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PubMed1994

Multiple structural proteins are required for both transcriptional activation and negative autoregulation of Caulobacter crescentus flagellar genes.

The periodic and sequential expression of flagellar (fla) genes in the Caulobacter crescentus cell cycle depends on their organization into levels I to IV of a regulatory hierarchy in which genes at the top of the hierarchy are expressed early in the cell cycle and are required for the later expression of genes below them. In these studies, we have examined the regulatory role of level II fliF operon, which is located near the top of the hierarchy. The last gene in the fliF operon, flbD, encodes a transcriptional factor required for activation of sigma 54-dependent promoters at levels III and IV and negative autoregulation of the level II fliF promoter. We have physically mapped the fliF operon, identified four new genes in the transcription unit, and determined that the organization of these genes is 5'-fliF-fliG-flbE-fliN-flbD-3'. Three of the genes encode homologs of the MS ring protein (FliF) and two switch proteins (FliG and FliN) of enteric bacteria, and the fourth encodes a predicted protein (FlbE) without obvious similarities to known bacterial proteins. We have introduced nonpolar mutations in each of the open reading frames and shown that all of the newly identified genes (fliF, fliG, flbE, and fliN) are required in addition to flbD for activation of the sigma 54-dependent flgK and flbG promoters at level III. In contrast, fliF, fliG, and flbE, but not fliN, are required in addition to flbD for negative autoregulation of the level II fliF promoter. The simplest interpretation of these results is that the requirements of FlbD in transcriptional activation and repression are not identical, and we speculate that FlbD function is subject to dual or overlapping controls. We also discuss the requirement of multiple structural genes for regulation of levels II and III genes and suggest that fla gene expression in C. crescentus may be coupled to two checkpoints in flagellum assembly.

Amino Acid Sequence↗

PubMed1972

Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes.

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of approximately 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively

Acrylamides↗

PubMed2004

In vitro characterization of FlgB, FlgC, FlgF, FlgG, and FliE, flagellar basal body proteins of Salmonella.

The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.

Bacterial Proteins↗

PubMed1977

[Studies on serological classification of Pseudomonas aeruginosa using the indirect fluorescent antibody technique (author's transl)].

An application mode of the indirect fluorescent antibody technique is described which allows the differentiation of soma and flagella of Ps. aeruginosa cells on the morphological and serological level simultaneously and the determination of the topic specificity of Ps. aeruginosa antisera. Antisera prepared by immunization of rabbits with formalinized semisolid agar cultures react with somatic and flagellar antigens (anti-OH-sera), while antisera made with heated cultures strain somatic antigens exclusively (anti-O-sera). Anti-OH-sera absorbed by heated cultures of the strain used for immunization lead to selective fluorescent staining of flagellar antigens (anti-H-sera). The reaction between somatic antigens and antisomatic antibodies is inhibited by coating substances which are removed by washing the cells. The masking effects of thermovariable and correlates with the viscosity of the cell suspension. The somatic antigens of washed cells are thermostabile, whereas the flagellar antigens are destroyed after heating at 60 degrees C. The strains representative of serogroups 0:1-0:12 of Habs show numerous uni- and bilateral relationships of the somatic antigens. However, group-specific reacting anti-O-sera are obtained by dilution of absorption of the overlapping antisomatic antibodies. The flagellae of the representative strains belong to two flagellae types: one type shows a complex, the other a uniform antigen structure. They do not include all flagellar antigens existing in Ps; aeruginosa;

Animals↗

PubMed1984

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.

Animals↗

PubMed1974

Plain and complex flagella of Pseudomonas rhodos: analysis of fine structure and composition.

Cells of Pseudomonas rhodos 9-6 produce two morphologically distinct flagella termed plain and complex, respectively. Fine structure analyses by electron microscopy and optical diffraction showed that plain flagellar filaments are cylinders of 13-nm diameter composed of globular subunits like normal bacterial flagella. The structure comprises nine large-scale helical rows of subunits intersecting four small-scale helices of pitch angle 25 degrees . Complex filaments have a conspicuous helical sheath, 18-nm wide, of three close-fitting helical bands, each about 4.7-nm wide, separated by axial intervals, 4.7 nm wide, running at an angle of 27 degrees . The internal core has similar but not identical substructure to plain filaments. Unlike plain flagella, the complex species is fragile and does not aggregate in bundles. Mutants bearing only one of two types of flagellum were isolated. Cells with plain flagella showed normal translational motion, and cells with complex flagella showed rapid spinning. Isolated plain flagella consist of a 37,000-dalton subunit separable into two isoproteins. Complex filaments consist of a 55,000-dalton protein; a second 43,000-dalton protein was assigned to complex flagellar hooks. The results indicate that plain and complex flagella are entirely different in structure and composition and that the complex type represents a novel flagellar species. Its possible mode of action is discussed.

Bacterial Proteins↗

PubMed1998

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells.

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF.

Animals↗

PubMed1990

Morphogenesis of the flagellum and its associated structures in Leishmania.

Electron microscopic study of the flagellar apparatus of promastigote and amastigote forms of Leishmania revealed major differences. Promastigotes were characterized by possessing a paraxial rod with a well developed lattice-like complex alongside the emerging axoneme enclosed within the flagellar sheath. The latter was anchored to the protozoan plasma membrane by focal or spot-like macular desmosomes at the emergence site. With cytodifferentiation to amastigotes there was a notable reduction in the axonemal size and its microtubular elements, absence of paraxial rod and a remarkable binding of the endocellular flagellum to its pocket through dense zonular desmosomal adhesions.

Animals↗

PubMed2003

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly.

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.

Cryoelectron Microscopy↗

PubMed1988

Flagellar systems in the euglenoid flagellates.

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.

Animals↗

PubMed2004

Kola acuminata proanthocyanidins: a class of anti-trypanosomal compounds effective against Trypanosoma brucei.

Human African trypanosomiasis is undergoing an alarming rate of recrudescence in many parts of sub-Saharan Africa. Yet, there is no successful chemotherapy for the disease due to a limited number of useful drugs, side effects and drawbacks of the existing medication, as well as the development of drug resistance by the parasite. Here we describe a new lead anti-trypanosomal compound isolated from Kola acuminata (Makasu). We purified a proanthocyanidin by chromatographic procedures and confirmed its homogeneity and structure by Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionisation Time-of-Flight mass spectrometry, respectively. In vitro, this compound potently induced growth arrest and lysis of bloodstream form trypanosomes in a dose- and time-dependent manner. In a mouse model, it exhibited a trypanostatic effect that extended the life of infected, treated animals up to 8 days post-infection against the 4 days for infected, untreated animals. The proanthocyanidin showed a low cytotoxicity against mammalian cells, whereas treated-BF showed massive enlargement of their flagellar pocket and lysosome-like structures caused by an intense formation of multivesicular bodies and vesicles within these organelles. The observed ultrastructural alterations caused rupture of plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Interestingly, the proanthocyanidin acted against BF but not procyclic form trypanosomes. This new anti-trypanosomal compound should be further studied to determine its efficacy and suitability as an anti-trypanosomal drug and may be used as a tool to define novel specific drug targets in BF trypanosomes.

Animals↗

PubMed1987

Identification of a gene cluster involved in flagellar basal body biogenesis in Caulobacter crescentus.

The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. A series of Tn5 insertion mutations in the flaD BC region were mapped. Complementation analysis of the Tn5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation.

Bacterial Proteins↗

PubMed2003

Does a structural bridge exist between the DNA and the specialized cytoplasmic organelles during the early part of their development? A mechanism for the positioning of flagella and possibly other cytoplasmic organelles.

Cell differentiation involves the development of a new cytoplasm containing a set of specialized organelles such as cilia and flagella which are placed in the cell with a predetermined orientation. Arguments are put forward to show that the orientation of the flagellar apparatus could be brought about by a macromolecular structural bridge between the nucleoid and the assembling flagellar apparatus, the orientation being determined by the spatial geometry inherent in the folding of the DNA. An analysis of differentiation in unicelled eukaryotes suggests that the same basic mechanism of a structural bridge could also apply to the orientation of their cilia and flagella and perhaps may have a more general application in the positioning of cytoplasmic organelles.

Animals↗

PubMed1971

Attachment of flagellar basal bodies to the cell envelope: specific attachment to the outer, lipopolysaccharide membrane and the cyoplasmic membrane.

A procedure is described for the purification of the Escherichia coli outer membrane (lipopolysaccharide or L membrane) with flagella still attached. The resulting lipopolysaccharide membrane was in the form of vesicles that had a trilaminar structure in thin section and contained about 55% lipopolysaccharide and 45% protein. T2 or T4 phage preadsorbed to E. coli were found attached to the purified lipopolysaccharide membrane. Flagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring). The cytoplasmic membrane in preparations from osmotically lysed E. coli spheroplasts or Bacillus subtilis protoplasts was specifically attached to flagella at the basal body ring farthest from the hook (the M ring). In the E. coli preparation, lipopolysaccharide membrane was also present and was attached to the L ring. From these data and a knowledge of the structure and dimensions of the E. coli flagellar basal body and cell envelope, a model for flagellar attachment is deduced.

Adenosine Triphosphatases↗

PubMed1982

Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function.

Reversion analysis of flagellar-motility mutants of Chlamydomonas reinhardtii yields an unusual class of intergenic suppressor mutations that restore flagellar activity to paralyzed radial-spoke or central-pair mutants without altering the structural or molecular defects associated with the original mutations. Four suppressors representing independent genetic loci were studied in detail. Two of the mutations, suppf1 and suppf2, restore flagellar motility to either radial-spoke or central-pair mutants of different genes. The mutants suppf3 and suppf 4 suppress flagellar paralysis associated only with mutants defective for the radial spokes. Analyses of the axonemal polypeptides of suppf1, suppf3 and suppf4 mutants indicate that the mutations restore flagellar activity to paralyzed radial-spoke or central-pair mutants by altering other components of the flagellar axoneme. suppf1 shows an altered electrophoretic migration for a 325,000 molecular weight polypeptide known to be a subunit of an outer-arm dynein. suppf3 and suppf4 are missing different axonemal polypeptides with molecular weights of 60,000 (in the case of suppf3), and 40,000 and 29,000 (in the case of suppf4). Genetic evidence has been obtained indicating that the polypeptides affected in suppf3 and suppf4 are components of a newly identified functional and/or structural compartment of the flagellar axoneme. The suppressor mutations described here reveal the operation of a control mechanism that inhibits the operations of flagellar movements in the presence of radial-spoke or central-pair defects. Suppressor mutations release the inhibition. The molecular defects of suppf1, suppf3 and suppf4 provide evidence that the inhibitory mechanism can be interrupted at two different levels of axonemal function.

Chlamydomonas↗

PubMed1984

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.

Animals↗

PubMedDate not supplied

Reinvestigation of spermatic flagella structure: the teleostean Cyprinodontidae.

The study of 7 genera and 15 species of teleostean fishes belonging to the family Cyprinodontidae shows a similar morphology of mobile spermatozoa and a wide diversity of structure of the spermatic flagellum. The flagellar membrane has one, two, or three lateral expansions depending on the species. Peripheral doublets of axonema show only the external arm, except for two species that completely lack them. Intratubular differentiations (ITD) are present in A or B tubules of peripheral doublets as in central tubules of some species, whereas others are totally devoid of them. The ITD can affect all doublets or preferentially doublets 1, 5, and 6. These variations may be due to neutral mutations.

Animals↗

PubMed1992

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.

Actins↗