Cloning and characterisation of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant

Peadar O'Gaora, Duncan Maskell, David Coleman, Mary Cafferkey, Gordon Dougan

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Abstract

A gene library of Yersinia enterocolitica 8081 was constructed in the cosmid vector pHC79. Recombinants containing the aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, were identified by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. All six recombinant plasmids which complemented aroA also complemented the serC mutation in E. coli K-12 strain KL282. Tn5 mutagenesis suggested serC encoding 3-phosphoserine aminotransferase was the proximal gene in an operon with aroA. The nucleotide sequence of a 3-kb HindII-EcoRV fragment encoding the two genes was determined. The serC and aroA open reading frames contain 362 and 428 codons, respectively, and the deduced amino acid sequences share 78% and81% homology, respectively, with the corresponding E. coli genes. Sequence inspection revealed no obvious terminators or promoters in the intergenic region. The cloned Y. enterocolitica aroA gene was inactivated in vitro and reintroduced into the parental Y. enterocolitica 8081 strain using the suicide vector pJM703.1. Stable aroA insertion mutants of Y. enterocolitica were isolated.

Original languageEnglish
Pages (from-to)23-30
Number of pages8
JournalGene
Volume84
Issue number1
DOIs
Publication statusPublished - 7 Dec 1989
Externally publishedYes

Keywords

  • complementation
  • cosmid
  • gene library
  • intergenic region
  • operon
  • Recombinant DNA
  • sequence homology

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