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Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production

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Summary

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75–2, 75–5 and 75–6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 as PehA protein, respectively, fused to the mature part of the periplasmic β-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75–5 and 75–6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp− mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR−), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR− mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR−1 as well as PehA−1 mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

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Author notes

  1. Hannu T. Saarilahti

    Present address: Department of Biochemistry, University of Helsinki, SF-00100, Helsinki, Finland

  2. Diana Flego

    Present address: Department of Plant Pathology, National University of Mar del Plata, Box 276, AR-7620, Balcarce, Argentina

Authors and Affiliations

  1. Department of Molecular Genetics, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Box 7003, S-750 07, Uppsala, Sweden

    Hannu T. Saarilahti, Minna Pirhonen, Maj-Britt Karlsson, Diana Flego & E. Tapio Palva

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  1. Hannu T. Saarilahti
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  2. Minna Pirhonen
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  3. Maj-Britt Karlsson
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Communicated by H. Hennecke

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Saarilahti, H.T., Pirhonen, M., Karlsson, MB. et al. Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production. Molec. Gen. Genet. 234, 81–88 (1992). https://doi.org/10.1007/BF00272348

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  • DOI: https://doi.org/10.1007/BF00272348

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