Characterisation of <i>Shigella</i> Spa33 and <i>Thermotoga</i> FliM/N reveals a new model for C‐ring assembly in T3SS

McDowell, Melanie A.; Marcoux, Julien; McVicker, Gareth; Johnson, Steven; Fong, Y.H.; Stevens, Rebecca; Bowman, Lesley A.H.; Degiacomi, Matteo T.; Yan, Jun; Wise, Adam; Friede, Miriam E.; Benesch, Justin L. P.; Deane, Janet E.; Tang, Christoph M.; Robinson, Carol V.; Lea, Susan M.

doi:10.1111/mmi.13267

Cited by 70 publications

(129 citation statements)

References 61 publications

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“…Our cryo-ET maps confirm that FliG, FliM and FliN interact in a 1:1:3 ratios, as suggested for the non-flagellar homolog Spa33 in Shigella (McDowell et al, 2016). This stoichiometry favors a model of the C-ring in which two homodimers of FliN creating a closed loop.…”

Section: Discussionsupporting

confidence: 80%

“…Our model supports the 1:1:3 (FliG, FliM, and FliN) model in lieu of the 1:1:4 model, as there is no additional density for a fourth FliN molecule (Lee et al, 2010;Roy et al, 2010;. The 1:3 model suggests a FliM-FliN heterodimer interacts with the FliN homodimer characterized by Mass spectroscopy, as had been modeled into a previously solved cryo-EM map (McDowell et al, 2016;Thomas et al, 2006). We used this information and Rosetta Protein-Protein docking to identify and refine these interfaces in the V. alginolyticus flagellar motor.…”

Section: Molecular Architectures Of the Ccw And Cw C-ringssupporting

confidence: 81%

“…The previous crystal structures and solution NMR studies showed FliMM interacting with FliG (Dyer et al, 2009;Lam et al, 2013;Vartanian et al, 2012). FliMC shows sequence homology to FliN and structural homology to the FliN homodimer, as shown from the structure of a crystallized FliMC-FliN fusion protein (McDowell et al, 2016;Notti et al, 2015). To place FliM in our structure we generated a FliMMC model using I-TASSER and oriented the domain orientation to fit into our cryo-ET map (Fig.…”

Section: Flim/flin Interactions Hold the C-ring Subunits Togethermentioning

confidence: 93%

“…The third protein, FliN (and FliY in some species), a small single-domain protein, dimerizes with FliM or itself to form the base of the C-ring (Brown et al, 2005). Proposed models for FliG, FliM and FliN assembly include 1:1:4 (Sarkar et al, 2010a;Sarkar et al, 2010b) or a 1:1:3 (McDowell et al, 2016) stoichiometry. Two FliN homodimers form a ring at the base of each C-ring subunit in the first model (Sarkar et al, 2010a), while a FliM:FliN heterodimer and a FliN homodimer create a spiral base of the C-ring in the second model (McDowell et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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The flagellar motor ofVibrio alginolyticusundergoes major structural remodeling during rotational switching

Carroll

Nishikino³

et al. 2020

Preprint

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The bacterial flagellar motor is an intricate nanomachine that switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants (CCW-biased fliG-G214S and CW-locked fliG-G215A) in Vibrio alginolyticus. Subtomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

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Section: Discussionsupporting

confidence: 80%

Section: Molecular Architectures Of the Ccw And Cw C-ringssupporting

confidence: 81%

Section: Flim/flin Interactions Hold the C-ring Subunits Togethermentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The flagellar motor ofVibrio alginolyticusundergoes major structural remodeling during rotational switching

Carroll

Nishikino³

et al. 2020

Preprint

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“…2i). After we discovered this, similar control was reported for SpaO homologues in the Salmonella SPI-2, Shigella and Yersinia T3SSs434445. Our solution was to unpack the genetics34 into independent long ( spaO ) and short ( spaOs ) genes that are physically separated in the refactored inv-spa operon (Fig.…”

Section: Resultsmentioning

confidence: 61%

Control of type III protein secretion using a minimal genetic system

et al. 2017

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Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology.

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The sorting platform in the type III secretion pathway: From assembly to function

Soto

Lara‐Tejero

2023

BioEssays

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The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe‐like apparatus composed of multiple building blocks, including both membrane‐associated and soluble proteins. The cytosolic components organize together in a chamber‐like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host‐pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

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Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C‐ring assembly in T3SS

Cited by 70 publications

References 61 publications

The flagellar motor ofVibrio alginolyticusundergoes major structural remodeling during rotational switching

The flagellar motor ofVibrio alginolyticusundergoes major structural remodeling during rotational switching

Control of type III protein secretion using a minimal genetic system

The sorting platform in the type III secretion pathway: From assembly to function

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