scholarly journals The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor

2010 ◽  
Vol 192 (21) ◽  
pp. 5609-5615 ◽  
Author(s):  
Hiroyuki Terashima ◽  
Masafumi Koike ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Basal Body ◽  
High Speed ◽  
Electron Microscopic Observation ◽  
Specific Protein ◽  
Ring Structure ◽  
Flagellar Motor ◽  
Wild Type ◽  
Electron Microscopic ◽  
E Coli ◽  
Motor Structure

ABSTRACT In Vibrio alginolyticus, the flagellar motor can rotate at a remarkably high speed, ca. three to four times faster than the Escherichia coli or Salmonella motor. Here, we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla− and Mot− phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of ΔflgT cells revealed a smaller LP ring structure compared to the wild type, and most of the T ring was lost. His6-tagged FlgT could be coisolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

scholarly journals Bld10p, a novel protein essential for basal body assembly in Chlamydomonas

2004 ◽  
Vol 165 (5) ◽  
pp. 663-671 ◽  
Author(s):  
Kumi Matsuura ◽  
Paul A. Lefebvre ◽  
Ritsu Kamiya ◽  
Masafumi Hirono
Keyword(s):  
Basal Body ◽  
Cell Biology ◽  
Early Stage ◽  
Rotational Symmetry ◽  
Coiled Coil ◽  
Electron Microscopic Observation ◽  
Basal Bodies ◽  
Mitotic Spindles ◽  
Electron Microscopic ◽  
Cytoplasmic Microtubules

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.

1985 ◽  
Vol 101 (5) ◽  
pp. 1903-1912 ◽  
Author(s):  
R L Wright ◽  
J Salisbury ◽  
J W Jarvik
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Nuclear Dna ◽  
Electron Microscopic Observation ◽  
Variable Number ◽  
Immunofluorescent Staining ◽  
Major Protein ◽  
Basal Bodies ◽  
Electron Microscopic ◽  
And Function

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus-basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.

scholarly journals Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation

2008 ◽  
Vol 190 (20) ◽  
pp. 6660-6667 ◽  
Author(s):  
Yong-Suk Che ◽  
Shuichi Nakamura ◽  
Seiji Kojima ◽  
Nobunori Kami-ike ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
High Speed ◽  
Proton Translocation ◽  
Energy Coupling ◽  
Flagellar Motor ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Torque Generation ◽  
Serovar Typhimurium ◽  
Bacterial Flagellar Motor ◽  
Protein Instability

ABSTRACT MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

scholarly journals Use of Gyrase Resistance Mutants To Guide Selection of 8-Methoxy-Quinazoline-2,4-Diones

2008 ◽  
Vol 52 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
Nadezhda German ◽  
Muhammad Malik ◽  
Jonathan D. Rosen ◽  
Karl Drlica ◽  
Robert J. Kerns
Keyword(s):  
Early Stage ◽  
Population Analysis ◽  
Resistance Mutation ◽  
Selection Criterion ◽  
Ring Structure ◽  
Wild Type ◽  
Mutant Selection ◽  
E Coli ◽  
Function Selection ◽  
Resistance Mutants

ABSTRACT A series of 1-cyclopropyl-8-methoxy-quinazoline-2,4-diones was synthesized and evaluated for lowering the ratio of the antimicrobial MIC in gyrase resistance mutants to that in the gyr + (wild type) using isogenic strains of Escherichia coli. Dione features that lowered this ratio were a 3-amino group and C-7 ring structure (3-aminomethyl pyrrolidinyl < 3-aminopyrrolidinyl < diazobicyclo < 2-ethyl piperazinyl). The wild-type MIC was also lowered. With the most active derivative tested, many gyrA resistance mutant types were as susceptible as, or more susceptible than, wild-type cells. The most active 2,4-dione derivatives were also more active with two quinolone-resistant gyrB mutants than with wild-type cells. With respect to lethality, the most bacteriostatic 2,4-dione killed E. coli at a rate that was affected little by a gyrA resistance mutation, and it exhibited a rate of killing similar to its cognate fluoroquinolone at 10× the MIC. Population analysis with wild-type E. coli applied to agar showed that the mutant selection window for the most active 2,4-dione was narrower than that for the cognate fluoroquinolone or for ciprofloxacin. These data illustrate a new approach to guide early-stage antimicrobial selection. Use of antimutant activity (i.e., ratio of the antimicrobial MIC in a mutant strain to the antimicrobial MIC in a wild-type strain) as a structure-function selection criterion can be combined with traditional efforts aimed at lowering antimicrobial MICs against wild-type organisms to more effectively afford lead molecules with activity against both wild-type and mutant cells.

scholarly journals Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

2020 ◽  
Author(s):  
Akihiro Kawamoto ◽  
Tomoko Miyata ◽  
Fumiaki Makino ◽  
Miki Kinoshita ◽  
Tohru Minamino ◽  
...  
Keyword(s):  
Ring Structure ◽  
Flagellar Motor ◽  
Native Structure ◽  
The Core ◽  
Ring Assembly ◽  
Native Ms ◽  
Torque Generation ◽  
High Resolution Structure ◽  
Motor Structure ◽  
Resolution Structure

AbstractThe bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor. It not only acts as the template for rod and C ring assembly but also houses the type III protein export gate for assembly of the rod, hook and filament. The cytoplasmic C ring, involved in torque generation and rotation switch, is directly attached to the MS ring, and a symmetry mismatch between 26-fold MS ring and 34-fold C ring had been a long puzzle as to whether this would play some role in motor function. Although this puzzle seemed to have been resolved by the recent high-resolution structure of the MS ring with 33-fold symmetry with a variation from 32-fold to 35-fold because the C ring also shows a similar symmetry variation, it still remained ambiguous whether their symmetries are matched in the native motor structure. Here we show that the native MS ring structure formed by full-length FliF is 34-fold with no symmetry variation whereas the C ring has a small symmetry variation, indicating a flexibility in C ring assembly to generate small symmetry mismatches. We also show two conformations of FliF in part of its periplasmic region to form the 34-subunit ring with 23-fold and 11-fold subsymmetries in the inner and middle M ring, respectively, to accommodate the export gate at the center of the M ring.

scholarly journals Mug28, a Meiosis-specific Protein of Schizosaccharomyces pombe, Regulates Spore Wall Formation

2010 ◽  
Vol 21 (12) ◽  
pp. 1955-1967 ◽  
Author(s):  
Akira Shigehisa ◽  
Daisuke Okuzaki ◽  
Takashi Kasama ◽  
Hideki Tohda ◽  
Aiko Hirata ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Electron Microscopic Observation ◽  
Specific Protein ◽  
Spore Wall ◽  
Electron Microscopic ◽  
Spore Walls ◽  
Green Fluorescent

The meiosis-specific mug28+ gene of Schizosaccharomyces pombe encodes a putative RNA-binding protein with three RNA recognition motifs (RRMs). Live observations of meiotic cells that express Mug28 tagged with green fluorescent protein (GFP) revealed that Mug28 is localized in the cytoplasm, and accumulates around the nucleus from metaphase I to anaphase II. Disruption of mug28+ generated spores with low viability, due to the aberrant formation of the forespore membrane (FSM). Visualization of the FSM in living cells expressing GFP-tagged Psy1, an FSM protein, indicated that mug28Δ cells harbored abnormal FSMs that contained buds, and had a delayed disappearance of Meu14, a leading edge protein. Electron microscopic observation revealed that FSM formation was abnormal in mug28Δ cells, showing bifurcated spore walls that were thicker than the nonbifurcated spore walls of the wild type. Analysis of Mug28 mutants revealed that RRM3, in particular phenylalanin-466, is of primary importance for the proper localization of Mug28, spore viability, and FSM formation. Together, we conclude that Mug28 is essential for the proper maturation of the FSM and the spore wall.

scholarly journals Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na+-Driven Flagellar Motor

2008 ◽  
Vol 190 (10) ◽  
pp. 3565-3571 ◽  
Author(s):  
Madoka Obara ◽  
Toshiharu Yakushi ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Electrostatic Interactions ◽  
Intramolecular Interactions ◽  
The Other ◽  
Flagellar Motor ◽  
Wild Type ◽  
Mutant Proteins ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Suppressor Analysis

ABSTRACT Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na+-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na+-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na+-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na+-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.

scholarly journals Structure of the molecular bushing of the bacterial flagellar motor

2020 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Fumiaki Makino ◽  
Tomoko Miyata ◽  
Tohru Minamino ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
Basal Body ◽  
High Speed ◽  
Rotational Symmetry ◽  
Ring Structure ◽  
Repulsive Force ◽  
Bacterial Flagellum ◽  
Ring Assembly ◽  
High Speed Rotation ◽  
Speed Rotation ◽  
Intersubunit Interactions

AbstractThe bacterial flagellum is a motility organelle, consisting of the basal body acting as a rotary motor, the filament as a helical propeller and the hook connecting these two as a universal joint1,2. The basal body contains three rings: the MS ring as the transmembrane core of the rotor; the C ring essential for torque generation and switching regulation; and the LP ring as a bushing supporting the distal rod for its rapid, stable rotation without much friction. The negatively charged surface of the distal rod suggested electrostatic repulsive force in supporting high-speed rotation of the rod as a drive shaft3, but the LP ring structure was needed to see the actual mechanisms of its bushing function and assembly against the repulsive force. Here we report the LP ring structure by electron cryomicroscopy at 3.5 Å resolution, showing 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners that explains the structural stability. The inner surface is charged both positively and negatively, and positive charges on the P ring presumably play important roles in its initial assembly around the rod in the peptidoglycan layer followed by the L ring assembly in the outer membrane.

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