Architecture of the type IVa pilus machine

Chang, Yi‐Wei; Rettberg, Lee A.; Treuner-Lange, Anke; Iwasa, Janet; Søgaard‐Andersen, Lotte; Jensen, Grant J.

doi:10.1126/science.aad2001

Cited by 376 publications

(395 citation statements)

References 99 publications

(93 reference statements)

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“…In two Vibrio cholerae cells (one from a C6706 lacZ mutant [41] and one from a ΔctxA ΔtcpB strain [15]) we observed clusters of nanospheres, hollow granules with thick walls (Fig. 4).…”

Section: Intracellular Structures (I) Nanospheresmentioning

confidence: 95%

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

et al. 2017

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Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated. Here we present a survey of previously uncharacterized structures that we observed in bacterial cells by electron cryotomography, structures that will initiate new lines of research investigating their identities and roles.KEYWORDS bacteria, electron cryotomography, bacterial ultrastructure, uncharacterized structures, electron microscopy T he history of cell biology has been punctuated by advances in imaging technology. In particular, the development of electron microscopy (EM) in the 1930s produced a wealth of new information about the ultrastructure of cells (1). For the first time, the structure of cell envelopes, internal organelles, cytoskeletal filaments, and even large macromolecular complexes like ribosomes became visible. A further advance came in the 1980s and 1990s with the development of electron cryotomography (ECT) (2), which allows small cells to be imaged intact in 3D in a near-native, "frozen-hydrated" state to "macromolecular" (ϳ5 nm) resolution, without the limitations and artifacts of more traditional specimen-preparation methods (3).ECT has helped reveal the previously unappreciated complexity of "simple" bacterial cells. Our group has been using ECT to study bacteria for more than a decade, generating more than 15,000 tomograms of 88 different species. These tomograms have revealed new insights into, among other things, the bacterial cytoskeleton (4-7), cell wall architecture (8, 9), morphogenesis (10), metabolism (11), motility (12-15),

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“…In two Vibrio cholerae cells (one from a C6706 lacZ mutant [41] and one from a ΔctxA ΔtcpB strain [15]) we observed clusters of nanospheres, hollow granules with thick walls (Fig. 4).…”

Section: Intracellular Structures (I) Nanospheresmentioning

confidence: 95%

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

et al. 2017

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“…4E and F), resembling the pore structures of the type II secretion system or the nonpiliated state of the type IV pilus system ( Fig. 4G and H) (42,45). In most electron microscopy (EM) reconstructions of pore-forming, nonpiliated secretins (of the type II or type III secretion system or the type IV pilus system), an apparently closed periplasmic gate was observed (42,45,46).…”

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confidence: 91%

Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium “Candidatus Pelagibacter ubique”

Zhao

Schwartz

Pierson

et al. 2017

Appl Environ Microbiol

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SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 Ϯ 0.011 m 3 . Strikingly, the periplasmic space occupied ϳ20% to 50% of the total cell volume in log-phase cells and ϳ50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/m 3 . Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

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“…The polymer incorporates major pilins (PilA) and a variety of minor pilins (5). The pilus polymer is anchored within the cell envelope by a membranespanning complex (6). Importantly, the length of the T4P is dynamic; elongation of T4P requires a cytoplasmic ATPase formed by PilB, and retraction requires its antagonist PilT (7).…”

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confidence: 99%

Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics

et al. 2017

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For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ϳ30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa. Finally, our data support the previously proposed model of slingshot-like "twitching" motility of P. aeruginosa. IMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic "twitching" behavior.

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Architecture of the type IVa pilus machine

Cited by 376 publications

References 99 publications

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography

Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium “Candidatus Pelagibacter ubique”

Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics

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