Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Feb;174(3):736–742. doi: 10.1128/jb.174.3.736-742.1992

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii.

M Kamekura 1, Y Seno 1, M L Holmes 1, M L Dyall-Smith 1
PMCID: PMC206149  PMID: 1732209

Abstract

The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker.

Full text

PDF
736

Selected References

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

  1. Cowan D. A., Smolenski K. A., Daniel R. M., Morgan H. W. An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C. Biochem J. 1987 Oct 1;247(1):121–133. doi: 10.1042/bj2470121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dunn R., McCoy J., Simsek M., Majumdar A., Chang S. H., Rajbhandary U. L., Khorana H. G. The bacteriorhodopsin gene. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6744–6748. doi: 10.1073/pnas.78.11.6744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. FLANNERY W. L. Current status of knowledge of halophilic bacteria. Bacteriol Rev. 1956 Jun;20(2):49–66. doi: 10.1128/br.20.2.49-66.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fusek M., Lin X. L., Tang J. Enzymic properties of thermopsin. J Biol Chem. 1990 Jan 25;265(3):1496–1501. [PubMed] [Google Scholar]
  5. GIBBONS N. E. The effect of salt concentrations on the biochemical reactions of some halophilic bacteria. Can J Microbiol. 1957 Mar;3(2):249–255. doi: 10.1139/m57-029. [DOI] [PubMed] [Google Scholar]
  6. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  7. Holmes M. L., Dyall-Smith M. L. A plasmid vector with a selectable marker for halophilic archaebacteria. J Bacteriol. 1990 Feb;172(2):756–761. doi: 10.1128/jb.172.2.756-761.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Holmes M. L., Nuttall S. D., Dyall-Smith M. L. Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol. 1991 Jun;173(12):3807–3813. doi: 10.1128/jb.173.12.3807-3813.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Izotova L. S., Strongin A. Y., Chekulaeva L. N., Sterkin V. E., Ostoslavskaya V. I., Lyublinskaya L. A., Timokhina E. A., Stepanov V. M. Purification and properties of serine protease from Halobacterium halobium. J Bacteriol. 1983 Aug;155(2):826–830. doi: 10.1128/jb.155.2.826-830.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kamekura M., Seno Y. A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1. Biochem Cell Biol. 1990 Jan;68(1):352–359. doi: 10.1139/o90-048. [DOI] [PubMed] [Google Scholar]
  11. Lin X., Tang J. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J Biol Chem. 1990 Jan 25;265(3):1490–1495. [PubMed] [Google Scholar]
  12. Norberg P., von Hofsten B. Proteolytic enzymes from extremely halophilic bacteria. J Gen Microbiol. 1969 Feb;55(2):251–256. doi: 10.1099/00221287-55-2-251. [DOI] [PubMed] [Google Scholar]
  13. Ogawa Y., Tatsumi H., Murakami S., Ishida Y., Murakami K., Masaki A., Kawabe H., Arimura H., Nakano E., Motai H. Secretion of Aspergillus oryzae alkaline protease in an osmophilic yeast, Zygosaccharomyces rouxii. Agric Biol Chem. 1990 Oct;54(10):2521–2529. [PubMed] [Google Scholar]
  14. 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]
  15. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  16. Tatsumi H., Ogawa Y., Murakami S., Ishida Y., Murakami K., Masaki A., Kawabe H., Arimura H., Nakano E., Motai H. A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae. Mol Gen Genet. 1989 Oct;219(1-2):33–38. doi: 10.1007/BF00261154. [DOI] [PubMed] [Google Scholar]
  17. Thomm M., Wich G., Brown J. W., Frey G., Sherf B. A., Beckler G. S. An archaebacterial promoter sequence assigned by RNA polymerase binding experiments. Can J Microbiol. 1989 Jan;35(1):30–35. doi: 10.1139/m89-005. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wells J. A., Ferrari E., Henner D. J., Estell D. A., Chen E. Y. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 1983 Nov 25;11(22):7911–7925. doi: 10.1093/nar/11.22.7911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Woese C. R., Kandler O., Wheelis M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. doi: 10.1073/pnas.87.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yanagida N., Uozumi T., Beppu T. Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. J Bacteriol. 1986 Jun;166(3):937–944. doi: 10.1128/jb.166.3.937-944.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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