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Cloning of theHelicobacter pylori recA gene and functional characterization of its product

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Abstract

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ70 promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA −) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.

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Authors and Affiliations

  1. Abteilung Infektionsbiologie, Max Planck-Institut für Biologie, Spemannstr, 34, D-72076, Tübingen, Germany

    Wolfgang Schmitt, Stefan Odenbreit, Dorothee Heuermann & Rainer Haas

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  1. Wolfgang Schmitt
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  2. Stefan Odenbreit
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  3. Dorothee Heuermann
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  4. Rainer Haas
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Communicated by W. Goebel

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Schmitt, W., Odenbreit, S., Heuermann, D. et al. Cloning of theHelicobacter pylori recA gene and functional characterization of its product. Molec. Gen. Genet. 248, 563–572 (1995). https://doi.org/10.1007/BF02423452

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  • DOI: https://doi.org/10.1007/BF02423452

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