Dataset Information

In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles.

ABSTRACT: The bacterial flagellum is a filamentous organelle extending from the cell surface. The axial structure of the flagellum consists of the rod, hook, junction, filament, and cap. The axial structure is formed by axial component proteins exported via a specific protein export apparatus in a well-regulated manner. Although previous studies have revealed the outline of the flagellar construction process, the mechanism of axial structure formation, including axial protein export, is still obscure due to difficulties in direct observation of protein export and assembly in vivo. We recently developed an in vitro flagellar protein transport assay system using inverted membrane vesicles (IMVs) and succeeded in reproducing the early stage of flagellar assembly. However, the late stage of the flagellar formation process remained to be examined in the IMVs. In this study, we showed that the filament-type proteins are transported into the IMVs to produce the filament on the hook inside the IMVs. Furthermore, we provide direct evidence that coordinated flagellar protein export and assembly can occur at the post-translational level. These results indicate that the ordered construction of the entire flagellar structure can be regulated by only the interactions between the protein export apparatus, the export substrate proteins, and their cognate chaperones.

SUBMITTER: Terashima H

PROVIDER: S-EPMC7022808 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles.

Terashima Hiroyuki H Tatsumi Chinatsu C Kawamoto Akihiro A Namba Keiichi K Minamino Tohru T Imada Katsumi K

Biomolecules 20200111 1

The bacterial flagellum is a filamentous organelle extending from the cell surface. The axial structure of the flagellum consists of the rod, hook, junction, filament, and cap. The axial structure is formed by axial component proteins exported via a specific protein export apparatus in a well-regulated manner. Although previous studies have revealed the outline of the flagellar construction process, the mechanism of axial structure formation, including axial protein export, is still obscure due ...[more]

PMID: 31940802

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Project description:The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE, resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC, the MS ring gene, fliF, and one of the core T3SS genes, fliR. These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.

Project description:BackgroundBacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ?10 nm water film vs. 20 to >200 nm OMV;).Methodology/principal findingsWe have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ?6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods.Conclusions/significanceNanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms.

Dataset Information

In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles.

Publications

In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles.

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