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Single-molecule dynamics show a transient lipopolysaccharide transport bridge

Nature volume 623, pages 814–819 (2023)Cite this article

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Abstract

Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet1,2,3. The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface4. LPS transport and assembly requires seven conserved and essential LPS transport components5 (LptA–G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface6,7, but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5–10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly.

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Fig. 1: Observation of an immobile state of Lpt proteins suggests that they can form a bridge in cells.
Fig. 2: LptA has immobile states that are independent of the bridging state, whereas LptB does not.
Fig. 3: Bridges break and form rapidly, with bridge formation facilitated by LPS.
Fig. 4: Model for coordinating intermembrane Lpt bridge formation with LPS transport.

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Data availability

The data supporting the findings of the study are available in this article and its Supplementary Information files. Raw images and trajectory files are available at https://dataverse.harvard.edu/dataverse/Lpt_tracking_data.

Code availability

Code used in this paper is available at https://github.com/ltoerk/Lpt-tracking-code, and lifetime analysis code available at https://bitbucket.org/garnerlab/squyres-2020/src/master/Lifetime%20Analysis/.

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Acknowledgements

The authors thank G. Squyres, M. Holmes and E. Fivenson for experimental advice; A. Polissi for the gift of the anti-LptA and anti-LptC antibodies; and T. Mitchison for providing feedback on the manuscript. This work was supported by NIH Grant R01 AI149778 and R01 AI081059 to D.K. Further, this work was funded by AI083365 from the NIH (National Institute for Allergy and Infectious Disease) and Investigator funds from the Howard Hughes Medical Institute to T.G.B.

Author information

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Lisa Törk, Caitlin B. Moffatt & Daniel Kahne

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

    Caitlin B. Moffatt, Ethan C. Garner & Daniel Kahne

  3. Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Thomas G. Bernhardt

  4. Howard Hughes Medical Institute, Boston, MA, USA

    Thomas G. Bernhardt

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Contributions

L.T. designed, performed and analysed experiments (all TIRFM experiments and data analyses, strain constructions, sucrose gradient fractionation and related immunoblots, growth curves, spot plate assay and LptC overexpression immunoblots), interpreted results and wrote the manuscript. C.B.M. performed experiments (silver stain and related immunoblots, Lpt–HaloTag fusion immunoblots and LptC(G153R) immunoblots) and contributed to writing the revised manuscript. E.C.G. provided expertise in the design of the single-molecule tracking experiments. T.G.B. provided expertise in the design of genetic tools for constructing the fusion strains. D.K. designed experiments, interpreted results and wrote the manuscript. All authors contributed to editing.

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Correspondence to Ethan C. Garner or Daniel Kahne.

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Extended data figures and tables

Extended Data Fig. 1 Lpt-HaloTag fusions are expressed and support cell growth.

a, α-LptA immunoblot of LT136 (expresses wild-type endogenous LptA (~20 kDa) and LptA-Halo (~53 kDa)), LT23 (expresses LptA-Halo) and TB28 (expresses wild-type endogenous LptA). b, α-LptB immunoblot of LT137 (expresses wild-type endogenous LptB (~25 kDa) and Halo-LptB (~55 kDa)), LT17 (expresses Halo-LptB), and TB28 (expresses wild-type endogenous LptB). c, α-LptC immunoblot of LT138 (expresses wild-type endogenous LptC (~20 kDa) and Halo-LptC (~55 kDa)), LT16 (expresses Halo-LptC) and TB28 (expresses wild-type endogenous LptC). d, α-LptD immunoblot of LT18 (expresses LptD-Halo) and TB28 (expresses wild-type endogenous LptD) without and with beta-mercaptoethanol (βme) treatment. Under nonreducing conditions, wild-type endogenous LptD is ~110 kDa and LptD-Halo is ~150 kDa. The addition of βme reduces disulfide bonds in LptD and causes wild-type endogenous LptD to run around 87 kDa, and LptD-Halo to run around 120 kDa. LptD-Halo shows the same response to βme addition as wild-type LptD, confirming that it can properly fold19. e, α-LptE immunoblot of LT61 (expresses wild-type endogenous LptE (~20 kDa) and Halo-LptE (~55 kDa)), LT63 (expresses Halo-LptE) and TB28 (expresses wild-type endogenous LptE). a-e, Shown results are representative of two independent experiments. LT136, LT137, LT138, and LT61 were included in blots as controls to identify bands. Uncropped gel images can be found in Supplementary Fig. 1. f, Growth curves of LT23 (LptA-Halo), LT17 (Halo-LptB), LT16 (Halo-LptC), LT18 (LptD-Halo), LT63 (Halo-LptE) and TB28 (WT, green).

Extended Data Fig. 2 Representative trajectories of Halo-tagged LptA, B, C, D, and E.

Trajectories were overlaid over the corresponding phase image. Each trajectory is represented in a different color. The color was chosen randomly.

Extended Data Fig. 3 Mean square displacement versus τ curves show inhomogeneous dynamics for LptA, B, and C.

20 randomly sampled MSD versus τ plots are shown for trajectories collected for LptA, B, C, E, and D. MSD plots for LptA, B and C trajectories show immobile, mobile and switching trajectories. LptD and E show mostly immobile trajectories.

Extended Data Fig. 4 Cumulative distribution function analysis shows two-state dynamics for LptA, B, and C.

Cumulative distribution function of displacements with Δt = 200 ms for single-molecule tracks of LptA, B, C, and D were plotted. (Fig. 1f) One state (red line) and two state (black line) dynamic models were fitted to the CDF plots. a, Residuals for the one-state dynamic model fit are plotted. b, Residuals for the two-state dynamic model fit are shown. For all three proteins, the two-state model resulted in the best fit without over fitting the curve. The results shown are representative of at least two independent experiments.

Extended Data Fig. 5 The mobility of LptA, LptB, and LptC changes in response to altering the levels of LptC* and LptC.

a, Immunoblots against LptC and RpoA are shown for LT16 containing pBAD33LptC(G153R) treated with different arabinose (ara) concentrations to express LptC(G153R) (LptC*) under the same conditions used for imaging. Molecular weight markers are given in kDa. b, Immunoblots against LptC, LptB, and LptA are shown for LT16 containing pBAD33LptC treated with different arabinose (ara) concentrations to express wild-type LptC. c, Confinement radius plots for LptA, B, and C without inducing (black line, LptC* low) and with inducing LptC(G153R) production with 40 mM arabinose (green line, LptC* high). n = low/high, LptA:3,552/8,330, LptB:6,773/2,324, LptC:18,938/30,560. d, Confinement radius plots for LptA, B, and C without inducing (black line, LptC low) and with inducing overproduction of wild-type LptC with 40 mM arabinose (green line, LptC high). n = low/high, LptA:2,371/12,773, LptB:1,311/2,747, LptC:8,864/11,788. e, Immunoblots of the fractionation of LptA-Halo containing strain, LT23, are shown. OMlight is a mixed membrane fraction and contains both IM and OM proteins (including the Lpt bridge), and OMheavy contains components fractionating only with the OM6. Results shown in this figure are representative of at least two independent experiments.

Extended Data Fig. 6 LptA, LptB, and LptC display biexponential decay kinetics.

Single exponential curves (red) and biexponential curves (black) were fitted to the lifetime plots of LptA, B, and C. (line = fit, dots = lifetime data). (Fig. 3b) Fitted values can be found in Extended Data Fig. 7. a, Residuals for the single exponential fits are shown (red). b, Residuals for the biexponential fits are shown (black). For all three proteins the biexponential fit resulted in the best fit of the lifetime distribution. Plots are representative of two independent experiments.

Extended Data Fig. 7 Bridge lifetime follows a biexponential decay.

a, Bleaching control measurement; Halo-LptC (LT16) lifetime plots measured with 500 ms exposure in 500 ms intervals (green, n = 708) compared to 500 ms exposure in 1 s intervals (blue, n = 1,186). The average of two independent experiments is shown with the standard deviations (error bars.) b, Results for the fitted values of the biexponential and exponential fits to the lifetime data of LptA, B, and C, and for the lifetime data measured for LptB with overproduction of the LptCA fusion. Provided values are the mean of two independent experiments and the standard deviation is given as error. c, Silver stain, α-LptA, α-LptD, α-LptE and α-RpoA blots of LT17 with (+/LPSlow) and without (-/wt) 0.5X MIC LpxC inhibitor (PF 5081090) treatment are shown. Two replicates are shown. The silver stain detects LPS levels. Molecular weight markers are given in kDa. d, Spot dilutions of LT17 on LB agar after LpxC inhibitor treatment (LPSlow) are shown in comparison to untreated LT17 (LPSwt). Three biological replicates are shown. e, Phase images of LT17 with (LPSlow) and without (LPSwt) LpxC inhibitor treatment are shown. Shown images are representative field of views of two independent experiments. Histograms of cell length and width, measured of LT17 cells (n=cells/independent experiments, LPSwt:300/2, LPSlow:300/2) with (blue) and without (green) LpxC inhibitor treatment are depicted. f, Confinement radius plots for Halo-LptB under wild-type conditions (black line, n = 2,110) and with LpxC inhibitor treatment (green line, n = 8,145). g, Confinement radius plots for Halo-LptB under wild-type conditions (black line, n = 3,637) and with overproduction of LptCA-fusion protein (40 mM arabinose, green line, n = 3,602). f, g, Results are representative of at least two independent experiments. h, i, Residuals of the exponential (red, h) and biexponential (black, i) fit to the LptCA-fusion lifetime distribution (Fig. 4b) are shown.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Tables 1–4.

Reporting Summary

Supplementary Tables

Supplementary Tables 5 and 6 which report all cumulative distribution fit results corresponding to Figs. 2c,e, 3d and 4d (Supplementary Table 5) and (immobile) tracks per cell surface values are reported corresponding to Fig. 2f,g (Supplementary Table 6).

Supplementary Video 1

Representative single-molecule video for LptD–Halo.

Supplementary Video 2

Representative single-molecule video for Halo–LptE.

Supplementary Video 3

Representative single-molecule video for LptA–Halo.

Supplementary Video 4

Representative single-molecule video for Halo–LptB.

Supplementary Video 5

Representative single-molecule video for Halo–LptC.

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Törk, L., Moffatt, C.B., Bernhardt, T.G. et al. Single-molecule dynamics show a transient lipopolysaccharide transport bridge. Nature 623, 814–819 (2023). https://doi.org/10.1038/s41586-023-06709-x

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