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. 1993 Feb;61(2):411–417. doi: 10.1128/iai.61.2.411-417.1993

Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein.

J Partridge 1, J King 1, J Krska 1, D Rockabrand 1, P Blum 1
PMCID: PMC302744  PMID: 8423071

Abstract

The dnaK (hsp70) gene from the facultative intracellular pathogen Erysipelothrix rhusiopathiae was cloned by heterologous DNA hybridization of a genomic library using the Escherichia coli dnaK gene as a probe. A 3.2-kb fragment which encoded an 1,800-bp open reading frame was recovered. The deduced amino acid sequence of this open reading frame shares 56% identity with the E. coli DnaK protein. Expression of the encoded protein in E. coli by using the phage T7 promoter/polymerase system resulted in accumulation of a unique 65-kDa protein. Western blot (immunoblot) analysis of extracts from a recombinant E. coli strain using anti-E. coli DnaK polyclonal antibodies confirmed that the cloned gene encodes a DnaK homolog. The recombinant E. rhusiopathiae DnaK protein was purified to 80% homogeneity by ATP affinity chromatography. The purified material hydrolyzed ATP with a specific activity of 100 nmol min-1 mg of protein-1. Analysis of total protein extracts from E. rhusiopathiae indicates that DnaK is a highly expressed protein in this organism.

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Selected References

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  1. Andersson S. G., Kurland C. G. Codon preferences in free-living microorganisms. Microbiol Rev. 1990 Jun;54(2):198–210. doi: 10.1128/mr.54.2.198-210.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Blum P., Ory J., Bauernfeind J., Krska J. Physiological consequences of DnaK and DnaJ overproduction in Escherichia coli. J Bacteriol. 1992 Nov;174(22):7436–7444. doi: 10.1128/jb.174.22.7436-7444.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blum P., Velligan M., Lin N., Matin A. DnaK-mediated alterations in human growth hormone protein inclusion bodies. Biotechnology (N Y) 1992 Mar;10(3):301–304. doi: 10.1038/nbt0392-301. [DOI] [PubMed] [Google Scholar]
  6. Brumbaugh J. A., Middendorf L. R., Grone D. L., Ruth J. L. Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5610–5614. doi: 10.1073/pnas.85.15.5610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buchmeier N. A., Heffron F. Induction of Salmonella stress proteins upon infection of macrophages. Science. 1990 May 11;248(4956):730–732. doi: 10.1126/science.1970672. [DOI] [PubMed] [Google Scholar]
  8. Galán J. E., Timoney J. F. Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae. Infect Immun. 1990 Sep;58(9):3116–3121. doi: 10.1128/iai.58.9.3116-3121.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hwang D. S., Kaguni J. M. Interaction of dnaA46 protein with a stimulatory protein in replication from the Escherichia coli chromosomal origin. J Biol Chem. 1988 Aug 5;263(22):10633–10640. [PubMed] [Google Scholar]
  10. Kornak J. M., Kuo C. C., Campbell L. A. Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog. Infect Immun. 1991 Feb;59(2):721–725. doi: 10.1128/iai.59.2.721-725.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Liberek K., Skowyra D., Zylicz M., Johnson C., Georgopoulos C. The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J Biol Chem. 1991 Aug 5;266(22):14491–14496. [PubMed] [Google Scholar]
  13. 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]
  14. Matin A., Auger E. A., Blum P. H., Schultz J. E. Genetic basis of starvation survival in nondifferentiating bacteria. Annu Rev Microbiol. 1989;43:293–316. doi: 10.1146/annurev.mi.43.100189.001453. [DOI] [PubMed] [Google Scholar]
  15. Morgan R. W., Christman M. F., Jacobson F. S., Storz G., Ames B. N. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8059–8063. doi: 10.1073/pnas.83.21.8059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murray P. J., Young R. A. Stress and immunological recognition in host-pathogen interactions. J Bacteriol. 1992 Jul;174(13):4193–4196. doi: 10.1128/jb.174.13.4193-4196.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. O'Brien R. L., Happ M. P., Dallas A., Palmer E., Kubo R., Born W. K. Stimulation of a major subset of lymphocytes expressing T cell receptor gamma delta by an antigen derived from Mycobacterium tuberculosis. Cell. 1989 May 19;57(4):667–674. doi: 10.1016/0092-8674(89)90135-9. [DOI] [PubMed] [Google Scholar]
  19. Ohki M., Tamura F., Nishimura S., Uchida H. Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product. J Biol Chem. 1986 Feb 5;261(4):1778–1781. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Spence J., Cegielska A., Georgopoulos C. Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation. J Bacteriol. 1990 Dec;172(12):7157–7166. doi: 10.1128/jb.172.12.7157-7166.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  24. Timoney J. The inactivation of Erysipelothrix rhuopathiae in macrophages from normal and immune mice. Res Vet Sci. 1969 May;10(3):301–302. [PubMed] [Google Scholar]
  25. Timoney J. The inactivation of Erysipelothrix rhusiopathiae in pig buffy-coat leucocytes. Res Vet Sci. 1970 Mar;11(2):189–190. [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Welch W. J., Feramisco J. R. Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol. 1985 Jun;5(6):1229–1237. doi: 10.1128/mcb.5.6.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Wood R. L., Harrington R., Jr Serotypes of Erysipelothrix rhusiopathiae isolated from swine and from soil and manure of swine pens in the United States. Am J Vet Res. 1978 Nov;39(11):1833–1840. [PubMed] [Google Scholar]

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