.1007/BF00332523. [DOI] [PubMed] [Google Scholar]
  43. Saito H., Uchida H. Initiation of the DNA replication of bacteriophage lambda in Escherichia coli K12. J Mol Biol. 1977 Jun 15;113(1):1–25. doi: 10.1016/0022-2836(77)90038-9. [DOI] [PubMed] [Google Scholar]
  44. Saito H., Uchida H. Organization and expression of the dnaJ and dnaK genes of Escherichia coli K12. Mol Gen Genet. 1978 Aug 4;164(1):1–8. doi: 10.1007/BF00267592. [DOI] [PubMed] [Google Scholar]
  45. Sakakibara Y. The dnaK gene of Escherichia coli functions in initiation of chromosome replication. J Bacteriol. 1988 Feb;170(2):972–979. doi: 10.1128/jb.170.2.972-979.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Shinnick T. M. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol. 1987 Mar;169(3):1080–1088. doi: 10.1128/jb.169.3.1080-1088.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Streips U. N., Polio F. W. Heat shock proteins in bacilli. J Bacteriol. 1985 Apr;162(1):434–437. doi: 10.1128/jb.162.1.434-437.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sussman M. D., Setlow P. Nucleotide sequence of a Bacillus megaterium gene homologous to the dnaK gene of Escherichia coli. Nucleic Acids Res. 1987 May 11;15(9):3923–3923. doi: 10.1093/nar/15.9.3923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Webb R., Reddy K. J., Sherman L. A. Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin. J Bacteriol. 1990 Sep;172(9):5079–5088. doi: 10.1128/jb.172.9.5079-5088.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wetzstein M., Dedio J., Schumann W. Complete nucleotide sequence of the Bacillus subtilis dnaK gene. Nucleic Acids Res. 1990 Apr 25;18(8):2172–2172. doi: 10.1093/nar/18.8.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wetzstein M., Schumann W. Nucleotide sequence of a Bacillus subtilis gene homologous to the grpE gene of E. coli located immediately upstream of the dnaK gene. Nucleic Acids Res. 1990 Mar 11;18(5):1289–1289. doi: 10.1093/nar/18.5.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wood H. E., Dawson M. T., Devine K. M., McConnell D. J. Characterization of PBSX, a defective prophage of Bacillus subtilis. J Bacteriol. 1990 May;172(5):2667–2674. doi: 10.1128/jb.172.5.2667-2674.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yamaguchi R., Matsuo K., Yamazaki A., Nagai S., Terasaka K., Yamada T. Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression. FEBS Lett. 1988 Nov 21;240(1-2):115–117. doi: 10.1016/0014-5793(88)80350-8. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Young D., Lathigra R., Hendrix R., Sweetser D., Young R. A. Stress proteins are immune targets in leprosy and tuberculosis. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4267–4270. doi: 10.1073/pnas.85.12.4267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zeilstra-Ryalls J., Fayet O., Georgopoulos C. The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol. 1991;45:301–325. doi: 10.1146/annurev.mi.45.100191.001505. [DOI] [PubMed] [Google Scholar]
  58. Zylicz M., Ang D., Georgopoulos C. The grpE protein of Escherichia coli. Purification and properties. J Biol Chem. 1987 Dec 25;262(36):17437–17442. [PubMed] [Google Scholar]

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