Abstract
The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.
Highlights
From the Unite de Genetiaue Moleculaire (Centre National de la Recherche Scientifique UA1149), Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France
Replacement ofthe Asp2residue by Asn, Glu,or Ser causedthe enzyme to fractionate with outer membrane-derivedvesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes
We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located
Summary
Coli,depending onwhether or not the producingstrains One of the very few lipoproteins that cross OthMe express the genes required for the is pullulanase (PulA), a n amylolyticenzymenormally prosecond step in pullulanase secretion. When they are duced by strains of Klebsiella (Michaelis et al, 1985; d'Enfert expressed, the enzyme is localized to the cell surface, et al, 1987a). Replacement ofthe Asp2residue by Asn, Glu,or Ser causedthe enzyme to fractionate with outer membrane-derivedvesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes.
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