Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM

Abstract

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.

FIG. 1.

Cellular location of GFP-PulL and GFP-PulM in E. coli K-12. (A) Localization of GFP-PulL in wild-type E. coli and (B) in a recombinant E. coli strain in which all pul genes except pulL are expressed from plasmid pCHAP1217. (E) Localization of GFP alone in E. coli. (F) Localization of GFP-PulL in an E. coli strain carrying a plasmid encoding all pul genes including pulL (pCHAP710). (C) Localization of GFP-PulM in wild-type E. coli and (D) in E. coli harboring pCHAP1359 (lacking pulM). (G) Localization of GFP-PulM in an E. coli strain containing pCHAP710 (all pul genes, including pulM). The gfp gene and the gfp-pul chimeras were integrated into the chromosome at the λatt site, and their expression was induced with 10 μM IPTG. Fluorescence images of live cells (right) were captured using a fluorescein isothiocyanate filter; corresponding phase-contrast images are presented in the panels on the left. The bar represents 2 μm.

FIG. 2.

Distribution of GFP-PulL and GFP-PulM is unaffected by PulM and PulL, respectively. Localization of fluorescent GFP-PulL in E. coli bearing pCHAP8040 (pulM) (A) and of fluorescent GFP-PulM in E. coli with pCHAP2235 (pulL) (B). Fluorescent images are on the right, and phase-contrast images are on the left. The bar represents 2 μm.

FIG. 3.

Other Pul proteins do not affect levels of GFP-PulL and GFP-PulM. Shown are SDS-PAGE and immunoblot analysis with antiserum against GFP, MalE-PulL, MalE-PulM, and SecG of soluble (S) and particulate (P) fractions of E. coli expressing gfp-pul chimeras. GFP-PulL was produced by strain PAP9106 with or without pCHAP1217 (encoding all Pul proteins except PulL). GFP-PulM was produced by strain PAP9108 with or without pCHAP1359 (encoding all Pul proteins except PulM). GFP chimeras are indicated on the right by asterisks. The prominent band around 43 kDa and indicated by a triangle is MalE (the antibodies were raised against MalE-PulL and MalE-PulM chimeras and reabsorbed against disrupted E. coli cells with abundant MalE). PulL and PulM proteins encoded by pCHAP1359 and pCHAP1217, respectively, are indicated by a circle. Expression of the gfp gene fusions was induced with 10 μM IPTG. All fractions loaded were derived from the same volume of bacterial suspension.

FIG. 4.

Overproduction of GFP-PulL and GFP-PulM leads to polar accumulation. Fluorescent images of live cells from an E. coli strain bearing pCHAP7508 (gfp-pulL) and pCHAP7509 (gfp-pulM) grown in the absence or presence (100 μM) of inducer IPTG. The bar represents 2 μm.

FIG. 5.

Cellular location of GFP-PulL and GFP-PulM in Klebsiella oxytoca. Localization of GFP-PulL and GFP-PulM was determined by fluorescence microscopy. Genes were constitutively expressed from pCHAP7508 (gfp-pulL), pCHAP7509 (gfp-pulM), and pDSW207 (gfp) in K. oxytoca strain UNF5023. The bar represents 2 μm.