Expression and biochemical analysis of the galacturonate transport protein or Erwinia chrysanthemi EC16
Haseloff, Brian James
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Erwinia chrysanthemi is a plant pathogen responsible for causing disease on a wide variety of plants. Pathogenicity is due in part to the degradation of pectin, an integral component of the plant cell wall. Pectin is degraded into monomers, dimers. And oligomers of galacturonate. These molecules are transported into the bacterium where they are metabolized and generate inducers for the production and secretion of the pectin degrading enzymes. The uptake of these molecules into the bacterium, therefore, plays an important role in the virulence potential of the bacterium. The exuT gene, encoding the protein for transport of galacturonate (GalUA) into E. chrysanthemi has been previously cloned and sequenced. In order to study this permease, the GalUA transporter was overexpressed and localized to the bacterial cytoplasmic membrane. The toplogy of the protein was mapped using information from computer algorithms, hydrophobic analysis, and gene fusion studies. These studies indicate that ExuT has nine transmembrane a-helices, four periplasmic domains, and four cytoplasmic domains The amino-terminus resides in the cytoplasm and the carboxy-terminus in the periplasm. Mutations in exuT were generated by marker exchange mutagenesis and characterized for their ability to grow on and transport GalUA. These mutants were also assessed for their ability to cause disease on plant tissue. The ^JCWJ mutants were unable to grow on GalUA as a sole carbon source and showed reduced uptake of the monomer The maceration of plant tissue by these mutants was delayed and reduced significantly when compared to the parent strain EC 16.