Current Microbiology

, Volume 21, Issue 5, pp 329–337 | Cite as

Cloning and expression of anAeromonas hydrophila chitinase gene inescherichia coli

  • Paul E. Roffey
  • John M. Pemberton


The gene encoding an extracellular chitinase fromAeromonas hydrophila has been cloned and expressed inEscherichia coli. Plasmid pJP2512 contained the smallest DNA insert (3.9 kb) producing chitinase. The chitinase gene is transcribed from its own promoter, producing a protein of Mr 96,000. The chitinase open reading frame, an estimated 2.6 kb in length, has been subcloned to a 3.0 kb fragment; however, this fragment does not carry the functional chitinase promoter. InE. coli the chitinase enzyme is unable to transverse the outer membrane, being secreted across the cytoplasmic membrane and accumulating in the periplasmic space.


Enzyme Outer Membrane Chitinase Cytoplasmic Membrane Chitinase Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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Literature Cited

  1. 1.
    Berkeley RCW (1979) Chitin, chitosan and their degradative enzymes. In: Berkeley RCW, Gooday GW, Ellwood DC (eds) Microbial polysaccharides and polysaccharases. London: Academic Press, pp 205–236Google Scholar
  2. 2.
    Burke V, Gracey M, Robinson J, Peck D, Beaman J, Bundall C (1983) The microbiology of childhood gastroenteritis:Aeromonas species and other infective agents. J Infect Dis 148:68–74Google Scholar
  3. 3.
    Cabib E (1987) The synthesis and degradation of chitin. In: Meister A (ed) Advances in enzymology, vol 59. New York: John Wiley and Sons, pp 59–101Google Scholar
  4. 4.
    Chakraborty T, Montenegro MA, Sanyal SC, Helmuth R, Bulling E, Timmis KN (1984) Cloning of Enterotoxin gene fromAeromonas hydrophila provides conclusive evidence of production of a cytotonic enterotoxin. Infect Immun 46:435–441Google Scholar
  5. 5.
    Chung CT, Miller RH (1988) A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res 16:3580Google Scholar
  6. 6.
    Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antiobiotic resistance in bacteria: genetic transformation ofE. coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114Google Scholar
  7. 7.
    Cornelis P, Digneffe C, Willmot K (1982) Cloning and expression of aBacillus coagulans amylase gene inEscherichia coli. Mol Gen Genet 186:507–511Google Scholar
  8. 8.
    Ferenci T, Silhavy TJ (1987) Sequence information required for protein translocation from the cytoplasm. J Bacteriol 169:5339–5342Google Scholar
  9. 9.
    Focareta T, Manning PA (1987) Extracellular proteins ofVibrio cholerae: molecular cloning, nucleotide sequencing and characterization of the deoxyribonuclease (DNase) together with its periplasmic location inEscherichia coli K-12. Gene 53:31–40Google Scholar
  10. 10.
    Freudl F, Schwartz H, Degen M, Henning U (1987) The signal sequence suffices to direct export of outer membrane protein Omp A ofEscherichia coli K-12. J Bacteriol 169:66–71Google Scholar
  11. 11.
    Gilardi GL (1971) Characterization of non-fermentative, non-fastidious Gram-negative bacteria encountered in medical bacteriology. J Appl Bacteriol 34:623–644Google Scholar
  12. 12.
    Gobius KS, Pemberton JM (1986) Use of the plasmid pULB113 (RP4::Min-mu) to construct a genomic map ofAeromonas hydrophila. Curr Microbiol 13:111–115Google Scholar
  13. 13.
    Gobius KS, Pemberton JM (1988) Molecular cloning, characterization and nucleotide sequence of an extracellular amylase gene fromAeromonas hydrophila. J Bacteriol 170:1325–1332Google Scholar
  14. 14.
    Gooday GW (1977) Biosynthesis of the fungal cell wall-mechanisms and implications. J Gen Microbiol 99:1–11Google Scholar
  15. 15.
    Gooday GW (1983) The microbial synthesis of cellulose, chitin and chitosan. Prog Indust Microbiol 18:85–127Google Scholar
  16. 16.
    Hazen TC, Raker ML, Esch GW, Fliermans CB (1978) Ultrastructure of red-sore lesion on largemouth bass (Micropterus salmoides): association of ciliateEpistylis sp and the bacteriumAeromonas hydrophila. J Protozool 25:351–355Google Scholar
  17. 17.
    Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298Google Scholar
  18. 18.
    Howard SP, Buckley JT (1985) Protein export by a Gramnegative bacterium: production of aerolysin byAeromonas hydrophila. J Bacteriol 161:1118–1124Google Scholar
  19. 19.
    Howard SP, Buckley JT (1986) Molecular cloning and expression inEscherichia coli of the structural gene for the hemolytic toxin aerolysin fromAeromonas hydrophila. Mol Gen Genet 204:289–295Google Scholar
  20. 20.
    Kaper JB, Lockman H, Colwell RR, Joseph SW (1981)Aeromonas hydrophila: ecology and toxigenicity of isolates from an estuary. J Appl Bacteriol 50:359–377Google Scholar
  21. 21.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  22. 22.
    Lejeune A, Dartois V, Colson C (1988) Characterization and expression inEscherichia coli of an endogluconase gene ofPseudomonas fluorescens subspcellulosa. Biochim Biophys Acta 950:204–214Google Scholar
  23. 23.
    Ljungh A, Wadstrom T (1983) Toxins ofVibrio parahaemolyticus andAeromonas hydrophila. J Toxicol Tox Rev 1:257–307Google Scholar
  24. 24.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, pp 431–433Google Scholar
  25. 25.
    Naumovski L, Friedberg EC (1982) Molecular cloning of eukaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD 3 gene ofSaccharomyces cerevisiae. J Bacteriol 152:323–331Google Scholar
  26. 26.
    O'Brien M, Colwell RR (1987) A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-β-d-glucosamide. Appl Environ Microbiol 53:1718–1720Google Scholar
  27. 27.
    Priefer U (1984) Characterization of plasmid DNA by agarose gel electrophoresis. In: Pühler A, Timmis KN (eds) Advanced molecular genetics. Berlin: Springer-Verlag, pp 26–37Google Scholar
  28. 28.
    Priefer U, Simon R, Pühler A (1984) Cloning with cosmids. In: Pühler A, Timmis KN (eds) Advanced molecular genetics. Berlin: Springer-Verlag, pp 190–201Google Scholar
  29. 29.
    Pugsley AP, Schwartz M (1985) Export and secretion of proteins by bacteria. FEMS Microbiol Rev 32:3–38Google Scholar
  30. 30.
    Quigley NB, Reeves PR (1987) Chloramphenicol resistance cloning vector based on pUC9. Plasmid 17:54–57Google Scholar
  31. 31.
    Rasmussen BA, Silhavy TJ (1987) The first 28 amino acids of mature Lam B are required for rapid and efficient export from the cytoplasm. Genes Dev 1:185–196Google Scholar
  32. 32.
    Rigney MM, Zilinsky JW, Rouf MA (1978) Pathogenicity ofAeromonas hydrophila in red-leg disease in frogs. Curr Microbiol 1:175–179Google Scholar
  33. 33.
    Rippey SR, Cabelli VJ (1980) Occurrence ofAeromonas hydrophila in limnetic environments: relationship of the organism to trophic state. Microbial Ecol 6:45–54Google Scholar
  34. 34.
    Robbins PW, Albright C, Benfield B (1988) Cloning and expression of aStreptomyces plicatus chitinase (Chitinase-63) inEscherichia coli. J Biol Chem 263:443–447Google Scholar
  35. 35.
    Roberts RL, Cabib E (1982)Serratia marcescens chitinase: one-step purification and uses for the determination of chitin. Anal Biochem 127:402–412Google Scholar
  36. 36.
    Russel M, Model P (1984) Replacement of thefip gene ofEscherichia coli by an inactive gene cloned on a plasmid. J Bacteriol 159:1034–1039Google Scholar
  37. 37.
    Seidler RJ, Allen DA, Lockman H, Colwell RR, Joseph SW, Daily OP (1980) Isolation, enumeration and characterization ofAeromonas from polluted water encountered in diving operations. Appl Environ Microbiol 39:1010–1018Google Scholar
  38. 38.
    Shieh HS (1987) Protection of Atlantic salmon against motile aeromonad septicemia withAeromonas hydrophila protease. Microbios Lett 36:133–138Google Scholar
  39. 39.
    Stelma GN (Jr), Johnson CH, Spaulding P (1986) Evidence for the direct involvement of the β-haemolysin inAeromonas hydrophila enteropathogenicity. Curr Microbiol 14:71–77Google Scholar
  40. 40.
    Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078Google Scholar
  41. 41.
    Teather RM, Wood PJ (1982) Use of congo red-polysaccharide interaction in the enumeration and characterization of celluloytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780Google Scholar
  42. 42.
    Tracey MV (1957) Chitin. Pure Appl Chem 7:1–14Google Scholar
  43. 43.
    Turnbull PC, Lee JV, Miliotis MD, Van de Walle S, Koornhof HJ, Jeffery L, Bryant TN (1984) Enterotoxin production in relation to taxonomic grouping and source of isolation ofAeromonas species. J Clin Microbiol 19:175–180Google Scholar
  44. 44.
    Vieira J, Messing J (1982) The pUC plasmids, and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic primers. Gene 19:259–268Google Scholar
  45. 45.
    Wharton D (1980) Nematode egg-shells. Parasitology 81:447–463Google Scholar
  46. 46.
    Whistler RL, Smart CL (1953) Chitin. In: Polysaccharide chemistry. New York: Academic Press, pp 395–405Google Scholar
  47. 47.
    Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag New York Inc 1990

Authors and Affiliations

  • Paul E. Roffey
    • 1
  • John M. Pemberton
    • 1
  1. 1.Department of MicrobiologyUniversity of QueenslandBrisbaneAustralia

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