Dataset Information

Bending stiffness characterization of Bacillus subtilis' flagellar filament.

ABSTRACT: The filament of a bacterial flagellum is a tube-like organelle made of a single protein-flagellin-and assembled into multiple polymorphic forms. The filament can be further discretized into four subunit domains (D0, D1, D2, and D3) along the radial direction. However, it remains unclear which subunit domain plays an important role in regulating the rigidity of the filament. In this article, we address how the absence of two outer subunit domains (D2 and D3) affects the bending stiffness of the bacterium B. subtilis' flagellar filament. We first shear off flagellar filaments from the cell body, anchor one of its ends to the wall of a microfluidic channel, and correlate the elongation of the filament with the driving background flow. A numerical model is then applied to determine the bending stiffness of the filament. We find that the bending stiffness does not change drastically when the filament transforms from normal to hyperextended forms, which is estimated to be 2-3 pN⋅μm². Furthermore, B. subtilis' flagellar filament has similar bending stiffness to Salmonella's, although the radius of the former is almost half of that of the latter, suggesting that the rigidity comes from the inner D0 and D1 subunit domains.

SUBMITTER: Shen X

PROVIDER: S-EPMC9247555 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Bending stiffness characterization of Bacillus subtilis' flagellar filament.

Shen Xinhui X Tran Phu N PN Tay Benjamin Z BZ Marcos

Biophysical journal 20220512 11

The filament of a bacterial flagellum is a tube-like organelle made of a single protein-flagellin-and assembled into multiple polymorphic forms. The filament can be further discretized into four subunit domains (D0, D1, D2, and D3) along the radial direction. However, it remains unclear which subunit domain plays an important role in regulating the rigidity of the filament. In this article, we address how the absence of two outer subunit domains (D2 and D3) affects the bending stiffness of the b ...[more]

PMID: 35550881

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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.

Project description:Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

Dataset Information

Bending stiffness characterization of Bacillus subtilis' flagellar filament.

Publications

Bending stiffness characterization of Bacillus subtilis' flagellar filament.

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