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PubMed2006

MotD of Sinorhizobium meliloti and related alpha-proteobacteria is the flagellar-hook-length regulator and therefore reassigned as FliK.

The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.

Amino Acid Sequence↗

PubMed1997

Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis.

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.

Bacterial Proteins↗

PubMed1978

Identification of the structural gene for the hook subunit protein of Escherichia coli flagella.

Previous studies showed that the structural gene for the flagellar hook subunit protein (molecular weight 42,000) was one of a group of flagellar genes located on the Escherichia coli genome near pyrC. Several lines of evidence indicate that the flaK gene is the structural gene for the hook subunit protein. Fla+ strains that were insensitive to chi infection could be isolated as revertants of an FlaK- amber mutant strain but from no other Fla- strain. The hook subunit proteins isolated from such chi-sensitive revertants of the FlaK- strain were shown to be antigenically and electrophoretically different from the hook protein isolated from the wild-type strain. Thus, reversion of a mutation in the flaK gene resulted in alteration of the structure of the hook protein. Furthermore, in programming experiments with hybrid lambda containing flagellar genes, lambdafla with flaK genetic activity programmed the synthesis of a 42,000-molecular weight protein, whereas lambdafla without flaK genetic activity did not.

Bacterial Proteins↗

PubMed1992

Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.

Adenosine Triphosphate↗

PubMed1986

Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure.

Unfertilized sea urchin eggs contain a Mg2+-ATPase which shares physical and enzymatic characteristics with dynein, the enzyme which powers ciliary and flagellar movement. To further investigate the homology of the egg ATPase and axonemal dynein, ATP-binding subunits in preparations of each of the enzymes were identified using a photoaffinity probe of ATP, 8-azido-ATP (8-N3ATP), and three high molecular weight (HMW) polypeptide components of the two enzymes were compared by one-dimensional peptide mapping. Two heavy chains (A and B) of both the flagellar and egg ATPases bound [alpha-32P]8-N3ATP. The labeling of the HMW bands was specifically inhibited by ATP or ADP. Both the cytoplasmic ATPase and flagellar dynein utilized 8-N3ATP as a substrate indicating that the reagent binds to the active site. The two HMW ATP-binding polypeptides and one other HMW component of the egg ATPase were compared to flagellar dynein heavy chains by peptide mapping. Digestion of the egg versus flagellar HMW polypeptides with Staphylococcus V8 protease or alpha-chymotrypsin produced a highly similar group of peptides, and each pair of heavy chains was qualitatively estimated to be over 85% homologous. These data support the identification of the egg ATPase heavy chains as components of a cytoplasmic dynein and suggest that the HMW polypeptides form active enzymatic sites in flagellar and egg dynein which are substantially homologous.

Actins↗

PubMed1999

Genetic and structural analyses of cytoplasmic filaments of wild-type Treponema phagedenis and a flagellar filament-deficient mutant.

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments, cfpA, was identified in various treponemal species. Treponema phagedenis cfpA was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from T. denticola, T. vincentii, and T. pallidum subsp. pertenue. Wild-type T. phagedenis and a PFF-deficient isolate were analyzed by electron microscopy to assess the structural relationship of the cytoplasmic filaments and the PFF. The number of cytoplasmic filaments per cell of T. phagedenis (mean, 5.7) was compared with the number of PFF at each end of the cell (mean, 4.7); the results suggest that there is no direct one-to-one correlation at the cell end. Moreover, a structural link between these structures could not be demonstrated. The cytoplasmic filaments were also analyzed by electron microscopy at different stages of cell growth; this analysis revealed that they are cleaved before or during septum formation and before the nascent formation of PFF. A PFF-deficient mutant of T. phagedenis possessed cytoplasmic filaments similar to those of the wild type, suggesting that intact PFF are not required for their assembly and regulation. The extensive conservation of CfpA among pathogenic spirochetes suggests an important function, and structural analysis suggests that it is unlikely that the cytoplasmic filaments and the flagellar apparatus are physically linked.

Bacterial Proteins↗

PubMed2003

A high-order trans-membrane structural linkage is responsible for mitochondrial genome positioning and segregation by flagellar basal bodies in trypanosomes.

In trypanosomes, the large mitochondrial genome within the kinetoplast is physically connected to the flagellar basal bodies and is segregated by them during cell growth. The structural linkage enabling these phenomena is unknown. We have developed novel extraction/fixation protocols to characterize the links involved in kinetoplast-flagellum attachment and segregation. We show that three specific components comprise a structure that we have termed the tripartite attachment complex (TAC). The TAC involves a set of filaments linking the basal bodies to a zone of differentiated outer and inner mitochondrial membranes and a further set of intramitochondrial filaments linking the inner face of the differentiated membrane zone to the kinetoplast. The TAC and flagellum-kinetoplast DNA connections are sustained throughout the cell cycle and are replicated and remodeled during the periodic kinetoplast DNA S phase. This understanding of the high-order trans-membrane linkage provides an explanation for the spatial position of the trypanosome mitochondrial genome and its mechanism of segregation. Moreover, the architecture of the TAC suggests that it may also function in providing a structural and vectorial role during replication of this catenated mass of mitochondrial DNA. We suggest that this complex may represent an extreme form of a more generally occurring mitochondrion/cytoskeleton interaction.

Animals↗

PubMed2006

Gap compression/extension mechanism of bacterial flagellar hook as the molecular universal joint.

Bacterial flagellar hook acts as a molecular universal joint, transmitting torque produced by the flagellar basal body, a rotary motor, to the flagellar filament. The hook forms polymorphic supercoil structures and can be considered as an assembly of 11 circularly arranged protofilaments. We investigated the molecular mechanism of the universal joint function of the hook by a approximately two-million-atom molecular dynamics simulation. On the inner side of the supercoil, protein subunits are highly packed along the protofilament and no gaps remain for further compression, whereas subunits are slightly separated and are hydrogen bonded through one layer of water molecules on the outer side. As for the intersubunit interactions between protofilaments, subunits are packed along the 6-start helix in a left-handed supercoil whereas they are highly packed along the 5-start helix in a right-handed supercoil. We conclude that the supercoiled structures of the hook in the left- and right-handed forms make maximal use of the gaps between subunits, which we call "gap compression/extension mechanism". Mutual sliding of subunits at the subunit interface accompanying rearrangements of intersubunit hydrogen bonds is interpreted as a mechanism to allow continuous structural change of the hook during flagellar rotation at low energy cost.

Amino Acid Sequence↗

PubMed1980

Ultrastructural aspects of basal body associated fibrous structures in green algae: a critical review.

Ultrastructural aspects of fibrous structures associated with basal bodies of green algae are critically discussed. It is apparent that variation among these structures is much greater than in microtubular flagellar root systems and it is therefore suggested that fibrous structures may be more useful than microtubular roots in elucidating phylogenetic relationships within the Chlorophyceae sensu Stewart and Mattox and the Prasinophyceae sensu Christensen. Two main types of fibrous structures are distinguished: (1) Connecting fibres (these connect different basal bodies); (2) Fibrous roots (these originate at basal bodies and terminate somewhere else in the cell). Fibrous roots are of two types: (a) microtubular-root associated striated fibres (striation pattern 25-35 nm; system I-fibres); (b) striated fibres composed of a bundle of filaments (filament diameter: 5-10 nm; striation pattern greater than 80 nm; system II fibres). Numbers, disposition and substructure of connecting fibres and fibrious roots are variable in different genera of green algae. In the experimental secretion new observations on fibrous roots in the ulvalean genus Enteromorpha as well as preliminary information on fibrous structures in Carteria obtusa and Bryopsis lyngbyei are included. Functional and evolutionary aspects of fibrous structures associated with the flagellar apparatus of green algae are discussed.

Chlorophyta↗

PubMed1995

Regulated underexpression and overexpression of the FliN protein of Escherichia coli and evidence for an interaction between FliN and FliM in the flagellar motor.

The FliN protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of regulated underexpression and overexpression of FliN in a fliN null strain. Cells that lack the FliN protein do not make flagella. When FliN is underexpressed, cells produce relatively few flagella and those made are defective, rotating at subnormal, rapidly varying speeds. These results are similar to what was seen previously when the flagellar protein FliM was underexpressed and unlike what was seen when the motility proteins MotA and MotB were underexpressed. Overexpression of FliN impairs motility and flagellation, as has been reported previously for FliM, but when FliN and FliM are co-overexpressed, motility is much less impaired. This and additional evidence presented indicate that FliM and FliN are associated in the flagellar motor, in a structure distinct from the MotA/MotB torque generators. A recent study showed that FliN might be involved in the export of flagellar components during assembly (A. P. Vogler, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). We show here that approximately 50 amino acid residues from the amino terminus of FliN are dispensable for function and that the remaining, essential part of FliN has sequence similarity to a part of Spa33, a protein that functions in transmembrane export in Shigella flexneri. Thus, FliN might function primarily in flagellar export, rather than in torque generation, as has sometimes been supposed.

Amino Acid Sequence↗

PubMed1986

Pairwise perturbation of flagellin subunits. The structural basis for the differences between plain and complex bacterial flagellar filaments.

Although plain and complex bacterial flagellar filaments differ in their physical properties and helical symmetry, they both appear to derive from a common underlying structure. Analysis of electron micrographs of complex filaments of Rhizobium lupini revealed that the unit cell has twice the length of that of plain filaments, with a corresponding reduction in helical symmetry whereby the six-start helical family present in plain filaments collapses into a three-start family. Mass per unit length measurements were made by scanning transmission electron microscopy. These, together with the unit cell dimensions and the molecular weight of the flagellin monomer, enabled the number of monomers per unit cell to be estimated. Whereas plain filaments have a single monomer per unit cell, complex filaments have two. These results suggest that complex filament structure differs from plain filament structure by a pairwise perturbation, or interaction, of the flagellin monomers. The additional bonding interactions involved in the perturbation in the complex filament may make it more rigid than the plain filament, which has no such perturbation.

Bacterial Proteins↗

PubMed1996

Flagellar assembly in Caulobacter crescentus: a basal body P-ring null mutation affects stability of the L-ring protein.

The P- and L-rings are structural components of the flagellar basal body that are positioned in the periplasmic space and outer membrane, respectively. In order to explore the mechanism of P- and L-ring assembly, we examined the effect of a null mutation in the gene encoding the P-ring subunit, FlgI, on the expression, stability, and subcellular localization of the L-ring subunit, FlgH, in Caulobacter crescentus. Transcription of the L-ring gene and synthesis of the L-ring protein were both increased in the P-ring null mutant. However, steady-state L-ring protein levels were dramatically reduced compared with those of wild type. This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. The instability of the L-ring protein was apparent throughout the cell cycle of the P-ring mutant and contrasted with the fairly constant level of L-ring protein during the cell cycle of wild-type cells. Low levels of the L-ring protein were detected exclusively in the cell envelope of cells lacking the P-ring, suggesting that, in the absence of P-ring assembly, L-ring monomers are unable to form multimeric rings and are thus subject to proteolysis in the periplasm.

Bacterial Proteins↗

PubMed1996

FliN is a major structural protein of the C-ring in the Salmonella typhimurium flagellar basal body.

The Salmonella typhimurium FliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function. We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition. The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111 +/- 13). This explains why single, missense fliN, fliG or fliM mutations, found in many non-motile strains, can alter the belled morphology. Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN. The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable. These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility. (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies. (3) FliN, together with FliM, account for most of the C-ring. As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export. Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick-freeze replica electron microscopy, may gate such export. Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles. This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.

Bacterial Proteins↗

PubMed1983

flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor.

The flaAII gene of Salmonella typhimurium has also been termed motC and cheV, because defective alleles may give rise to a nonflagellate, paralyzed, or nonchemotactic phenotype. We isolated a temperature-sensitive motility mutant (MY1) and have found that the mutation occurs in the flaAII gene. In temperature-jump experiments, MY1 could be converted from highly motile to paralyzed within 0.5 s, demonstrating that flaAII is a structural gene whose product is immediately essential for motor rotation. The mutant, although chemotactic at permissive temperatures (less than 36 degrees C), had a higher clockwise rotational bias than did the wild type; it can therefore be regarded simultaneously as motC(Ts) and cheV (tumbly). The only previously reported S. typhimurium cheV mutant was smooth-swimming. A shift toward counterclockwise bias accompanied loss of rotational speed in the restrictive temperature range. This result, by analogy with known proton motive force effects on motor switching, further indicates a central role of the flaAII (motC, cheV) protein in the energy transduction and switching process. Since there is no evidence associating it with the isolable entity known as the basal body, it may reside at the cytoplasmic face of the flagellar motor.

Energy Transfer↗

PubMed1982

The functional analysis of the flagellar apparatus in green algae.

The flagellar apparatus of green algae is a complex organelle of great structural diversity. Diversity exists with respect to numbers of flagella, flagellar appendages (e.g., scales), origin of flagella in relation to cell symmetry, basal body and transitional region ultrastructure, fibrous structures interconnecting basal bodies and flagellar root systems. The flagellar apparatus performs several different functions in a single cell and also probably functions differently in different green algal genera. In addition to being an organelle which moves the cell through an aqueous environment the flagellar apparatus reacts to the mechanical, chemical, gravitational and light stimulation of the cell. It also plays an important role during sexual fusion of isogamous, anisogamous and oogamous species of green algae. Furthermore it contains several microtubule-organising centres (MTOCs) and in most flagellated green algae these MTOCs organise the whole microtubular system of the cell. In this review the different strategies which are currently employed to evaluate the functional significance of various structures of the flagellar apparatus in green algae are discussed. Emphasis is given to those structures which are not directly engaged in generating the motive force of flagella.

Cell Membrane↗

PubMed1990

Transposon-induced non-motile mutants of Vibrio cholerae.

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.

DNA Transposable Elements↗

PubMed2000

Mutational analysis of genes encoding the early flagellar components of Helicobacter pylori: evidence for transcriptional regulation of flagellin A biosynthesis.

We investigated the roles of fliF, fliS, flhB, fliQ, fliG, and fliI of Helicobacter pylori, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of flaA mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in H. pylori flagellar assembly.

Bacillus subtilis↗

PubMed1992

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.

Animals↗