Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 25;286(12):9977-84.
doi: 10.1074/jbc.M110.212688. Epub 2011 Feb 1.

Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27

Janin Burkhardt  1 , Janet Vonck, Beate Averhoff
Affiliations

Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27

Janin Burkhardt et al. J Biol Chem. .

Abstract

Secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type IV pili, DNA and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. PilQ of the thermophilic bacterium Thermus thermophilus HB27 is a member of the secretin family required for natural transformation. Here we report the isolation, structural, and functional analyses of a unique PilQ from T. thermophilus. Native PAGE, gel filtration chromatography, and electrophoretic mobility shift analyses indicated that PilQ forms a macromolecular homopolymeric complex that binds dsDNA. Electron microscopy showed that the PilQ complex is 15 nm wide and 34 nm long and consists of an extraordinary stable "cone" and "cup" structure and five ring structures with a large central channel. Moreover, the electron microscopic images together with secondary structure analyses combined with structural data of type II protein secretion system and type III protein secretion system secretins suggest that the individual rings are formed by conserved domains of alternating α-helices and β-sheets. The unprecedented length of the PilQ complex correlated well with the distance between the inner and outer membrane of T. thermophilus. Indeed, PilQ was found immunologically in both membranes, indicating that the PilQ complex spans the entire cell periphery of T. thermophilus. This is consistent with the hypothesis that PilQ accommodates a PilA4 comprising pseudopilus mediating DNA transport across the outer membrane and periplasmic space in a single-step process.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Detection of a PilQ complex in T. thermophilus HB27. A and B, 3–12% polyacrylamide gradient SDS-PAGE of crude extracts of HB27 wild type (wt) and pilQ mutant (pilQ::kat) (A) followed by immunoblotting with antiserum against PilQ (B). C, membranes of HB27 were treated with 10% SDS and incubated at 100 °C. Samples were taken at the indicated time points and subjected to Western blot analyses with PilQ antibodies.
FIGURE 2.
FIGURE 2.
Subcellular localization of PilQ. T. thermophilus crude extracts were separated in soluble (S) and membrane (M) fractions. Total membranes were separated in IM and OM fractions. The fractions were subjected to SDS-PAGE in a 3–12% polyacrylamide gradient gel followed by immunoblotting with PilQ antibodies.
FIGURE 3.
FIGURE 3.
Purification of the PilQ complex. Protein fractions of the different steps of the purification procedure were analyzed on a 3–12% polyacrylamide gradient SDS-PAGE gel. Lane 1, crude extract; lane 2, membrane fraction; lane 3, solubilized proteins; lane 4, pooled PilQ-containing fractions of the density gradient; lane 5, pooled PilQ-containing fractions of the anion exchange chromatography (Q-Sepharose); lane 6, pooled PilQ-containing fractions of the gel filtration (Superose 6).
FIGURE 4.
FIGURE 4.
Subunit composition of the PilQ complex. A and B, the purified PilQ complex was treated with hot phenol and subjected to 3–12% polyacrylamide gradient SDS-PAGE (A) and immunoblotting (B). Lane 1, untreated PilQ complex; lane 2, PilQ complex treated with hot phenol. C, the purified untreated PilQ complex was subjected to Blue native-PAGE followed by immunoblotting.
FIGURE 5.
FIGURE 5.
Electron microscopic analysis of PilQ. A, class averages from a data set of 2000 PilQ particles after multivariate statistical analysis and classification. B and C, class averages of SDS-treated PilQ from a data set of 4000 particles. B, side views; C, top views. The scale bar represents 20 nm for all panels.
FIGURE 6.
FIGURE 6.
DNA binding of the PilQ complex measured by bandshift analyses. Reaction mixtures contained 1 nm (30,000 cpm) of dsDNA. DNA was incubated with 5, 20, 50, 100, and 200 nm purified PilQ complex at 37 °C (A) and 68 °C (B), respectively. Competitor DNA was added to 200 nm PilQ complex in 20-fold excess (lane C). 200 nm BSA was used as negative control.
FIGURE 7.
FIGURE 7.
Secondary structure prediction of secretins. The PredictProtein server was used to predict potentially β-sheet (gray boxes) and α-helical (black boxes) regions within the secretins shown. Conserved C-terminal domains are indicated as dark gray boxes A, B, C, and D (61) for PilQ of T. thermophilus; the processing site is indicated by an arrow. T. t., T. thermophilus; K. o., K. oxytoca; E. c., E. coli; P. a., Pseudomonas aeruginosa; S. e., Salmonella enterica; Y. p., Yersinia pestis; EPEC, enteropathogenic E. coli; BFP, bundle-forming pilus system; N. m. Neisseria meningitidis.
See this image and copyright information in PMC

References

    1. Johnsborg O., Eldholm V., Håvarstein L. S. (2007) Res. Microbiol. 158, 767–778 - PubMed
    1. Averhoff B. (2009) FEMS Microbiol. Rev. 33, 611–626 - PubMed
    1. Averhoff B. (2004) J. Bioenerg. Biomembr. 36, 25–33 - PubMed
    1. Hobbs M., Mattick J. S. (1993) Mol. Microbiol. 10, 233–243 - PubMed
    1. Tønjum T., Freitag N. E., Namork E., Koomey M. (1995) Mol. Microbiol. 16, 451–464 - PubMed

MeSH terms

LinkOut - more resources