title: Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane.
abstract: The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.
authors.affiliation: Institut Pasteur, Molecular Genetics Unit and CNRS URA2172, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Nienke
authors.initials: N
authors.lastname: Buddelmeijer
authors.forename: Martin
authors.initials: M
authors.lastname: Krehenbrink
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pecorari
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D018832:Molecular Chaperones; C061377:PulD protein, Klebsiella pneumoniae