Article Figures & Data
Figures
- FIG 1
Schematic model for V. cholerae T6SS structure and secretion mechanism. In the growing phase of the duty cycle, the sheath and shaft assemble at the membrane. In the contraction phase of the duty cycle, the TssBC sheath undergoes conformational change, contracting the sheath and driving the Hcp/VgrG shaft through the recipient cell membrane. In the shrinking phase of the duty cycle, ClpV binds the N terminus of TssC which is exposed only in the contracted conformation. The ATPase activity of the TssC-bound ClpV then proceeds to disassemble the sheath.
- FIG 2
(A) Phase-contrast image of a polymicrobial microcolony consisting of both P. aeruginosa and Burkholderia thailandensis cells. (B) Cell identity is inferred by cytoplasmic fluorescence of cyan fluorescent protein (CFP) expressed in B. thailandensis cells. (C) ClpV-mCherry and Fha-GFP visualized by fluorescence microscopy in a single frame. (D) Cells are segmented from the phase-contrast image. Cell identity is inferred from CFP fluorescence and foci are detected in mCherry and GFP channels. B. thailandensis and P. aeruginosa cells are pink and blue, respectively. Foci with neighboring B. thailandensis cells are green; those without are blue. (E) Kymograph showing fluorescence intensity of Fha (blue) and ClpV (red) long-axis dynamics over a 4-min interval. (F) Fha (blue) and ClpV (red) focus intensity dynamics. Arrows show the corresponding events in the kymograph and intensity plots.
- FIG 3
Example data demonstrating measurement of ClpV firing lifetimes (ts) and time between spikes (tp). There are five spikes in this trace. (Top) Representative ClpV firing for a single cell at one focus region. Inset shows image of cell with focus area indicated by outline. Here and in subsequent figures, each trace is representative of one focus area in one cell. (Bottom) Comparisons of ClpV lifetimes. Mean lifetime of the ClpV spike is 37 s, n ≈ 2,200; measurement of time between spikes, n ≈ 1,100.
- FIG 4
(A) ClpV and TssB normalized fluorescence intensities over time in single cell (2-s frame rate). Fluorescence intensity fluctuates, and focus motion is observed, but the focus remains diffraction limited, as seen in panel B. Cell images for the region indicated by the dashed boundary are shown in panel B. (B) Typical single-focus data show a representative coordinated ClpV and TssB fluorescence intensity traces for one focus region in ΔretS background. (C) Population-level analysis reveals average lag times between ClpV and TssB traces. n ≈ 280.
- FIG 5
Representative intensity traces of ClpV and Fha. (Top) Fha levels start increasing a few seconds (2 to 60 s) before ClpV begins to fire. After the firing has terminated, Fha returns to a baseline level. (Bottom) Fha disappears and returns. Due to the diffuse nature of the Fha focus, an automated approach to determining lifetime and lag time was not reliable. The intensity traces shown above were representative of the traces examined by eye (n ≈ 1,760).
- FIG 6
(A) Representative intensity/position/speed graph for one region. Frame rate, 2 s. Position is measured by projecting coordinates onto best-fit line. (B) Example spread of ClpV data and Gaussian-distributed data. Major and minor axes are indicated in black. (C) Correlation coefficients between speed and intensity in wild-type cells (n ≈ 650). There is generally a negative correlation between speed and intensity. Note that the right tail of the distribution is primarily due to uncertainty in resolving the focus centroid at lower intensities, leading to imperfections in calculating the speed.
- FIG 7
Schematic model for P. aeruginosa T6SS structure and secretion mechanism. As the TssBC sheath begins formation, TagJ associates with the sheath. TagJ in turns complexes with ClpV, which allows ClpV to associate with the TssBC sheath without any sheath conformational changes. ClpV can thus localize to the T6SS apparatus within a few seconds after the initiation of TssB localization. ClpV then disassembles the TssBC sheath, generating a force similar to that seen in microtubule growth and decay, which is used to deliver effectors to the neighboring cell (20). This is in direct contrast to ClpV and TssBC in the T6SS of V. cholerae, which require a conformational change to bind (4).
Tables
- TABLE 1
Strains used in this study
Organism Genotype Source or reference Pseudomonas aeruginosa PAO1 clpV-GFP 17 tssB-sfGFP clpV-mCherry This study ΔretS tssB-sfGFP clpV-mCherry This study fha1-sfGFP clpV-mCherry This study Burkholderia thailandensis attTn7::Tp-CFP 14
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