Spirochetes flagellar collar protein FlbB has astounding effects in orientation of periplasmic flagella,bacterial shape,motility, and assembly of motors in Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, is a highly motile spirochete, and motility, which is provided by its periplasmic flagella, is critical for every part of the spirochete's enzootic life cycle. Unlike externally flagellated bacteria, spirochetes possess a unique periplasmic flagellar structure called the collar. This spirochete‐specific novel component is linked to the flagellar basal body; however, nothing is known about the proteins encoding the collar or their function in any spirochete. To identify a collar protein and determine its function, we employed a comprehensive strategy that included genetic, biochemical, and microscopic analyses. We found that BB0286 (FlbB) is a novel flagellar motor protein, which is located around the flagellar basal body. Deletion of bb0286 has a profound effect on collar formation, assembly of other flagellar structures, morphology, and motility of the spirochete. Orientation of the flagella toward the cell body is critical for determination of wild‐type spirochete's wave‐like morphology and motility. Here, we provide the first evidence that FlbB is a key determinant of normal orientation of the flagella and collar assembly.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR POSITION IN THE COLONIAL GREEN ALGA PLATYDORINA CAUDATA (CHLOROPHYCEAE)1

The ultrastructure of the flagellar apparatus in pre-inversion and inversion stages of Platydorina resembles that of Chlamydomonas in having 180° rotational symmetry and clockwise absolute orientation. Basal bodies are in a “V” configuration and connected by one distal and two proximal fibers. Alternating two- and four-membered microtubular rootlets are cruciately arranged. During maturation, the basal bodies rotate and separate, and 180° rotational symmetry is lost. Simultaneously, each proximal fiber detaches from one of the functional basal bodies, and the distal fiber detaches from both. The mature apparatus has widely separated and nearly parallel basal bodies. Flagellar orientation in Platydorina is completed just after inversion and a flattening of the colony called intercalation, resulting in the pairs of flagella of neighboring cells extending from the colony in opposite directions in an alternating fashion. Flagellar orientation and separated basal bodies minimize the interference between the flagella of neighboring cells. Basal bodies and rootlets of the two intercalated halves of a colony rotate, resulting in the effective strokes of the flagella of every cell being towards the colonial posterior. The flagella of each cell beat with an effective stroke in the direction of the two inner rootlets. The flagella have an asymmetrical ciliary type beat. The rotated, separated, and parallel basal bodies, together with the nearly parallel rootlets probably are adaptations for movement of this colonial volvocalean alga. The flagellar apparatus in immature stages of Platydorina lends support to the suggestion that the alga has evolved from a Chlamydomonas-like ancestor.     

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS‐DegU two‐component system. Here we report a role for flagella in the regulation of the K‐state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU‐P, which inhibits the expression of ComK, the master regulator for the K‐state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hag^A233V) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU‐P levels through an unknown signaling mechanism. This flagellar‐load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K‐state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.     

Bacillus subtilis Bactofilins Are Essential for Flagellar Hook- and Filament Assembly and Dynamically Localize into Structures of Less than 100 nm Diameter underneath the Cell Membrane

Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

The Uni2 phosphoprotein is a cell cycle regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.     

A phylogenetic analysis of the purple photosynthetic bacteria

It is proposed that gliding motility in bacteria is based on rotary assemblies located in the cell envelope and that these assemblies may be analogous to basal regions of bacterial flagella. This proposal rests on the following evidence: (i) Structures resembling flagellar basal regions have been demonstrated in cells ofCytophaga johnsonae andFlexibacter columnaris, and such structures are absent from one nonmotile mutant ofF. columnaris. (ii) The effects of inhibitors of energy metabolism on gliding motility are identical with their effects on prokaryotic fiagellar motility. (iii) The active movement of latex spheres along surfaces of gliding bacteria appears to depend on mechanisms responsible for motility and can be explained by the presence of rotary surface assemblies.     

The flagellar apparatus of the golden algaSynura uvella: Four absolute orientations

Summary Flagellated vegetative cells of the colonial golden algaSynura uvella Ehr, were examined using serial sections. The two flagella are nearly parallel as they emerge from a flagellar pit near the apex of the cell. The photoreceptor is restricted to swellings on the flagella in the region where they pass through the apical pore in the scale case and the swellings are not associated with the cell membrane or an eyespot. A unique ring-like structure surrounds the axonemes of both flagella at a level just above the transitional helix. The basal bodies are interconnected by three striated, fibrous bands. Four short (<100 nm) microtubules lie between the basal bodies at their proximal ends. Two rhizoplasts extend down from the basal bodies and separate into numerous fine striated bands which lie over the nucleus. Three- and four-membered microtubular roots arise from the rhizoplasts and extend apically together. As the roots reach the cell anterior, the three-membered root bends and curves clockwise to form a large loop around the flagella; the four-membered root bends anticlockwise and terminates under the distal end of the three-membered root as it completes the loop. There are four absolute orientations, termed Types 1–4, in which the flagellar apparatus can occur. With each orientation type the positions of the Golgi body, nucleus, rhizoplasts, chloroplasts and microtubular roots change with respect to the flagella, basal bodies and photoreceptor. Two new basal bodies appear in pre-division cells, and three short microtubules appear in a dense substance adjacent to each new basal body. Based upon the positions of new pre-division basal bodies, a hypothesis is proposed to explain why there are four orientations and how they are maintained through successive cell divisions.     

The flagellar apparatus structure inMicrospora (Chlorophyceae) confirms a close evolutionary relationship with unicellular green algae

The spatial configuration of the flagellar apparatus of the biflagellate zoospores of the green algal genusMicrospora is reconstructed by serial sectioning analysis using transmission electron microscopy. Along with the unequal length of the flagella, the most remarkable characteristics of the flagellar apparatus are: (1) the subapical emergence of the flagella (especially apparent with scanning electron microscopy); (2) the parallel orientation of the two basal bodies which are interconnected by a prominent one-piece distal connecting fiber; (3) the unique ultrastructure of the distal connecting fiber composed of a central tubular region which is bordered on both sides by a striated zone; (4) the different origin of the d-rootlets from their relative basal bodies; (5) the asymmetry of the papillar region which together with the subapical position of the basal bodies apparently cause the different paths of corresponding rootlets in the zoospore anterior; (6) the presence of single-membered d-rootlets and multi-membered s-rootlets resulting in a 7-1-7-1 cruciate microtubular root system which, through the different rootlet origin, does not exhibit a strict 180° rotational symmetry. It is speculated that the different basal body origin of the d-rootlets is correlated with the subapical implant of flagella. It is further hypothesized that in the course of evolution the ancestors ofMicrospora had a flagellar papilla that has migrated from a strictly apical position towards a subapical position. Simultaneously, ancestral shift of flagella along the apical cell body periphery has taken place as can be concluded from the presence of an upper flagellum overlying a lower flagellum in the flagellar apparatus ofMicrospora. The basic features of the flagellar apparatus of theMicrospora zoospore resemble those of the coccoid green algal generaDictyochloris andBracteacoccus and also those of the flagellate green algal genusHeterochlamydomonas. This strengthens the general supposition thatMicrospora is evolutionarily closely related to taxa which were formerly classified in the traditionalChlorococcales.     

FLAGELLAR MOTION AND FINE STRUCTURE OF THE FLAGELLAR APPARATUS IN CHLAMYDOMONAS

The biflagellate alga Chlamydomonas reinhardi was studied with the light and electron microscopes to determine the behavior of flagella in the living cell and the structure of the basal apparatus of the flagella. During normal forward swimming the flagella beat synchronously in the same plane, as in the human swimmer's breast stroke. The form of beat is like that of cilia. Occasionally cells swim backward with the flagella undulating and trailing the cell. Thus the same flagellar apparatus produces two types of motion. The central pair of fibers of both flagella appear to lie in the same plane, which coincides with the plane of beat. The two basal bodies lie in a V configuration and are joined at the top by a striated fiber and at the bottom by two smaller fibers. From the area between the basal bodies four bands of microtubules, each containing four tubules, radiate in an X-shaped pattern, diverge, and pass under the cell membrane. Details of the complex arrangement of tubules near the basal bodies are described. It seems probable that the connecting fibers and the microtubules play structural roles and thereby maintain the alignment of the flagellar apparatus. The relation of striated fibers and microtubules to cilia and flagella is reviewed, particularly in phytoflagellates and protozoa. Structures observed in the transitional region between the basal body and flagellar shaft are described and their occurrence is reviewed. Details of structure of the flagellar shaft and flagellar tip are described, and the latter is reviewed in detail.     

ULTRASTRUCTURE OF THE FLAGELLAR APPARATUS OF PYROBOTRYS (CHLOROPHYCEAE)1

The flagellar apparatus of Pyrobotrys has a number of features that are typical of the Chlorophyceae, but others that are unusual for this class. The two flagella are inserted at the apex, but they extend to the side of the cell toward the outside of the colony, here designated as the ventral side. Four basal bodies are present, two of which extend into flagella. Four microtubular rootlets alternate between the functional and accessory basal bodies. In each cell, the two ventral rootlets are nearly parallel, but the dorsal rootlets are more widely divergent. The rootlets alternate between two and four microtubules each. A striated distal fiber connects the two functional basal bodies in the plane of the flagella. Two additional, apparently nonstriated, fibers connect the basal bodies proximal to the distal fiber. Another striated fiber is associated with each four-membered rootlet near its insertion into the flagellar apparatus. A fine periodic component is associated with each two-membered rootlet. A rhizoplast-like structure extends into the cell from each of the functional basal bodies. The arrangement of these components does not reflect the 180° rotational symmetry that is usually present in the Chlorophyceae, but appears to be derived from a more symmetrical ancestor. It is suggested that the form of the flagellar apparatus is associated with the unusual colony structure of Pyrobotrys.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.     

Drosophila Bld10 Is a Centriolar Protein That Regulates Centriole, Basal Body, and Motile Cilium Assembly

Cilia and flagella play multiple essential roles in animal development and cell physiology. Defective cilium assembly or motility represents the etiological basis for a growing number of human diseases. Therefore, how cilia and flagella assemble and the processes that drive motility are essential for understanding these diseases. Here we show that Drosophila Bld10, the ortholog of Chlamydomonas reinhardtii Bld10p and human Cep135, is a ubiquitous centriolar protein that also localizes to the spermatid basal body. Mutants that lack Bld10 assemble centrioles and form functional centrosomes, but centrioles and spermatid basal bodies are short in length. bld10 mutant flies are viable but male sterile, producing immotile sperm whose axonemes are deficient in the central pair of microtubules. These results show that Drosophila Bld10 is required for centriole and axoneme assembly to confer cilium motility.     

COMPARATIVE ULTRASTRUCTURE OF ZOOSPORES WITH PARALLEL BASAL BODIES FROM THE GREEN ALGAE DICTYOCHLORIS FRAGRANS AND BRACTEACOCCUS SP.

The chlorococcalean algae Dictyochloris fragrans and Bracteacoccus sp. produce naked zoospores with two unequal flagella and parallel basal bodies. Ultrastructural features of the flagellar apparatus of these zoospores are basically identical and include a banded distal fiber, two proximal fibers, and four cruciately arranged microtubular rootlets with only one microtubule in each dexter rootlet. In D. fragrans, each proximal fiber is composed of two subfibers, one striated and one nonstriated, and each sinister rootlet is composed of five microtubules (4/1), decreasing to four away from the basal bodies. In Bracteacoccus sp., each proximal fiber is a single unit, the sinister rootlets are four (3/1) or rarely five (4/1) microtubules, and each basal body is associated with an unusual curved structure. The basic features of the flagellar apparatus of the zoospores of these two algae resemble those of Heterochlamydomonas rather than most other chlorococcalean algae that have equal length flagella, basal bodies in the V-shape arrangement, and clockwise absolute orientation. It is proposed that these algae with unequal flagella and parallel basal bodies have a shared common ancestry within the green algae.     

Basal apparatus behaviour during cellular division (sporulation) in the coccoid green algaChlorosarcina

Summary We studied the basal body cycle (including basal body segregation, duplication, migration, and reorientation) in dividing cells of the colonial coccoid green algaChlorosarcina stigmatica using serial thin sections. Although flagella are lacking, all cells examined possess a rudimentary flagellar apparatus composed of two basal bodies linked by a distal striated fibre, two probasal bodies, and four cruciately arranged microtubular roots (2-4-2-4 type). Basal body segregation occurs at preprophase, during which two half-basal apparatuses (each consisting of one basal body, one probasal body, and a left and a right root) migrate into opposite directions. The segregation axis is defined by the two left roots which remain closely associated during segregation and slide along each other. The segregation axis is parallel to the axis of chromosome separation, and perpendicular to the plane of subsequent cell division. Duplication of basal apparatus components does not occur until telophase when daughter basal apparatuses migrate towards the plane of division. At cytokinesis which is effected by the unilateral ingrowth of a septum, each daughter basal apparatus rotates 90° and becomes associated with the new septum.Abbreviations BA basal (body) apparatus - NBBC nucleus-basal body connector     

Chlamydomonas flagella

Chlamydomonas reinhardtii Dangard has been widely adopted as a model system for studies of eukaryotic cilia and flagella. Here I review recent progress in understanding flagellar ultrastructure, the mechanisms that generate and regulate flagellar beating and gliding motility, the flagellar assembly process, basal body structure and function, and adhesion-based signaling, all advanced by work with this single-celled organism.