A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel.

Bacterial strains and plasmids

Bacterial strains used in this study are derived from L. pneumophila strains LP01 and LP02 and are listed in Table S1 . L. pneumophila was grown on charcoal yeast extract (CYE) plates in 37 °C as described previously 21 . L. pneumophila strains were constructed by standard recombinant DNA and allelic exchange procedures using the plasmid pSR47S as described previously 22 . sfGFP or mCherry were inserted downstream the Dot/Icm genes and separated by a DNA linker encoding Arg-Thr-Gly-Gly-Ala-Ala.

Fluorescent microscopy imaging and processing Imaging of L. pneumophila expressing

Dot/Icm fluorescent fusions was carried out by resuspension of 2 day heavy patches in water, after which they were spotted on a thin pad of 1% agarose, covered with a cover slip and immediately imaged at room temperature. Fluorescence micrographs were captured using a Nikon Eclipse TE2000-S inverted microscope equipped with a Spectra X light engine from Lumencor, CoolSNAP EZ 20MHz digital monochrome camera from Photometrics and a Nikon Plan Apo100× objective len (1.4 numerical aperture) under the control of SlideBook™ 6.0 (Intelligent Imaging Innovations). Samples were imaged using a 196 mW 485 nm or a 260 mW 560 nm LED lights, with typical exposure times of 100-400 ms and 2×2 binning. Time lapses of DotB-sfGFP were acquired using continuous illumination. Polarity scores were calculated with SlideBook™ by measuring the ratio between the variance and the mean of the fluorescence signal at region of interest located between the pole and the cell center 22 . DotB-sfGFP and DotO-sfGFP time laps micrographs ( Figure 2C ) were acquired using a Nikon TE2000 spinning disc confocal microscope equipped with a Nikon Plan Apo100× objective (1.4 numerical aperture) and restored by the NearestNeighbors deconvolution algorithm 23 of SlideBook™.

Fluorescent recovery after photobleaching

FRAP was performed on a Nikon TiE by fiber coupling a 405-nm solid state laser into the FRAP arm of a TIRF/FRAP illuminator, which focuses the laser to a single diffraction-limited spot on the sample. Imaging was accomplished in wide-field by illuminating the sample with the 470/24 line of a SpectraX solid-state light source laser delivering a wavelength of 405 nm. The photobleached area was determined with SlideBook™ software; bleaching was achieved with minimal intensities to avoid possible phototoxic effects. Fluorescence intensity measurements were corrected for non-specific photobleaching. The premise for the correction is that the overall intensity of non-bleached areas (of different cells in the same field) should remain constant over time. Intensity measurements were multiplied by the inverse of the ratio of fluorescence at a given time point over fluorescence at the initial time point.

Intracellular growth assay

Replication of L. pneumophila in RAW 264.7 macrophage-like cells was determined over 2 days using a standard assay that has been described previously 37 . Briefly, RAW 264.7 macrophage-like cells in 24 well tissue culture dishes were infected with L. pneumophila at a multiplicity of infection of 1. One hour after infection the wells were washed with PBS and fresh tissue culture medium was added to each wells. Colony-forming units were determined from individual wells after the PBS washes and at 2-days to measure intracellular replication.

Immunoblot analysis and co-immunoprecipitation

For immunoblot analysis, L. pneumophila cultures were grown for 48 h at 37 °C on CYE plates, resuspended in water and adjusted to an OD 600 of 1.2. Cells from 300 μl were collected by centrifugation at 21,000g for 1 min and resuspended in 300 μl Laemmli sample buffer and boiled for 10 min. 15 μl were loaded for western blot analysis using a primary polyclonal anti GFP antibody (GenScript) followed by incubation with secondary antibody conjugated to horseradish peroxidase (Sigma). Proteins were visualized by using an ECL detection kit (Amersham Biosciences). For co-immunoprecipitation analysis, L. pneumophila cultures were grown for 48 h at 37 °C on CYE plates. Cultures were resuspended in water and adjusted to an OD 600 of 6. Cells from 10 ml were collected by centrifugation at 11,000g for 10 min, re-suspended in 1.5 ml of in lysis buffer (50 mM Tris-HCl pH 8) with protease inhibitor cocktail (Roche Diagnostics), EDTA (2mM) and lysozyme (0.2 mg ml −1 , Sigma) and incubated for 1 h on ice prior to sonication. Lysates were centrifuged 10 min at 10,000 g and filtered to remove unlysed cells before the supernatant was incubated for 1 h at 4 °C with a polyclonal anti GFP antibody (GenScript). Samples were then incubated over night with protein A magnetic beads (Novex) and washed 10 times with the lysis buffer. Finally, beads, were resuspended in 30 μl Laemmli sample buffer and boiled for 10 min. 10 μl were loaded for western blot analysis using a primary polyclonal anti DotO antibody (provided by Ralph Isberg).

Preparation of Frozen-Hydrated Specimens

Bacterial cultures were grown 48 h at 37 °C on CYE agar plates. Bacteria were removed from the plates and suspended in water, then mixed with 10 nm colloidal gold particles (used as fiducial markers in image alignment) and deposited onto freshly glow-discharged, holey carbon grids for 1 min. The grids were blotted with filter paper and rapidly frozen in liquid ethane, using a gravity-driven plunger apparatus as described previously 24 , 25 .

Cryo-ET Data Collection and 3-D Reconstructions

The frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct detection device (Gatan K2 Summit). The microscope was operated at 300 kV with a magnification of ×15,500, resulting in an effective pixel size of 2.5 Å at the specimen level. We used SerialEM 26 to collect low-dose, single-axis tilt series with dose fractionation mode at about 5 μm defocus and a cumulative dose of ~50 e − /Å 2 distributed over 35 stacks. Each stack contains ~8 images. Over 2,000 tilt series were collected from −51° to 51° with increment of 3°. We used Tomoauto 25 to facilitate data processing which includes drift correction of dose-fractionated data using Motioncorr 27 and assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment and contrast transfer function correction of tilt series by IMOD 28 , and reconstruction of tilt series into tomograms by Tomo3D 29 . Each tomographic reconstruction is 3,710 × 3,838 × 2,400 pixels and ~130Gb in size. In total, 2,062 tomographic reconstructions from 7 different strains were generated ( Supplementary Information Table 2 ).

Sub-tomogram averaging and correspondence analysis

We used tomographic package I3 (0.9.9) for sub-tomogram analysis as described previously 30 . A total of 10,291 Type IVB secretion machines (400 × 400 × 400 voxels) were visually identified and then extracted from 2,062 cryo-tomographic reconstructions. Two of the three Euler angles of each Type IVB secretion machine were estimated based on the orientation of each particle in the cell envelope. To accelerate image analysis, 4×4×4 binned sub-tomograms (100×100×100 voxels) were used for initial alignment. The alignment proceeds iteratively with each iteration consisting of three parts in which references and classification masks are generated, sub-tomograms are aligned and classified, and finally class averages are aligned to each other. Classification focusing on core complex showed 13-fold symmetry feature, so in the following processing a 13-fold symmetry was imposed to assist the sub-tomograms alignment. Further classification focusing on the cytoplasmic complex showed a hexagonal structure in four different classes. After multiple cycles of alignment and classification for 4×4×4 binned sub-tomograms, we used 2×2×2 binned sub-tomograms for refinement.

Fourier shell correlation

(FSC) between the two independent reconstructions was used to estimate the resolution of the averaged structures ( Supplementary Information Fig. 4 ).

3-D Visualization and Molecular Modeling

We used IMOD to visualize the maps and also to generate 3-D surface rendering of L. pneumophila cell and UCSF Chimera 31 to visualize subtomogram averages in 3-D and molecular modeling. The pseudo DotO C-terminal structure was modeled using VirB4 (PDB 4AG5) as template, each of 12 DotO C-terminal structures was manually fitted onto the each of distal half of 12 rods. Since no DotB structure is available, we used a homohexamer of PilT (PDB 3JVV) fit into the distal short cylinder with its C terminal toward cytoplasm.

Statistical analysis

All experiments were performed at least twice, unless otherwise noted. Differences in the distribution of polarity scores were determined using a non-parametric test (Mann–Whitney), as most of samples displayed long tailed distributions. P < 0.05 was considered significant. For most microscopy experiments, roughly 200 cells were analyzed. For FRAP experiments means were compared using a two-sided Student’s t-test. No statistical methods were used to predetermine sample size, and the researchers were not blinded to sample identity.

Data Availability

Density maps and coordinate data that support the Dot/Icm structure determined by cryo-ET have been deposited in EMDB (EMD-7611, EMD-7612). The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files.

Supplementary Material 1 2