Biochemical characterization of the flagellar stator-associated inner membrane protein FliL fromVibrio alginolyticus

Kumar, Aman; Isumi, Miyu; Sakuma, Mayuko; Zhu, S. J.; Nishino, Yuuki; Onoue, Yasuhiro; Kojima, Seiji; Miyanoiri, Yohei; Imada, Katsumi; Homma, Michio

doi:10.1093/jb/mvw076

Cited by 7 publications

(14 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The particle weight value estimated from the mobility of the gel-filtration column calibrated using globular proteins of a known mass gave the value of approximately 11.2 kDa, which suggested that H. pylori FliL-C is monomeric in solution under the tested conditions. This result is in agreement with the previous report on the periplasmic domain of Vibrio alginolyticus FliL, which is also primarily monomeric in solution in the concentration range used in this study (27).…”

Section: Crystallizationsupporting

confidence: 94%

“…However, it is anticipated that determination of the crystal structure of its soluble periplasmic domain will provide a critical insight into its function through identification of functional homologues with a similar fold, as protein structure is more conserved than sequence. The production of the crystals of the periplasmic domain of FliL from Vibrio alginolyticus was reported (27), but no structure is available yet. Here, we report the cloning, purification, crystallization and the preliminary X-ray crystallographic analysis of the C-terminal periplasmic domain of H. pylori FliL.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Purification, crystallization and preliminary X-ray crystallographic studies on the C-terminal domain of the flagellar protein FliL from Helicobacter pylori

Chan

Machuca

Rahman

et al. 2018

BST

View full text Add to dashboard Cite

FliL is an inner membrane protein, occupying a position between the rotor and the stator of the bacterial flagellar motor. Its proximity to, and interactions with, the MS (membrane and supramembranous) ring, the switch complex and the stator proteins MotA/B suggests a role in recruitment and/or stabilization of the stator around the rotor, although the precise role of FliL in the flagellum remains to be established. In this study, recombinant C-terminal domain of Helicobacter pylori FliL (amino-acid residues 81-183) has been expressed in Escherichia coli and purified to > 98% homogeneity. Purified recombinant protein behaved as a monomer in solution. Crystals were obtained by the hanging-drop vapour-diffusion method using ammonium phosphate monobasic as a precipitant. These crystals belong to space group P1, with unit-cell parameters a = 62.5, b = 82.6, c = 97.8 Å, α = 67.7, ꞵ = 83.4, γ = 72.8°. A complete data set has been collected to 2.8 Å resolution using synchrotron radiation. This is an important step towards elucidation of the function of FliL in the bacterial flagellar motor.

show abstract

Section: Crystallizationsupporting

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Purification, crystallization and preliminary X-ray crystallographic studies on the C-terminal domain of the flagellar protein FliL from Helicobacter pylori

Chan

Machuca

Rahman

et al. 2018

BST

View full text Add to dashboard Cite

show abstract

“…Since the protein diffusion on membrane usually dominated by the number of transmembrane segment 56 , it’s unusual to measure such a low diffusion rate for a single transmembrane FliL protein. According to the recent biochemical characterization 57 , FliL without transmembrane domain would interact with each other to form oligomer. These results highly suggest that FliL protein may diffuse on the membrane as an oligomer.…”

Section: Discussionmentioning

confidence: 99%

FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy

Lin

Zhu

Kojima

et al. 2018

Sci Rep

Self Cite

View full text Add to dashboard Cite

Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.

show abstract

“…The motor of this spirochete also possesses a particular structure, called the collar, to which FliL is associated (18). Furthermore, biochemical and X-ray crystallography studies in V. alginolyticus suggested that rings of FliL might associate around stator units (19,20). From these and other studies, it may be concluded that FliL is a small membrane protein that is part of the flagellar basal body and that might have a role in stabilizing the stator (21).…”

mentioning

confidence: 90%

“…In addition to the paucity of studies on the role of FliL in alphaproteobacteria, there are no comparative works in bacterial species possessing two flagellar systems except for recent studies in V. alginolyticus (19,20). Since B. diazoefficiens uses its two flagellar systems for swimming (2), this species brings an opportunity to compare the role of FliL in each system for swimming.…”

mentioning

confidence: 99%

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System

et al. 2020

Self Cite

View full text Add to dashboard Cite

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction. IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system’s fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

show abstract

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL fromVibrio alginolyticus

Cited by 7 publications

References 22 publications

Purification, crystallization and preliminary X-ray crystallographic studies on the C-terminal domain of the flagellar protein FliL from <i>Helicobacter pylori </i>

Purification, crystallization and preliminary X-ray crystallographic studies on the C-terminal domain of the flagellar protein FliL from <i>Helicobacter pylori </i>

FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System

Contact Info

Product

Resources

About