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Chapter 1 : Electron Cryotomography of Bacterial Secretion Systems

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Abstract:

The envelope of bacterial cells consists of at least one—and often two—membranes, a cell wall, and possibly a surface layer. This envelope allows cells to differentiate themselves from their environment and handle the resulting osmotic pressure, but it also presents a significant obstacle. Anything a cell wishes to export, from a motility appendage to a plasmid, needs to be ushered across this barrier. To accomplish this, bacteria have evolved a battery of secretion systems. Secretion systems are often constructed from dozens of protein building blocks embedded in the cell’s envelope. The size, complexity, and location of these machines make them a particular challenge for structural characterization. High-resolution structure determination techniques such as X-ray crystallography and transmission electron microscopy (TEM)-based single-particle reconstruction (SPR) require objects to be purified from their cellular environment. This is problematic for membrane-associated proteins, which embed in the lipid bilayer by means of exposed hydrophobic patches. These patches can be protected during purification by adding detergents to the solvent, but often structural alterations still occur. Secretion systems are also unusually large targets and frequently lose peripheral or loosely-associated components during purification. In addition, they often cross two membranes and are avidly linked to the cell wall.

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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Figures

Image of Figure 1
Figure 1

T2SS. ECT revealed the structures of the T4aP in and , enabling high-resolution structures to be placed into the map to produce a hypothetical model of the machine. Further studies revealed structural convergence of the related T4bP in and T2SS in , with a similar periplasmic ring (arrows) formed by nonhomologous proteins in each system. Note in this and subsequent figures how bacterial secretion systems often locally distort the cell envelope . Images are reprinted from the following with permission: panel A left and middle, reference ; panel A right, reference ; panel B, reference ; and panel C, reference .

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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Image of Figure 2
Figure 2

T3SS. ECT and subtomogram averaging revealed the structures of diverse T3SSs, including the flagellar motors of many bacteria (three examples, from , , and ) and the injectisome from Typhimurium . Structures shown from left to right: EMD-3154, EMD-3155, and EBD-3150 from reference and EMD-2667 from reference .

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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Image of Figure 3
Figure 3

T4SS. ECT revealed the structure of the Dot/Icm T4SS, including a central channel extending from the inner membrane, and enabled components to be placed in the map. It also revealed the similar structure of the T4SS (inset), as well as novel, related OM tubes. Images are reprinted from the following with permission: panel A, reference (but see also reference ); panel B, reference .

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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Figure 4

T5SS. ECT revealed the stick-like form of the CDI T5SS (arrows) and helped elucidate its mechanism, in which half of CdiA extends out from the cell surface . Upon target binding, the remaining half of CdiA is exported to deliver the toxin to the target cell. Images reprinted with permission from reference .

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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Figure 5

T6SS. ECT revealed the contractile mechanism of the T6SS and its structure in . It also revealed higher-order arrays of T6SS in and a T6SS-related metamorphosis-associated contractile structure (MAC) in . Images are reprinted from the following with permission: panels A and B, reference ; panel D, reference .

Citation: Oikonomou C, Jensen G. 2019. Electron Cryotomography of Bacterial Secretion Systems, p 1-12. In Sandkvist M, Cascales E, Christie P (ed), Protein Secretion in Bacteria. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PSIB-0019-2018
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