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. 2014 Jan;196(2):378-90.
doi: 10.1128/JB.01094-13. Epub 2013 Nov 1.

Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus

Carmen Friedrich  1 , Iryna Bulyha, Lotte Søgaard-Andersen
Affiliations

Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus

Carmen Friedrich et al. J Bacteriol. 2014 Jan.

Abstract

Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.

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Figures

FIG 1
FIG 1
PilN, PilO, PilP, and Tgl are required for T4P-dependent motility in M. xanthus. (A) Model of the T4P machinery. The color code for the proteins is similar to the one used for the genes in the genetic map. PilD is not included in the model of the machinery. Genes and proteins are not drawn to scale. OM, outer membrane; IM, inner membrane. All the pil genes are clustered at the same locus, but not all pil genes are shown (49). tgl is not part of the pil cluster. (B) Motility phenotypes of the indicated mutants and complementation strains. Strains in the same column all contain the same in-frame deletion. Strains were incubated at 32°C for 24 h on 0.5% agar–0.5% CTT. Scale bar, 1 mm. (C) Immunoblots of T4PS protein accumulation. Total cell lysates from exponentially growing cultures were separated by SDS-PAGE (proteins from 7 × 107 cells loaded per lane) and analyzed by immunoblotting using specific antibodies as indicated. For PilQ, only the heat- and detergent-resistant oligomer is shown. The middle lane in each blot contains lysate from the relevant in-frame deletion mutant, and the last lane contains lysate from the relevant complementation strain.
FIG 2
FIG 2
T4PS protein accumulation in in-frame deletion mutants. (A) Cells were grown and analyzed as described for Fig. 1C using specific antibodies as indicated. (B) Summary of observed effects on protein stability. Green and red indicate no effect or negative effect, respectively.
FIG 3
FIG 3
Localization of PilN, PilO, PilP, PilQ, and Tgl. Cells from exponentially growing cultures were transferred to a 1.5% agar pad on a microscope slide and imaged by fluorescence microscopy in the case of the fluorescent fusion proteins of PilO, PilP, PilQ, and Tgl. For native PilN, PilP, and Tgl, cells were fixed and imaged by immunofluorescence microscopy using anti-PilN, anti-PilP, and anti-Tgl antibodies, respectively. Upper and lower rows show fluorescence and phase-contrast (Ph) microscopy images, respectively. Scale bar, 5 μm.
FIG 4
FIG 4
Localization of proteins of the T4PS. (A) Cells were analyzed as indicated for Fig. 3. Immunofluorescence microscopy was used to analyze the localization of PilB, PilC, PilM, and PilN. PilO, PilP, PilQ, and PilT were localized using the indicated sfGFP/YFP-tagged fusion proteins. Patterns of fluorescence signals were grouped into three categories. The histograms illustrate the distribution of localization patterns of the indicated proteins in the indicated in-frame deletion mutants (n = 57 to 317). *, in-frame deletion mutants in which the relevant native protein does not accumulate (compare with Fig. 2). Strains marked N/A (not applicable) were not analyzed. (B) Summary of observed effects on protein localization. Green and red indicate no effect or negative effect, respectively. Gray combinations were not analyzed. (C) Motility assays of the indicated pilM mutants and localization of the indicated PilM proteins. Upper row, motility assay of the indicated strains; scale bar, 1 mm. Cells were treated as indicated for Fig. 1B. Middle and lower rows, fluorescence (YFP) and phase-contrast (Ph) microscopy images of the indicated strains; scale bar, 2 μm. Cells were treated as indicated for Fig. 3.
FIG 5
FIG 5
PilP and PilQ as well as PilN and PilO interact directly. (A) PilN and PilO interact directly. The indicated proteins were mixed and applied to amylose and Ni-NTA matrices as indicated. Shown are relevant sections of Coomassie blue G-250-stained polyacrylamide gels of the flowthrough (F), wash (W), and elution (E) fractions with PilNΔ42-His6 (23 kDa) and MalE-PilOΔ37 (62 kDa). (B) PilP and PilQ interact directly. The experiments were carried out and are presented as for panel A, except that gel sections with MalE-PilPΔ20 (60 kDa) and PilQ20–656-His6 (69 kDa) are shown. (C) Summary of protein-protein interactions analyzed. Green, combinations of one or two proteins in which interactions were observed; red, combinations of two proteins in which no interactions were observed; gray, combinations were not analyzed.
FIG 6
FIG 6
PilQ localizes polarly late during or immediately after cell division. M. xanthus cells from an exponentially growing culture were applied directly on a coverslip with a 0.1% CTT agarose pad and imaged by fluorescence and DIC microscopy at 20-min intervals. Arrows indicate newly formed polar clusters. Strain used, SA6060 (ΔaglZ/PpilA-tgl pilQ-sfGFP). The ΔaglZ mutation blocks gliding motility. Scale bar, 5 μm.
FIG 7
FIG 7
Model of assembly of the M. xanthus T4P machinery. See the text for details. The color code is as described for Fig. 1A.
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