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Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

Gerc AJ, Diepold A, Trunk K, Porter M, Rickman C, Armitage JP, Stanley-Wall NR, Coulthurst SJ - Cell Rep (2015)

Bottom Line: The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells.Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo.We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

No MeSH data available.


Related in: MedlinePlus

TssH, TssJ, and TssL Show Differential Distribution and Mobility from TssB and Each Other(A–D) Representative set of fluorescence images of TssB-mCh (A), TssL-mCh (B), TssH-mCh (C), and TssJ-mCh (D) acquired at 100-ms (top), 1-s (middle), or 10-s intervals (bottom). From left to right: three sequential frames and an overlay are shown. In the overlay, the signal from the three frames is colored consecutively red, green, and blue; any signal present in all three frames will appear white, whereas movement will result in the appearance of color.(E and F) Example time courses of TssB-mCh (E) and TssH-mCh (F) acquired over a longer time; the cells shown in lower panels of parts A and C, respectively, are shown in 10-s intervals over a total of 4 min. Images were manually corrected for xy drift and photobleaching with the outline of the cell shown in red in the first image. The corresponding uncropped series of raw images are shown as Movies S2 and S3. Scale bars, 1 μm.See also Figure S2.
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fig4: TssH, TssJ, and TssL Show Differential Distribution and Mobility from TssB and Each Other(A–D) Representative set of fluorescence images of TssB-mCh (A), TssL-mCh (B), TssH-mCh (C), and TssJ-mCh (D) acquired at 100-ms (top), 1-s (middle), or 10-s intervals (bottom). From left to right: three sequential frames and an overlay are shown. In the overlay, the signal from the three frames is colored consecutively red, green, and blue; any signal present in all three frames will appear white, whereas movement will result in the appearance of color.(E and F) Example time courses of TssB-mCh (E) and TssH-mCh (F) acquired over a longer time; the cells shown in lower panels of parts A and C, respectively, are shown in 10-s intervals over a total of 4 min. Images were manually corrected for xy drift and photobleaching with the outline of the cell shown in red in the first image. The corresponding uncropped series of raw images are shown as Movies S2 and S3. Scale bars, 1 μm.See also Figure S2.

Mentions: Having established the overall behavior of our T6SS at the single-cell level using the relatively well-characterized TssB protein, we compared the properties of the other fusion proteins with TssB-mCh. As above, initial observation using “snapshot” imaging revealed distinct localization patterns for each protein (Figures 1D–1G). To examine the localization of the different T6SS components over different timescales, we used fluorescence microscopy to visualize bacteria expressing the functional mCherry-labeled components with frame rates of 100 ms, 1 s, and 10 s. TssB foci (Figure 4A) were stable and immobile on short timescales (up to a few seconds), while some changes in both the intensity and the localization could be observed over 10-s intervals (bottom row). TssL was found to be more dynamic than TssB (Figure 4B). Interestingly, while the positions of TssL spots remained relatively fixed, especially for brighter foci, spot intensities fluctuated over the 100-ms range, especially in weaker spots. TssH is even more dynamic than TssB and TssL (Figure 4C). However, while there is considerable movement for diffuse TssH and small foci, bright foci could be stable for more than 10 s (bottom row). Strikingly, the relatively weak TssJ patches moved considerably over seconds (Figure 4D). Of note, for TssB and TssH, cases were observed in which bright foci appeared and disappeared in multiple cycles. While TssB foci tended to disappear after 1 to 2 min and form at another position (Figure 4E; Movie S2), bright TssH foci were sometimes found to cycle between focal and diffuse fluorescence with a period of about 50 s for each state (Figure 4F; Movie S3).


Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

Gerc AJ, Diepold A, Trunk K, Porter M, Rickman C, Armitage JP, Stanley-Wall NR, Coulthurst SJ - Cell Rep (2015)

TssH, TssJ, and TssL Show Differential Distribution and Mobility from TssB and Each Other(A–D) Representative set of fluorescence images of TssB-mCh (A), TssL-mCh (B), TssH-mCh (C), and TssJ-mCh (D) acquired at 100-ms (top), 1-s (middle), or 10-s intervals (bottom). From left to right: three sequential frames and an overlay are shown. In the overlay, the signal from the three frames is colored consecutively red, green, and blue; any signal present in all three frames will appear white, whereas movement will result in the appearance of color.(E and F) Example time courses of TssB-mCh (E) and TssH-mCh (F) acquired over a longer time; the cells shown in lower panels of parts A and C, respectively, are shown in 10-s intervals over a total of 4 min. Images were manually corrected for xy drift and photobleaching with the outline of the cell shown in red in the first image. The corresponding uncropped series of raw images are shown as Movies S2 and S3. Scale bars, 1 μm.See also Figure S2.
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Related In: Results  -  Collection

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fig4: TssH, TssJ, and TssL Show Differential Distribution and Mobility from TssB and Each Other(A–D) Representative set of fluorescence images of TssB-mCh (A), TssL-mCh (B), TssH-mCh (C), and TssJ-mCh (D) acquired at 100-ms (top), 1-s (middle), or 10-s intervals (bottom). From left to right: three sequential frames and an overlay are shown. In the overlay, the signal from the three frames is colored consecutively red, green, and blue; any signal present in all three frames will appear white, whereas movement will result in the appearance of color.(E and F) Example time courses of TssB-mCh (E) and TssH-mCh (F) acquired over a longer time; the cells shown in lower panels of parts A and C, respectively, are shown in 10-s intervals over a total of 4 min. Images were manually corrected for xy drift and photobleaching with the outline of the cell shown in red in the first image. The corresponding uncropped series of raw images are shown as Movies S2 and S3. Scale bars, 1 μm.See also Figure S2.
Mentions: Having established the overall behavior of our T6SS at the single-cell level using the relatively well-characterized TssB protein, we compared the properties of the other fusion proteins with TssB-mCh. As above, initial observation using “snapshot” imaging revealed distinct localization patterns for each protein (Figures 1D–1G). To examine the localization of the different T6SS components over different timescales, we used fluorescence microscopy to visualize bacteria expressing the functional mCherry-labeled components with frame rates of 100 ms, 1 s, and 10 s. TssB foci (Figure 4A) were stable and immobile on short timescales (up to a few seconds), while some changes in both the intensity and the localization could be observed over 10-s intervals (bottom row). TssL was found to be more dynamic than TssB (Figure 4B). Interestingly, while the positions of TssL spots remained relatively fixed, especially for brighter foci, spot intensities fluctuated over the 100-ms range, especially in weaker spots. TssH is even more dynamic than TssB and TssL (Figure 4C). However, while there is considerable movement for diffuse TssH and small foci, bright foci could be stable for more than 10 s (bottom row). Strikingly, the relatively weak TssJ patches moved considerably over seconds (Figure 4D). Of note, for TssB and TssH, cases were observed in which bright foci appeared and disappeared in multiple cycles. While TssB foci tended to disappear after 1 to 2 min and form at another position (Figure 4E; Movie S2), bright TssH foci were sometimes found to cycle between focal and diffuse fluorescence with a period of about 50 s for each state (Figure 4F; Movie S3).

Bottom Line: The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells.Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo.We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

No MeSH data available.


Related in: MedlinePlus