Dataset Information

Assembly Order of Flagellar Rod Subunits in Bacillus subtilis.

ABSTRACT: Bacterial flagella contain an axle-like rod that transits the cell envelope and connects the transmembrane basal body to the extracellular hook and filament. Although the rod is a crucial component of the flagellum, its structure and assembly are poorly understood. Previous reports defining the order of rod assembly in Gram-negative bacteria suggest that the rod requires five proteins to successfully assemble, but assembly intermediates have not been well characterized due to metastability and periplasmic proteolysis. Bacillus subtilis is a Gram-positive, genetically tractable model bacterium that synthesizes flagella and lacks a true periplasm. Here, we genetically, biochemically, and cytologically determine the assembly order of the flagellar rod proteins from cell proximal to distal as FliE, FlgB, FlgC, FlhO, and FlhP. We further show that, under conditions in which rod structure cannot be completed, assembly intermediates are both metastable and subject to proteolysis. Finally, we support previous results that FliE serves as both a structural assembly platform for the rod and as an enhancer of flagellar type III secretion.IMPORTANCE Bacteria rotate propeller-like flagella to find and colonize environmental niches. The flagellum is a complex machine, and the understanding of its structure is still incomplete. Here, we characterize and biochemically define the assembly order of the subunits that make up the axle-like rod. The rod is a critical structure for the assembly of subsequent components and is central to our understanding of how the flagellum is anchored but still free spinning within the context of the cell envelope.

SUBMITTER: Burrage AM

PROVIDER: S-EPMC6222195 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Assembly Order of Flagellar Rod Subunits in Bacillus subtilis.

Burrage Andrew M AM Vanderpool Eric E Kearns Daniel B DB

Journal of bacteriology 20181106 23

Bacterial flagella contain an axle-like rod that transits the cell envelope and connects the transmembrane basal body to the extracellular hook and filament. Although the rod is a crucial component of the flagellum, its structure and assembly are poorly understood. Previous reports defining the order of rod assembly in Gram-negative bacteria suggest that the rod requires five proteins to successfully assemble, but assembly intermediates have not been well characterized due to metastability and p ...[more]

PMID: 30201778

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Project description:While the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied in Escherichia coli and Salmonella, less is known about the switch complex in Bacillus subtilis or other Gram-positive species. Two component proteins (FliG and FliM) are shared between E. coli and B. subtilis, but in place of the protein FliN found in E. coli, the B. subtilis complex contains the larger protein FliY. Notably, in B. subtilis the signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action in E. coli Here, we have examined the architecture and function of the switch complex in B. subtilis using targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, the B. subtilis switch complex appears to be organized similarly to that in E. coli The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that of E. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences from E. coli involve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCE Flagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex in Bacillus subtilis or other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas in E. coli or Salmonella CheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of the B. subtilis switch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control in B. subtilis and other Gram-positive species.

Dataset Information

Assembly Order of Flagellar Rod Subunits in Bacillus subtilis.

Publications

Assembly Order of Flagellar Rod Subunits in Bacillus subtilis.

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