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. 2014 Mar 11;111(10):E953-61.
doi: 10.1073/pnas.1322889111. Epub 2014 Feb 20.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Katja Siewering  1 , Samta Jain, Carmen Friedrich, Mariam T Webber-Birungi, Dmitry A Semchonok, Ina Binzen, Alexander Wagner, Stuart Huntley, Jörg Kahnt, Andreas Klingl, Egbert J Boekema, Lotte Søgaard-Andersen, Chris van der Does
Affiliations

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Katja Siewering et al. Proc Natl Acad Sci U S A. .

Abstract

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Projection maps of single-particle EM analysis of the PilQ complex from N. gonorrhoeae. Projection maps of class averages of single-particle EM images obtained from membranes isolated from the WT (A and E), the ΔtsaP strain (C and G), and the ΔtsaP/tsaP+ strain (D and H) grown in the presence of 1 mM IPTG are shown. (B and F) Class averages of single-particle EM images of the solubilized and purified His8–PilQ complex. Projection maps without (A–D) and with (E–H) 14-fold imposed symmetry are depicted. I, II, and III indicate the inner ring, the peripheral ring, and the spikes, respectively. (Scale bar: 10 nm.)
Fig. 2.
Fig. 2.
Identification of TsaP (NGFG_01788). (A) Coomassie-stained SDS/PAGE of the nonsolubilized fraction of SB3-12–treated OMs. Analysis by MS identified PilQ, TsaP (NGFG_01788), elongation factor Tu (EF-Tu), OM protein I (OMP I), OM protein III (OMP III), and a peroxiredoxin 2 (Per2) family protein. (B) Domain structure of TsaP. The signal sequence (ss) and LysM domain are depicted.
Fig. 3.
Fig. 3.
Membrane binding of TsaP depends on PilQ. (A) Immunoblot analysis of equal amounts of total cell extracts of the WT, ΔpilQ, ΔtsaP, and ΔtsaP/tsaP+ strains grown in the presence of 1 mM IPTG using α-TsaP and α-PilE antibodies. (B) Upper part of Coomassie-stained SDS/PAGE of nonphenol-treated OM fractions isolated from the indicated N. gonorrhoeae strains. (C, Left) Coomassie-stained SDS/PAGE of phenol-treated membrane fractions from the indicated strains. (C, Right) Immunoblot analysis of the same samples using α-TsaP, α-PilQ, and α-PilE antibodies. (D) Total membranes (TM) derived from N. gonorrhoeae WT (Left) and the ΔpilQ mutant (Right) were treated twice for 30 min with 7.5 M urea. After centrifugation, the supernatants (W1 and W2) and the resuspended membrane pellets (P) were analyzed by immunoblot analysis using α-TsaP antibodies.
Fig. 4.
Fig. 4.
Deletion of TsaP leads to formation of membrane protrusions containing T4P in N. gonorrhoeae. An EM analysis of WT, ΔtsaP, and ΔtsaP/tsaP+ strains grown in the presence of 1 mM IPTG was performed. Cells were applied to carbon-coated copper grids, washed twice with double-distilled water, and subsequently stained with uranyl acetate before investigation via EM. T4P (black arrows) and membrane blebs (black arrowheads) are shown. (Inset) Membrane protrusions (white arrows) observed in the ΔtsaP mutant are filled with T4P. (Scale bars: main images, 200 nm; Inset, 50 nm.)
Fig. 5.
Fig. 5.
Identification of genes encoding TsaP homologs and T4aPS-related genes in different genomes. A reciprocal BLAST analysis was performed for six proteins representative of T4aPSs (PilQ, PilT, PilF, PilM, PilN, and PilO), as well as for TsaP, to identify the different proteins in 450 proteobacterial genomes. Results were plotted on the 16S RNA phylogenetic tree. Colored boxes indicate the presence of a TsaP ortholog or the presence of at least four of the six proteins representative of T4aPSs.
Fig. 6.
Fig. 6.
Characterization of TsaP of M. xanthus. (A) PilQMX accumulates independently of TsaPMX. Equal amounts of total cell extracts of the indicated strains were separated by SDS/PAGE and analyzed by immunoblots with the α-PilQMX antibody. The upper and lower bands correspond to multimeric and monomeric PilQMX, respectively. (B) TsaPMX accumulation depends on PilQMX (as in A but analyzed with the α-TsaPMX antibody). TsaPMX and TsaPMX-mCherry are indicated. (C) TsaPMX is important for T4P-dependent motility. The indicated M. xanthus strains were incubated at 32 °C for 24 h on 0.5% agar/0.5% CTT medium. (Scale bar: 1 mm.) (D) Lack of TsaPMX reduces the number of T4P. Cells from exponentially growing cultures were visualized by EM after staining with uranyl acetate. (Scale bars: 1 μm.) (E) Histogram summarizes the number of T4P per cell of the indicated strains (n = 19–55). Mean values and SDs for each strain are indicated. (F) TsaPMX localizes preferentially to the cell poles and is dependent on PilQMX, whereas bipolar PilQ localization is independent of TsaPMX. (Top) Fluorescence microscopy and phase-contrast images (Insets) of WT, ΔpilQMX, and ΔtsaPMX strains expressing TsaPMX-mCherry. (Middle and Bottom) Fluorescence microscopy and phase-contrast images of fixed cells probed with α-TsaPMX or α-PilQMX antibodies. (Scale bar for main figure and Inset: 5 μm.)
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