Helicobacter pylori CheZHP and ChePep form a novel chemotaxis‐regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non‐phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ_HP, localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ_HP localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ_HP for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ_HP determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ_HP and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.     

FLAGELLAR HAIRS IN PRASINOPHYTES (CHLOROPHYTA): ULTRASTRUCTURE AND DISTRIBUTION ON THE FLAGELLAR SURFACE1

The flagellar hair ultrastructure of 16 strains of species of the prasinophycean genera Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis, Tetraselmis, Scherffelia, Pterosperma, and Pyraminonas was examined in detail by whole-mount electron microscopy. The flagellar hairs of all genera displayed a high degree of ultrastructural complexity that was completely conserved within each strain. In all strains, flagellar hairs occurred on the sides of the flagella (lateral hairs); in several strains, special flagellar hairs also were found on the flagellar tips (tip hairs; absent in the Chlorodendrales and in Nephroselmis). Two groups of lateral hairs were distinguished: 1) T-hairs (“Tetraselmis-type” flagellar hairs), characterized by a smooth, tubular shaft of ca. 15 nm diameter and an overall length of 0.5–1.3 μm, and 2) P_t-hairs (“Pterosperma-type lateral flagellar hairs”), which were considerably longer (ca. 1.5–5.4 μm), characterized by a thick shaft of ca. 30 nm diameter, which was covered with a layer of regularly spaced small particles of ca. 10 nm diameter. In both groups of flagellar hairs, a strain-specific number of subunits (1–101) in linear arrangement was attached to the distal end of the shaft. Tip hairs were either structurally related to T-hairs (Mamiellales, Pseudoscourfieldia) or represented a separate group, P_t-hairs (“Pterosperma-type flagellar tip hairs”; Pterosperma, Pyramimonas). In four genera (Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis), both groups of lateral hairs occurred together on the same cell. Interestingly in these taxa the P_t-hairs were exclusively attached to the shorter immature flagella (no. 2), but, in contrast, in Mantoniella and Pseudoscourfieldia the tip hairs were restricted to the longer mature flagellum (no. 1). Thus, flagella of different developmental status differ in their hair-scale complement. The occurrence, distribution, and ultrastructure of flagellar hairs can be used to identify and classify prasinophytes at all taxonomic levels.     

Transmembrane signaling in cilia and flagella

Summary Ciliary and flagellar membranes are dynamic. Ciliary and flagellar membranes have diverged widely during evolution and perform many specialized functions. Transmembrane signaling is an important component of the function of ciliary and flagellar surfaces in general. In this review, I discuss some of the functions performed by ciliary and flagellar surfaces and I present three different ciliary and flagellar signaling systems associated with rather different dynamic events performed by ciliary and flagellar surfaces. Two of these are associated withChlamydomonas flagella and one is associated with vertebrate olfactory cilia. Calcium regulation of protein phosphorylation appears to be important in regulating glycoprotein movements in theChlamydomonas flagellar membrane. Changes in levels of cAMP and cAMP-dependent protein phosphorylation are clearly central to the signaling associated with mating events in gametic flagella ofChlamydomonas, although calcium clearly has an important, if poorly understood, role to play. There is no known role for G proteins in flagellar membrane events inChlamydomonas. In contrast, mammalian olfactory cilia possess an odorant activated, G protein regulated adenylate cyclase and conductance channels that are directly gated by cyclic nucleotides. A second class of odorants that do not affect adenylate cyclase activity appear to act through G protein activated phospholipase C and changes in IP₃ second messenger levels. These examples demonstrate the diversity in the signaling pathways associated with ciliary and flagellar membranes.Abbreviations CaPK-2 calcium-dependent protein kinase - db-cAMP dibutyryl cAMP - Fab fragment antigen binding - IgE immunoglobulin E - IP₃ myo-inositol trisphosphate - IP₄ myo-inositol tetrakisphosphate - OBP odorant binding protein - PIP₂ phosphoinositol bisphosphate - TFP trifluoperazine - WGA wheat germ agglutinin     

Dual stator dynamics in the Shewanella oneidensis MR‐1 flagellar motor

The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion‐dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR‐1 expresses two distinct types of stator units: the Na⁺‐dependent PomA₄B₂ and the H⁺‐dependent MotA₄B₂. Here, we have explored the stator unit dynamics in the MR‐1 flagellar system by using mCherry‐labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na⁺‐levels. In 200 mM Na⁺, the numbers of PomAB and MotAB units in wild‐type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na⁺. Significantly, the average swimming speed of MR‐1 cells at low Na⁺ conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H⁺ and Na⁺ driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na⁺ levels.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

DISTINCTIVE LECTIN LABELING OF THE FLAGELLAR APPARATUS OF TETRASELMIS (PRASINOPHYCEAE) AND DUNALIELLA (CHLOROPHYCEAE)1

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH₂ moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.     

Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

The flagella of ‘Candidatus Liberibacter asiaticus’ and its movement in planta

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJ_Atu but not flgJ_Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ_Las interacts with FlgB but not with FliE. Ectopic expression of flaA_Las in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.     

The role of microtubules during the cytokinetic events of cell wall ontogenesis and papilla development inCarteria crucifera

Summary An ultrastructural study of cytokinesis, cell wall ontogenesis, and papilla development/form inCarteria crucifera Korsh. andChloromonas rosae Ettl was undertaken. After typical phycoplast-mediated cytokinesis, wall ontogenesis begins at the level of Golgi apparatus activation and secretion to the outside of the daughter cells of fibrillar wall precursors which self assemble into the typical chlamydomonad wall (sensuRoberts 1974). As wall ontogenesis approaches the flagellar region of the cell, several precisely timed events occur: flagellar apparatus formation, flagellar emergence, protoplasmic extension in the future papilla area underlined by series of parallel aligned microtubules, wall formation (at least the W₂–W₆ layers), retraction of the protoplasmic extension and loss of underlying microtubules, and final wall modification (gap filling by W₁ material) to yield the characteristic wall papilla. The transient cytoplasmic extensions mimic the shape of the future wall papilla and are maintained, at least inCarteria, by underlying microtubules. Structural and developmental properties of the papilla are characterized and phylogenetic implications are discussed.This research was supported by National Science Foundation Grant DEB 78-0554.     

Differential effects of sodium ions on motility in the homoacetogenic bacteriaAcetobacterium woodii andSporomusa sphaeroides

The strictly anaerobic homoacetogenic bacteria Acetobacterium woodii and Sporomusa sphaeroides differ with respect to their energy metabolism. Since growth as well as acetate and ATP formation of A. woodii is strictly dependent on Na⁺, but that of S. sphaeroides is not, the question arose whether these organisms also use different coupling ions for mechanical work, i.e. flagellar rotation. During growth on fructose in the presence of Na⁺ (50 mM), cells of A. woodii were vigorously motile, as judged by light microscopy. At low Na⁺ concentrations (0.3 mM), the growth rate decreased by only 15%, but the cells were completely non-motile. Addition of Na⁺ to such cultures restored motility instantaneously. Motility, as determined in swarm agar tubes, was strictly dependent on Na⁺; Li⁺, but not K⁺ partly substituted for Na⁺. Of the amilorides tested, phenamil proved to be a specific inhibitor of the flagellar motor of A. woodii. Growth and motility of S. sphaeroides was neither dependent on Na⁺ nor inhibited by amiloride derivatives. These results indicate that flagellar rotation is driven by Δμ_Na ⁺ in A. woodii, but by Δμ_H ⁺ in S. sphaeroides. Received: 30 May 1995 / Accepted: 31 August 1995     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

Flagellar apparatus ultrastructure inMesostigma viride (Prasinophyceae)

The ultrastructure of the flagellar apparatus ofMesostigma viride Lauterborn (Prasinophyceae) has been studied in detail with particular reference to absolute configurations, numbering of basal bodies, basal body triplets and flagellar roots. The two basal bodies are interconnected by three connecting fibers (one distal fiber = synistosome, and two proximal fibers). The flagellar apparatus shows 180° rotational symmetry; four microtubular flagellar roots and two system II fibers are present. The microtubular roots represent a 4-6-4-6-system. The left roots (1s, 2s) consist of 4 microtubules, each with the usual 3 over 1 root tubule pattern. Each right root (1d, 2d) is proximally associated with a small, but typical multi-layered structure (MLS). The latter displays several layers corresponding to the S₁ (the spline microtubules: 5–7), and presumably the S₂—S₄ (the lamellate layers) of the MLS of theCharophyceae. At its proximal origin (near the basal bodies) each right root originates with only two microtubules, the other spline microtubules being added more distally. The structural and positional information obtained in this study strongly suggest that one of the right roots (1d) ofMesostigma is homologous to the MLS-root of theCharophyceae and sperm cells of archegoniate land plants. Thus the typical cruciate flagellar root system of the green algae and the unilateral flagellar root system of theCharophyceae and archegoniates share a common ancestry. Some functional and phylogenetic aspects of MLS-roots are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Alteration of flagellar phenotype of Escherichia coli strain P12b, the standard type strain for flagellar antigen H17, possessing a new non-fliC flagellin gene flnA, and possible loss of original flagellar phenotype and genotype in the course of subculturing through semisolid media

A practically important phenomenon, resulting in the loss of the original flagellar phenotype (genotype) of bacteria, is described in the Escherichia coli H17 type strain P12b possessing two distinct genes for H17 and H4 flagellins, respectively. By PCR, sequencing, and phylogenetic investigation, the H17 gene (originally expressed) was considered a new non-fliC flagellin gene and assigned flnA, while the H4 gene (originally cryptic) was reaffirmed as fliC. H17 and H4 flagella differed morphologically. The phenomenon consisted in the replacement of H17 cells by H4 cells during subculturing through certain semisolid media and resulted from the excision of flnA _H17 entirely or in part. The substitution rate depended on the density and nutrient composition of media and reached 100% even after a single passage through 0.3% LB agar. Such phenomenon can lead to an unexpected loss of original H17 phenotype. Our review of the literature showed that the loss of the original flagellar genotype (phenotype) of P12b has occurred in some laboratories while the authors continued to consider their cultures H17. We showed how to distinguish these alternative flagellin genotypes using popular fliC primers. Attention was also paid to possible discrepancies between serological and molecular results in flagellar typing of E. coli.     

ENTOSIPHON SULCATUM (EUGLENOPHYCEAE): FLAGELLAR ROOTS OF THE BASAL BODY COMPLEX AND RESERVOIR REGION1,2

The flagellar root system of Entosiphon sulcatum (Dujardin) Stein (Euglenophyceae) is described and compared with kinetoplastid and other euglenoid systems. An asymmetric pattern of three microtubular roots, one between the two flagellar basal bodies and one on either side (here called the intermediate, dorsal, and ventral roots), is consistent within the euglenoid flagellates studied thus far. The dorsal root is associated with the basal body of the anterior flagellum (F₁) and lies on the left dorsal side of the basal body complex. Originating between the two flagellar basal bodies, and associated with the basal body of the trailing flagellum (F₂), the intermediate root is morphologically distinguished by fibrils interconnecting the individual microtubules to one another and to the overlying reservoir membrane. The intermediate root is often borne on a ridge projecting into the reservoir. The ventral root originates near the F₂ basal body and lies on the right ventral side of the cell. Fibrillar connections link the membrane of F₂ with the reservoir membrane at the reservoir-canal transition level. A large cross-banded fiber joins the two flagellar basal bodies, and a series of smaller striated fibers links the anterior accessory and flagellar basal bodies. Large nonstriated fibers extend from the basal body complex posteriorly into the cytoplasm.     

SYNUROPHYCEAE CLASSIS NOV., A NEW CLASS OF ALGAE

The silica-scaled algae (Synuraceae, Chrysophyceae sensu lato) are compared to other Chrysophyceae, Phaeophyceae and Bacillariophyceae with occasional comparisons to other chlorophyll c-containing algae, scaled protozoa and oomycete fungi. The silica-scaled algae have several unique characters which separate them from the above groups and based upon these differences a new order, Synurales ord. nov., and a new class, Synurophyceae class. nov., are described. The major distinguishing characters of the Synurophyceae class. nov. are: they have chlorophylls a and c₁ but lack chlorophyll c₂; their flagellar apparatus includes a microtubular root that loops around two parallel flagella and a flagellar root system which occurs in four absolute orientations; the photoreceptor consists of paired flagellar swellings which are not associated with the cell membrane and chloroplast; no eyespot is present; the nuclear envelope is not or is only weakly associated with the chloroplast endoplasmic reticulum. The Synurophyceae class. nov. are about equally distinct from the Chrysophyceae sensu stricto, Phaeophyceae and Bacillariophyceae when the class level characters are compared. Although the Phaeophyceae have a long history of being placed by themselves in the division Phaeophyta, and the Bacillariophyceae and Chrysophyceae have recently been placed alone in the Bacillariophyta and Chrysophyta, respectively, the similarities found among these classes suggest these algae are not so distinct that they require separate divisions. Tentatively, therefore, the Synurophyceae are placed in the division Phaeophyta along with the Bacillariophyceae and Chrysophyceae sensu stricto.     

Absolute orientation of the flagellar apparatus of Hibberdia magna comb. nov. (Chrysophyceae)

The first flagellum of Hibberdia magna comb. nov. bears mastigonemes that have both short and long lateral filaments attached to the tubular shaft. The second flagellum is very short (ca. 850 nm) and is directed posteriorly approximately 160° from the first flagellum. Three microtubular flagellar roots (R₁, R₂ and R₄) and a rhizoplast (= striated root) are present. The R₁ root consists of four microtubules that arise near the right surface of the first flagellum basal body; the R₁ root extends to the dorsal side of the cell and then curves back along the left side of the cell. Cytoskeletal microtubules are nucleated from the R₁ root including one loose cluster of cytoskeletal microtubules that extends down the left side of the cell adjacent to the contractile vacuole. The R₂ root is a single microtubule that arises along the left surface of the first flagellum basal body and extends to the left side of the cell. The R₄ root consists of three microtubules that arise along the left side of the second flagellum basal body. A helical band wraps around two microtubules at the proximal end of the R₄ root. Two of the three R₄ root microtubules extend along the left side of the second flagellum, curve around to the right side of that flagellum and terminate. No R₃ root was found. The orientation of the basal bodies of Hibberdia gen. nov. is similar to that of the Xanthophyceae and Oomycetes. There are apparent homologies in the R₁, R₂ and R₄ roots of Hibberdia and these and other protists, but only Hibberdia lacks a R₃ root. Three long flagella are present in preprophase but later one is endocytosized and the axoneme extends to the posterior of the cell. During metaphase the nuclear envelope is more or less intact except at the poles; the flagellar apparatuses are at the poles and the spindle microtubules originate near the basal bodies. Two stages are known in the life history: 1) a capsoidlike state with non-swimming flagellate cells inside a colonial gel, and 2) a free-swimming single-celled monad state. Vegetative cell division occurs in both stages. The flagellar apparatus, the cell division process and the life history combined with the previously described unique light-harvesting antheraxanthin make H. magna distinct from other algae. A new genus, Hibberdia gen. nov., a new family, Hibberdiaceae fam. nov. and a new order, Hibberdiales, ord. nov. are described.     

How is the flagellar length of mature sperm determined? : II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.