Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jan;175(1):176–181. doi: 10.1128/jb.175.1.176-181.1993

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

H Tsujibo 1, H Orikoshi 1, H Tanno 1, K Fujimoto 1, K Miyamoto 1, C Imada 1, Y Okami 1, Y Inamori 1
PMCID: PMC196111  PMID: 8416892

Abstract

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.

Full text

PDF
177

Images in this article

178Image
on p.178

Selected References

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

  1. ARTMAN M. The production of hydrogen sulphide from thiosulphate by Escherichia coli. J Gen Microbiol. 1956 Apr;14(2):315–322. doi: 10.1099/00221287-14-2-315. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Filloux A., Bally M., Ball G., Akrim M., Tommassen J., Lazdunski A. Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBO J. 1990 Dec;9(13):4323–4329. doi: 10.1002/j.1460-2075.1990.tb07881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Harpster M. H., Dunsmuir P. Nucleotide sequence of the chitinase B gene of Serratia marcescens QMB1466. Nucleic Acids Res. 1989 Jul 11;17(13):5395–5395. doi: 10.1093/nar/17.13.5395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hirst T. R., Holmgren J. Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J Bacteriol. 1987 Mar;169(3):1037–1045. doi: 10.1128/jb.169.3.1037-1045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Howard S. P., Buckley J. T. Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila. J Bacteriol. 1983 Apr;154(1):413–418. doi: 10.1128/jb.154.1.413-418.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jones J. D., Grady K. L., Suslow T. V., Bedbrook J. R. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J. 1986 Mar;5(3):467–473. doi: 10.1002/j.1460-2075.1986.tb04235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kamei K., Yamamura Y., Hara S., Ikenaka T. Amino acid sequence of chitinase from Streptomyces erythraeus. J Biochem. 1989 Jun;105(6):979–985. doi: 10.1093/oxfordjournals.jbchem.a122791. [DOI] [PubMed] [Google Scholar]
  9. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  10. Kuranda M. J., Robbins P. W. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem. 1991 Oct 15;266(29):19758–19767. [PubMed] [Google Scholar]
  11. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  12. LUNT M. R., KENT P. W. A chitinase system from Carcinus maenas. Biochim Biophys Acta. 1960 Nov 4;44:371–373. doi: 10.1016/0006-3002(60)91581-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Legrand M., Kauffmann S., Geoffroy P., Fritig B. Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6750–6754. doi: 10.1073/pnas.84.19.6750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Malcolm B. A., Rosenberg S., Corey M. J., Allen J. S., de Baetselier A., Kirsch J. F. Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc Natl Acad Sci U S A. 1989 Jan;86(1):133–137. doi: 10.1073/pnas.86.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Metraux J. P., Burkhart W., Moyer M., Dincher S., Middlesteadt W., Williams S., Payne G., Carnes M., Ryals J. Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proc Natl Acad Sci U S A. 1989 Feb;86(3):896–900. doi: 10.1073/pnas.86.3.896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reyss I., Pugsley A. P. Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. Mol Gen Genet. 1990 Jul;222(2-3):176–184. doi: 10.1007/BF00633815. [DOI] [PubMed] [Google Scholar]
  18. Robbins P. W., Trimble R. B., Wirth D. F., Hering C., Maley F., Maley G. F., Das R., Gibson B. W., Royal N., Biemann K. Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J Biol Chem. 1984 Jun 25;259(12):7577–7583. [PubMed] [Google Scholar]
  19. Roberts R. L., Cabib E. Serratia marcescens chitinase: one-step purification and use for the determination of chitin. Anal Biochem. 1982 Dec;127(2):402–412. doi: 10.1016/0003-2697(82)90194-4. [DOI] [PubMed] [Google Scholar]
  20. Sawai T., Takahashi I. [Assay methods for beta-lactamase activity and their application (author's transl)]. Tanpakushitsu Kakusan Koso. 1978;23(5):391–400. [PubMed] [Google Scholar]
  21. Stark M. J., Boyd A., Mileham A. J., Romanos M. A. The plasmid-encoded killer system of Kluyveromyces lactis: a review. Yeast. 1990 Jan-Feb;6(1):1–29. doi: 10.1002/yea.320060102. [DOI] [PubMed] [Google Scholar]
  22. Takayanagi T., Ajisaka K., Takiguchi Y., Shimahara K. Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochim Biophys Acta. 1991 Jul 12;1078(3):404–410. doi: 10.1016/0167-4838(91)90163-t. [DOI] [PubMed] [Google Scholar]
  23. Takegawa K., Mikami B., Iwahara S., Morita Y., Yamamoto K., Tochikura T. Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. Eur J Biochem. 1991 Nov 15;202(1):175–180. doi: 10.1111/j.1432-1033.1991.tb16359.x. [DOI] [PubMed] [Google Scholar]
  24. Watanabe T., Oyanagi W., Suzuki K., Ohnishi K., Tanaka H. Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. J Bacteriol. 1992 Jan;174(2):408–414. doi: 10.1128/jb.174.2.408-414.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Watanabe T., Suzuki K., Oyanagi W., Ohnishi K., Tanaka H. Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. J Biol Chem. 1990 Sep 15;265(26):15659–15665. [PubMed] [Google Scholar]
  26. Wortman A. T., Somerville C. C., Colwell R. R. Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe. Appl Environ Microbiol. 1986 Jul;52(1):142–145. doi: 10.1128/aem.52.1.142-145.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yu C., Lee A. M., Bassler B. L., Roseman S. Chitin utilization by marine bacteria. A physiological function for bacterial adhesion to immobilized carbohydrates. J Biol Chem. 1991 Dec 25;266(36):24260–24267. [PubMed] [Google Scholar]
  28. Zobell C. E., Rittenberg S. C. THE OCCURRENCE AND CHARACTERISTICS OF CHITINOCLASTIC BACTERIA IN THE SEA. J Bacteriol. 1938 Mar;35(3):275–287. doi: 10.1128/jb.35.3.275-287.1938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES