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. 2020 Oct 7;11(1):5054.
doi: 10.1038/s41467-020-18803-z.

PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus

Anke Treuner-Lange  1 , Yi-Wei Chang  2   3 , Timo Glatter  1 , Marco Herfurth  1 , Steffi Lindow  1 , Georges Chreifi  2 , Grant J Jensen  2   4 , Lotte Søgaard-Andersen  5
Affiliations

PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus

Anke Treuner-Lange et al. Nat Commun. .

Abstract

Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. PilY1 and minor pilins are essential for T4aP-dependent motility.
a Architectural model of T4aPM. The OM pore (PilQ secretin and TsaP) connect to PilP. PilP connect to a lower periplasmic ring (globular domains of PilN and PilO), which connect to a cytoplasmic ring (PilM). PilN/O and PilM generate a cage, which is occupied by PilC. PilB and PilT associate with PilC in a mutually exclusive manner during extension and retraction, respectively,,. For simplicity, PilB and PilT are not shown separately. Bent arrows, incorporation and removal from the pilus base of PilA during extension and retraction, respectively. Proteins labeled with single letters have the Pil prefix. b Schematic of M. xanthus gene clusters encoding PilY1 and minor pilins. Numbers below genes, distances between genes. Black lines below, genes deleted in cluster deletion mutants. c PilY1 and minor pilins are essential for T4aP-dependent motility. Scale bar, 1 mm. Strains motile by T4aP generate flares at the colony edge while strains non-motile by T4aP generate smooth-edged colonies. d Minor pilins and PilY1 are essential for T4aP formation. T4aP sheared off from ~15 mg cells were separated by SDS-PAGE and probed with α-PilA antibodies (top rows). Middle row, 40 µg of protein from total cell extracts were separated by SDS-PAGE and probed with α-PilA antibodies (middle rows) and, after stripping, with α-LonD antibodies as a loading control (bottom rows). e Minor pilins and PilY1 are essential for T4aP extension. Samples were prepared as in d. For better comparison, only 5% of T4aP sheared from the hyper-piliated ∆pilT strain (asterisk) and 25% of protein (hash) was applied. f Accumulation levels of T4aPM proteins. Protein from 3 × 108 cells was loaded per lane. In lane labeled ∆, protein from relevant in-frame deletion mutants was loaded. Gaps between lanes indicate lanes that were deleted for presentation purposes. g Bipolar localization of mCherry-PilM as a read-out for assembly of T4aPM. Scale bar, 5 µm. The results for the deletions of fimUpilVpilW en bloc in c–e were previously published and are included for comparison.
Fig. 2
Fig. 2. PilA and cluster_3 minor pilins interact.
a BACTH analysis of PilA and cluster_3 minor pilin interactions. T25 and T18 were fused to the N-terminus of the indicated mature, full-length proteins. Positive control (plus) in upper left corner, T25-Zip + T18-Zip; T18 and T25, negative control (minus). Full-length AglQ fused to T25 and T18 served as a specificity control. Left panel, representative images of E. coli BTH101 expressing the indicated protein fusions. For every tested interaction pair, four clones were tested with similar results. Middle panel, relative activity of β-galactosidase shown as mean ± standard deviation (n = 4) for the pairs on the left. Right panel, schematic summarizing observed interactions. b Fractionation of BTH101 E. coli cells expressing the indicated T18 fusions. Cleared cell lysates were fractionated into soluble (S) and membrane (M) fractions. αN N-terminal α-helix in mature pilins, TM trans-membrane helix in AglQ. Proteins from the same number of cells were loaded per lane. Right panel, schematics of domain organization and calculated molecular sizes of the corresponding proteins.
Fig. 3
Fig. 3. Cluster_3 minor pilins and PilY1.3 interact.
a T4aP-dependent motility assays for strains with tagged PilY1.3 or PilW3. Strains of the indicated genotypes were incubated 24 h. Scale bar, 1 mm. b Accumulation levels of PilY1.3-FLAG and GFP-FLAG. Protein from 3 × 108 cells were loaded per lane and blots probed with α-FLAG antibodies and α-LonD antibodies (loading control). c PilY1.3 and cluster_3 minor pilins interact. Pull-down experiments with α-FLAG matrix on cell extracts from strain of the indicated genotype expressing PilY1.3-FLAG or GFP-FLAG (negative control). Samples from two biological replicates and two negative controls were analyzed by LFQ-MS; mean iBAQ values and log2-fold enrichment in PilY1.3-FLAG samples compared to GFP-FLAG samples were calculated; enrichment for PilY1.3-FLAG was imputed. Columns represent mean of log2-fold enrichment (n = 2); dots represent the corresponding data points; n.d., not detected in PilY1.3-FLAG samples. d, e Accumulation levels of PilY1.3-sfGFP and PilW3-sfGFP. Protein from 3 × 108 cells was loaded per lane and blots were probed with α-GFP antibodies and α-LonD antibodies (loading control). Gap between lanes indicate lanes that were deleted for presentation purposes. f PilY1.3-sfGFP and PilW3-sfGFP localize bipolarly in the presence of assembled T4aPM. Insets are enlargements of the boxed areas. Note that the PilW3-sfGFP clusters formed in the ∆pilQ strain were smaller and of lower intensity than in WT. N > 100 cells per strain; localization patterns in percentage indicated in schematics (bipolar, unipolar, diffuse (top to bottom)). Scale bars, 5 µm. g PilY1.3-sfGFP and cluster_3 minor pilins interact. Pull-down experiments with α-GFP matrix and cell extracts of cells of the indicated genotype containing PilY1.3-sfGFP or GFP-FLAG (negative control). Samples were analyzed as in c; enrichment was imputed for PilY1.3-sfGFP. h PilW3-sfGFP and cluster_3 proteins interact. Pull-down experiments with α-GFP matrix on cell extracts of the indicated genotype containing PilW3-sfGFP or GFP-FLAG (negative control). Samples were analyzed as in c; enrichment was imputed for PilW3-sfGFP.
Fig. 4
Fig. 4. PilA, minor pilins, and PilY1 make up the short stem and plug.
a Structures (subtomogram averages) of T4aPM in strains of the indicated genotypes. Top row, central slices through subtomogram averages of empty T4aPM. Lower row, first panel describes densities observed in subtomogram average of empty WT T4aPM (see also Fig. 1a) as described in ref. , panels 2–5 show differences between T4aPM in mutants versus the WT (lacking densities, yellow), and panels 6 and 7 the difference between T4aPM in SA7791 (∆1∆2cluster ∆pilY1.3 + PilY1.3-sfGFP) and SA8764 (∆1∆2cluster ∆pilY1.3 + PilY1.3∆vWFA-FLAG) versus T4aPM in ∆1∆2cluster structure (lacking densities, yellow; extra density, red). Panels 1, 3, and 4 in both rows are from ref. (reprinted with permission from AAAS). Scale bar, 10 nm. b Model of T4aPM priming complex. Panel 1, component map of non-piliated T4aPM with priming complex and proteins assigned to the densities in the subtomogram average of the non-piliated WT T4aPM; for simplicity, PilB and PilT are not shown separately. Panel 2, hypothetical structural model of priming complex after low-pass filtering to 3-nm resolution and placed into the subtomogram average of non-piliated WT T4aPM.
Fig. 5
Fig. 5. Minor pilins and PilY1 are present in T4aP.
a Cluster_3 minor pilins and PilY1.3 are enriched in purified T4aP. T4aP were purified from the hyper-piliated ∆pilT mutant and the non-piliated ∆pilB∆pilT mutant (negative control). Samples from two biological replicates were analyzed by LFQ-MS, mean iBAQ values were determined, and log2-fold enrichment in ∆pilT samples compared to the negative control was calculated. For PilW3, enrichment was calculated using imputed values. Columns represent mean of log2-fold enrichment (n = 2), dots represent the corresponding data points. b iBAQ values from negative control were subtracted from iBAQ values of the ∆pilT samples from a; subsequently, these values were normalized relative to an iBAQ value of 10,000 for PilA in the ∆pilT sample. The columns represent the mean of normalized IBAQ values (n = 2), dots represent the corresponding data points. c Cryo-ET images of T4aP tip structures. First panel, T4aP extending from cell surface (arrow) and with the kinked structure at the tip; scale bar, 50 nm. Second panel, magnification of area in stippled box in first panel illustrating kinked structure without a density gap; scale bar, 10 nm. Third panel, the same pilus tip image as in the second panel, with double-headed arrows indicating the directions of length (L), width (W), and kink angle (θ) measurements. The result of measurements on the 31 available T4aP tip is listed underneath the panel (mean ± standard deviation). Fourth panel, kinked tip structure with a density gap between terminal globular density and the tip of the pilus shaft; scale bar, 10 nm.
Fig. 6
Fig. 6. Three functions of the PilY1/minor pilin complex.
Left panel, architectural model of non-piliated T4aPM with ten core proteins and the priming complex composed of PilY1, four minor pilins, and one PilA subunit. Right panel, architectural model of piliated T4aPM with ten core proteins and the priming complex at the pilus tip; in the piliated T4aPM, PilB and PilT associate with PilC in a mutually exclusive manner during extension and retraction, respectively. For simplicity, PilB and PilT are not shown separately. Proteins labeled with single letters have the Pil prefix. Color code for PilY1 and minor pilins as in Fig. 4b.
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