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. 2003 Jun;185(11):3416-28.
doi: 10.1128/JB.185.11.3416-3428.2003.

Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides

Guillaume Vignon  1 , Rolf Köhler, Eric Larquet, Stéphanie Giroux, Marie-Christine Prévost, Pascal Roux, Anthony P Pugsley
Affiliations

Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides

Guillaume Vignon et al. J Bacteriol. 2003 Jun.

Abstract

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).

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Figures

FIG. 1.
FIG. 1.
Immunofluorescence microscopy of E. coli K-12 (control) and derivatives carrying pCHAP231 {all pul genes [PulG], pCHAP1216 [pCHAP231 ΔpulG] and pCHAP4010 [gspG+] [GspG] or pCHAP1216 and pCHAP1380 [xcpG(T)+] [XcpT]} harvested from L-agar plates containing maltose to induce expression of the pul secreton genes carried by pCHAP231. The primary antibodies used were directed against the specific pseudopilins (anti-PulG was used for the control cells shown). Bacteria are stained red with propidium iodide (Molecular Probes), and bundled pili appear green because of the binding of primary antibodies, followed by Alexa Green 488-labeled secondary antibodies. Note that pili attach more tightly than bacteria to the poly-l-lysine-coated glass slides and that the bacteria wash off during processing to leave unattached pili on the slide.
FIG. 2.
FIG. 2.
Shearing (A) and immunofluorescence microscopy (B) of Klebsiella strains ATCC 15050 and UNF5023 with or without additional copies of the pulG gene on pCHAP1205. The bacteria were grown on LB agar containing maltose and IPTG (for strains with pCHAP1205). In the shearing analysis (A), sheared and cell-associated proteins were separated by SDS-PAGE and immunodetected with antibodies against PulG and the integral outer membrane protein LamB. E. coli K-12 strains carrying pCHAP231 and pCHAP1216 (pCHAP231 ΔpulG) were included as positive and negative controls, respectively. Details of the immunofluorescence study were as for Fig. 1.
FIG. 3.
FIG. 3.
Immunofluorescence microscopy of E. coli K-12 strain MC4100 hns carrying pCHAP4278 (gspAB gspC-O+) and pCHAP4010 (gspG) of the same strain without plasmids. The primary antibodies were directed against GspG. Other details are as for Fig. 1.
FIG. 4.
FIG. 4.
Immuno-TEM of E. coli K-12 carrying plasmids pCHAP1216 (pul+ ΔpulG) and pCCP2229 (outGEch) or pCCP2245 (outGEca) and grown on agar containing IPTG and maltose. Pili were labeled with PulG antibodies, followed by secondary antibodies tagged with 10-nm gold beads.
FIG. 5.
FIG. 5.
Phylogenetic analysis of representative proteins in the PulG family with ClustalX. Proteins whose assembly into pili by the Pul secreton is tested here are shown in bold characters. The other proteins are EptG encoded by a plasmid found in E. coli strain O157 (44), XcpG(T) from Burkholderia cepacia, HxcG(T) from P. aeruginosa (1), LspG from Legionella pneumophila (42), and GspG from Xylella fastidiosa. This figure was prepared by Dominique Vidal-Ingigliardi.
FIG. 6.
FIG. 6.
Immuno-TEM of E. coli K-12 carrying plasmids pCHAP1216 (pul+ ΔpulG) and pCHAP4278 (pulG-His6). Pili were labeled with PulG antibodies, followed by secondary antibodies labeled with 10-nm gold beads.
FIG. 7.
FIG. 7.
SDS-PAGE analysis of affinity-purified PulG-His6 pili. Proteins were stained with Coomassie brilliant blue.
FIG. 8.
FIG. 8.
TEM of purified PulG-His6 pili after negative staining with 2% uranyl acetate. A sample of tobacco mosaic virus (18-nm diameter) was included in the sample in the left panel as a size marker.
FIG. 9.
FIG. 9.
Effects of increased levels of pseudopilins on the ability of E. coli K-12 to assemble PulG pili. Strain PAP7501 carrying pCHAP231 and additional plasmids bearing pseudopilin genes under lacZp control were grown on LB agar containing maltose and IPTG. Harvested cells were subjected to shearing analysis (A) or examined by immuno-TEM with primary antibodies against PulG and secondary antibodies with 10-nm gold beads (B). Proteins blotted onto nitrocellulose membranes (A) were probed with antibodies against PulG and LamB. The band migrating slightly above PulG does not react with PulG antibodies. Control cells for the shearing experiment carried pCHAP1216, the ΔpulG derivative of pCHAP231 (lane 2; ΔG). In these samples, the band migrating faster than PulG is the truncated PulG protein that is not assembled into pili. The plasmids bearing the minor pilin genes were pCHAP1205 (pulG), pCHAP1331 (pulH), pCHAP1351 (pulI), pCHAP1328 (pulJ), and pCHAP1271 (pulK). K* (lane 8) indicates that the cells carry pCHAP5236, which is the same as pCHAP1271 except that the pulK gene has a mutation converting the methionine at position +5 to glutamate. In the samples from the strain overproducing PulG (lane 3), the band migrating more slowly that PulG is the PulG dimer that is not dissociated in SDS. Panel C shows overproduction of PulK arising from the presence of the pulK gene on pCHAP1271 (lanes 4 and 5). Lane 1 shows the level of PulK in cells with pCHAP231. Lanes 2 and 3 are controls lacking the entire pul gene cluster or with the pCHAP231 derivative pCHAP1325 (pulK::kan-2), respectively. The immunoblot of total cell extracts of maltose-grown PAP7501 strains bearing the indicated plasmids was probed with antibodies against PulK that react with an unrelated protein migrating at the position of PulK in strains lacking this protein.
FIG. 10.
FIG. 10.
Immuno-TEM of E. coli producing PulG pili without (left) or with (right) a c-myc tag at the C terminus of the pseudopilin PulG. The primary antibodies used were specific for the c-myc epitope, which is present in PulG encoded by pCHAP5246 but absent from PulG encoded by pCHAP1362 (and therefore was not labeled; arrows).
FIG. 11.
FIG. 11.
(A to D) Immuno-SEM of E. coli strain PAP9001 immunogold labeled with anti-PulG antibodies. Except for the main image in panel C (see details), all SEM pictures were taken with SEI and backscatter detectors. (A) Bacteria expressing all of the pul genes except pulG (PAP9001/pCHAP1216). Pili are not detected, and only background labeling is observed. (B) Bacteria expressing all of the pul genes, including pulG (pCHAP231). Specific labeling is observed on pili that interconnect the bacteria. (C) Bacteria expressing all of the pul genes except pulD (encoding the secretin; pCHAP1226). The main image was obtained with an SEI detector alone for clearer resolution of the bacterial cell surface. The image of the same bacterium in the top inset was taken with backscatter detectors. (D) Bacteria expressing all of the pul genes except pulD (pCHAP1226) and overexpressing pulG (pCHAP1205). Note the reappearance of surface pili because of higher-level pulG expression, despite the absence of secretin (compare panels C and D). (E and F) Immuno-TEM of bacteria similar to those in panels C and D.
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