title: Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12.
abstract: Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.
authors.affiliation: Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: d'Enfert
authors.forename: I
authors.initials: I
authors.lastname: Reyss
authors.forename: C
authors.initials: C
authors.lastname: Wandersman
authors.forename: A P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006026:Glycoside Hydrolases / Q000378:metabolism*; D015151:Immunoblotting; D007763:Lac Operon / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; C560058:PulD protein, E coli; D011993:Recombinant Fusion Proteins; C061378:PulC protein, Klebsiella pneumoniae; C061377:PulD protein, Klebsiella pneumoniae; D000469:Alkaline Phosphatase; D006026:Glycoside Hydrolases; C100181:pullulanase