scholarly journals BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Xu ◽  
Bo Hu ◽  
David A. Flesher ◽  
Jun Liu ◽  
Md A. Motaleb
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Lytic Enzyme ◽  
Enzyme Protein ◽  
Lytic Transglycosylase ◽  
Filament Assembly ◽  
Cryo Electron Tomography ◽  
Mutant Strains ◽  
Periplasmic Flagella ◽  
Dual Domain

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

scholarly journals Cryo-electron tomography of periplasmic flagella in Borrelia burgdorferi reveals a distinct cytoplasmic ATPase complex

PLoS Biology ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. e3000050 ◽  
Author(s):  
Zhuan Qin ◽  
Jiagang Tu ◽  
Tao Lin ◽  
Steven J. Norris ◽  
Chunhao Li ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Periplasmic Flagella

scholarly journals Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

2009 ◽  
Vol 191 (16) ◽  
pp. 5026-5036 ◽  
Author(s):  
Jun Liu ◽  
Tao Lin ◽  
Douglas J. Botkin ◽  
Erin McCrum ◽  
Hanspeter Winkler ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Rotational Symmetry ◽  
Flagellar Motor ◽  
Cell Axis ◽  
Ring Assembly ◽  
Flagellar Motors ◽  
Cryo Electron Tomography ◽  
Flagellar Rotation

ABSTRACT The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

scholarly journals The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Shiwei Zhu ◽  
Tatsuro Nishikino ◽  
Seiji Kojima ◽  
Michio Homma ◽  
Jun Liu
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Bacterial Species ◽  
Vibrio Alginolyticus ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Outer Membranes ◽  
Cryo Electron Tomography ◽  
Periplasmic Flagella ◽  
First Time

ABSTRACT The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, whereas spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here, we use cryo-electron tomography to visualize the polar sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio-specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes. IMPORTANCE Flagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella, such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

scholarly journals Hypothetical Protein BB0569 Is Essential for Chemotaxis of the Lyme Disease Spirochete Borrelia burgdorferi

2015 ◽  
Vol 198 (4) ◽  
pp. 664-672 ◽  
Author(s):  
Kai Zhang ◽  
Jun Liu ◽  
Nyles W. Charon ◽  
Chunhao Li
Keyword(s):  
Escherichia Coli ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Electron Tomography ◽  
Model Organism ◽  
Hypothetical Protein ◽  
Content Type ◽  
Cryo Electron Tomography

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferihas five putative methyl-accepting chemotaxis proteins (MCPs). In this report, we provide evidence that a hypothetical protein, BB0569, is essential for the chemotaxis ofB. burgdorferi. While BB0569 lacks significant homology to the canonical MCPs, it contains a conserved domain (spanning residues 110 to 170) that is often evident in membrane-bound MCPs such as Tar and Tsr ofEscherichia coli. Unlike Tar and Tsr, BB0569 lacks transmembrane regions and recognizable HAMP and methylation domains and is similar to TlpC, a cytoplasmic chemoreceptor ofRhodobacter sphaeroides. An isogenic mutant ofBB0569constantly runs in one direction and fails to respond to attractants, indicating that BB0569 is essential for chemotaxis. Immunofluorescence, green fluorescent protein (GFP) fusion, and cryo-electron tomography analyses demonstrate that BB0569 localizes at the cell poles and is required for chemoreceptor clustering at the cell poles. Protein cross-linking studies reveal that BB0569 forms large protein complexes with MCP3, indicative of its interactions with other MCPs. Interestingly, analysis ofB. burgdorferimcpmutants shows that inactivation of eithermcp2ormcp3reduces the level of BB0569 substantially and that such a reduction is caused by protein turnover. Collectively, these results demonstrate that the domain composition and function of BB0569 are similar in some respects to those of TlpC but that these proteins are different in their cellular locations, further highlighting that the chemotaxis ofB. burgdorferiis unique and different from theEscherichia coliandSalmonella entericaparadigm.IMPORTANCESpirochete chemotaxis differs substantially from theEscherichia coliandSalmonella entericaparadigm, and the basis for controlling the rotation of the bundles of periplasmic flagella at each end of the cell is unknown. In recent years,Borrelia burgdorferi, the causative agent of Lyme disease, has been used as a model organism to understand spirochete chemotaxis and its role in infectious processes of the disease. In this report, BB0569, a hypothetical protein ofB. burgdorferi, has been investigated by using an approach of genetic, biochemistry, and cryo-electron tomography analyses. The results indicate that BB0569 has a distinct role in chemotaxis that may be unique to spirochetes and represents a novel paradigm.

scholarly journals Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew Akamatsu ◽  
Ritvik Vasan ◽  
Daniel Serwas ◽  
Michael A Ferrin ◽  
Padmini Rangamani ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Force Production ◽  
Multiscale Model ◽  
Membrane Tension ◽  
Actin Assembly ◽  
Actin Network ◽  
Physical Constraints ◽  
Filament Assembly ◽  
Attachment Sites ◽  
Cryo Electron Tomography

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.

scholarly journals Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Tao Lin ◽  
Lihui Gao ◽  
Xiaowei Zhao ◽  
Jun Liu ◽  
Steven J. Norris
Keyword(s):  
Cell Division ◽  
Borrelia Burgdorferi ◽  
Type Iii Secretion ◽  
Electron Tomography ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Flagellar Motor ◽  
Content Type ◽  
Type Iii ◽  
Cryo Electron Tomography

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferimigrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI inB. burgdorferihave not been reported. In this study,fliHandfliItransposon mutants were utilized to dissect the mechanism of theBorreliatype III secretion system. ThefliHandfliImutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants infliHandfliIwere incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of eitherfliHorfliIresulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation offliHandfliImutants intransrestored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of eitherfliHorfliIinB. burgdorferiresults in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins.IMPORTANCEMany bacteria are able to rapidly transport themselves through their surroundings using specialized organelles called flagella. In spiral-shaped organisms called spirochetes, flagella act like inboard motors and give the bacteria the ability to bore their way through dense materials (such as human tissue) in a corkscrew manner. In this article, we studied how two proteins, called FliH and FliI, are important for the production of full-length flagella in the Lyme disease spirocheteBorrelia burgdorferi. Mutants with defective production of FliH and FliI have reduced flagellar length and motility; this deficiency in turn affects many aspects ofB. burgdorferi's biology, including the ability to undergo cell division and cause disease in mammals. Using a microscopic computed tomography (CT) scan approach called cryo-electron tomography, the structure that contains FliH and FliI was defined in the context of the flagellar motor, providing clues regarding how this amazing nanomachine is assembled and functions.

scholarly journals In situ structures of periplasmic flagella reveal a distinct cytoplasmic ATPase complex in Borrelia burgdorferi

2018 ◽  
Author(s):  
Zhuan Qin ◽  
Akarsh Manne ◽  
Jiagang Tu ◽  
Zhou Yu ◽  
Kathryn Lees ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Structural Framework ◽  
Structural Rigidity ◽  
Membrane Bound ◽  
Novel Therapeutic Strategies ◽  
Periplasmic Flagella ◽  
Distinct Morphology

ABSTRACTPeriplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific Type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we combine cryo-electron tomography and mutagenesis approaches to characterize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We define the fT3SS machine by systematically characterizing mutants lacking key component genes. We discover that a distinct cytosolic ATPase complex is attached to the flagellar C-ring through multiple spoke-like linkers. The ATPase complex not only strengthens structural rigidity of the C-ring, but also undergoes conformational changes in concert with flagellar rotation. Our studies provide structural framework to uncover the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the bases for the development of novel therapeutic strategies against several pathogenic spirochetes.

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