Role of flagellar hydrogen bonding in Salmonella motility and flagellar polymorphic transition

Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bistable forms L‐type or R‐type, having slightly different conformations and inter‐protofilaments interactions. By mixing different ratios of L‐type and R‐type protofilaments, flagella adopt multiple filament polymorphs and promote bacterial motility. In this study, we investigated the hydrogen bonding networks at the flagellin crystal packing interface in Salmonella enterica serovar typhimurium (S. typhimurium) by site‐directed mutagenesis of each hydrogen bonded residue. We identified three flagellin mutants D108A, N133A and D152A that were non‐motile despite their fully assembled flagella. Mutants D108A and D152A trapped their flagellar filament into inflexible right‐handed polymorphs, which resemble the previously predicted 3L/8R and 4L/7R helical forms in Calladine’s model but have never been reported in vivo. Mutant N133A produces floppy flagella that transform flagellar polymorphs in a disordered manner, preventing the formation of flagellar bundles. Further, we found that the hydrogen bonding interactions around these residues are conserved and coupled to flagellin L/R transition. Therefore, we demonstrate that the hydrogen bonding networks formed around flagellin residues D108, N133 and D152 greatly contribute to flagellar bending, flexibility, polymorphisms and bacterial motility.     

Reversible inhibition of Chlamydomonas flagellar surface motility

Chlamydomonas exhibits force transduction in association with its flagellar surface; this can be visualized by the saltatory movements of attached polystyrene microspheres. This flagellar surface motility has been quantitated by determining the percentage of attached microspheres in motion at the time of observation (60% in the case of control cells at 25 degrees C). A number of experimental treatments reversibly inhibit flagellar surface motility. These include an increase in sodium or potassium chloride concentration, a decrease in temperature, or a decrease in the free calcium concentration in the medium. Many of the conditions that result in inhibition of flagellar surface motility also result in an induction of flagellar resorption. Although both flagellar stability and flagellar surface motility are dependent on the availability of calcium, the two processes are separable; under appropriate conditions, flagellar surface motility can occur at normal levels on flagella that are resorbing. Inhibition of protein synthesis results in a gradual loss of both the binding of microspheres to the flagellum and the flagellar surface motility. After resumption of protein synthesis, both binding and movement return to control levels. The effect of the inhibition of protein synthesis is interpreted in terms of selective turnover of certain components within the intact flagellum, one or more of these components being necessary for the binding of the microspheres and their subsequent movement. If this turnover is inhibited by keeping the cells below 5 degrees C, the absence of protein synthesis no longer has an effect on microsphere attachment and motility, when measured immediately after warming the cells to 25 degrees C.     

Polar flagellar motility of the Vibrionaceae.

Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 microm/s. The sodium motive force powers rotation of the filament, which acts as a propeller. The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane. The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus. The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species. This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae. In addition to its propulsive role, the single polar flagellum of V. parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression. Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces. Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery.     

Effects of deuterium oxide on bacterial flagellar motility

Summary Low concentrations of ²H₂O had either no observable influence, or a slightly stimulatory effect, on the translational movement of flagellated bacteria. High ²H₂O levels had marked, but transient, effects, expressed as a retardation of movement, especially on peritrichous forms. Polarly flagellated bacteria rapidly recovered from ²H₂O effects, whereas peritrichous organisms possessed only a limited recovery capacity.Flagellar regeneration and resumption of normal motion were retarded in a 1.5% (w/v) Trypticase Soy-²H₂O medium, and again this was more evident in peritrichous bacteria. The overall length of flagella regenerated in the presence of ²H₂O differed noticeably from those regenerated in its absence. There was no flagellar regeneration in 99.8% ²H₂O.The morphological appearance of ²H₂O-treated bacteria suggests that deuterium oxide acts as if it is a mild plasmolytic agent whose effects are readily reversible in most cases.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

Origins of flagellar gene operons and secondary flagellar systems

Forty-one flagellated species representing 11 bacterial phyla were used to investigate the origin of secondary flagellar systems and the structure and formation of flagellar gene operons over the course of bacterial evolution. Secondary (i.e., lateral) flagellar systems, which are harbored by five of the proteobacterial species considered, originated twice, once in the alphaproteobacterial lineage and again in the common ancestor of the Beta- and Gammaproteobacteria. The order and organization of flagellar genes have undergone extensive shuffling and rearrangement among lineages, and based on the phylogenetic distributions of flagellar gene complexes, the flagellar gene operons existed as small, usually two-gene units in the ancestor of Bacteria and have expanded through the recruitment of new genes and fusion of gene units. In contrast to the evolutionary trend towards larger flagellar gene complexes, operon structures have been highly disrupted through gene disassociation and rearrangements in the Epsilon- and Alphaproteobacteria. These results demonstrate that the genetic basis of this ancient and structurally conserved organelle has been subject to many lineage-specific modifications.     

Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+ -binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.     

Mechanisms of flagellar motility deduced from backward-swimming bull sperm

Under certain conditions of cryopreservation, bull spermatozoa undergo an interesting structural alteration. The sperm tail becomes bent back on itself to form a hairpin shape. The bend in the tail occurs at a very precise point, 11 microns behind the neck, and it causes the tail to become kinked. Flagellar microtubules and dense fibers become broken and the ninefold symmetry of the flagellum is greatly distored. Although the portion of the flagellum between the kink and the sperm head does not propagate a wave, the distal portion of the flagellum propagates a base-to-tip wave, causing the spermatozoan to progress backward. These observations suggest that the mammalian spermatozoon does not need basal structures to propagate a flagellar wave.     

Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation

There is substantial evidence that cAMP-dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP-dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP-dependent phosphorylation and changes in axonemal function are still unknown. Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoattractants involves changes in asymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established. Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.     

Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility

The c‐di‐GMP‐binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c‐di‐GMP‐bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re‐annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ‐mediated downregulation of motility is fine‐tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c‐di‐GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c‐di‐GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c‐di‐GMP‐dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella‐driven swimming and swarming motility.     

Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.     

Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

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Gliding movement in Peranema trichophorum is powered by flagellar surface motility

A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10–12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility. Received: 13 March 1998 / Revised version: 17 April 1998 / Accepted: 17 April 1998     

Autophagic elimination of ribosomes during spermiogenesis provides energy for flagellar motility

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