Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae.
Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.
d'Enfert
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Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
Chapon
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Pugsley
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AP
D002462:Cell Membrane / Q000201:enzymology; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D011499:Protein Processing, Post-Translational
D008074:Lipoproteins; D046911:Macromolecular Substances; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases; C100181:pullulanase