Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila

Ashok Chopra, R. Pham, C. W. Houston

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage λEMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli[pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56°C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and λ bacteriophage libraries. We localized this heat-stable (56°C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Original languageEnglish (US)
Pages (from-to)87-91
Number of pages5
JournalGene
Volume139
Issue number1
DOIs
StatePublished - Feb 11 1994

Fingerprint

Aeromonas hydrophila
Enterotoxins
Organism Cloning
Cosmids
Genes
Clone Cells
Cricetulus
Ovary
Hot Temperature
DNA
Escherichia coli
Bacteriophages
Aeromonas
Genomic Library
Sodium Dodecyl Sulfate
Methionine
Cyclic AMP
Polyacrylamide Gel Electrophoresis
Plasmids
Peptides

Keywords

  • Aeromonas trota
  • cAMP
  • CHO cell elongation
  • cholera toxin
  • Phage T7 RNA polymerase/promoter system
  • Southern blot analysis

ASJC Scopus subject areas

  • Genetics

Cite this

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila. / Chopra, Ashok; Pham, R.; Houston, C. W.

In: Gene, Vol. 139, No. 1, 11.02.1994, p. 87-91.

Research output: Contribution to journalArticle

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abstract = "A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage λEMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli[pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56°C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and λ bacteriophage libraries. We localized this heat-stable (56°C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.",
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