Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM.
The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.
Molecular Genetics Unit, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Nienke
N
Buddelmeijer
Olivera
O
Francetic
Anthony P
AP
Pugsley
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002458:Cell Fractionation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D017930:Genes, Reporter; D006026:Glycoside Hydrolases / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D041121:Klebsiella oxytoca / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence; D021381:Protein Transport
D001426:Bacterial Proteins; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase