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PubMed1999

Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1.

The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or 'blob'. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.

Animals↗

PubMed2000

Spirochete periplasmic flagella and motility.

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.

Borrelia burgdorferi Group↗

PubMed1996

Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium.

The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG C) for lipoylation and signal peptide cleavage. We have confirmed the previous finding (M. Homma, K. Ohnishi, T. Iino, and R. M. Macnab, J. Bacteriol. 169:3617-3624, 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca. 25 kDa. flgH alleles with an in-frame deletion or a 3' truncation still permitted processing. The deletion permitted partial restoration of motility in complementation tests, whereas the truncation did not. Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH. When cells transformed with a plasmid containing the flgH gene were grown in the presence of [3H]palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used. Hook-basal body complexes from cells grown in the presence of [3H]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PeIB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form. With a low-copy-number plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type. Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated [3H]palmitate at low levels, perhaps through acylation of the N-terminal alpha-amino group. We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body. We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.

Alleles↗

PubMed1988

Effects of mot gene expression on the structure of the flagellar motor.

Direct freezing procedures have enabled us to visualize distinctive intramembrane particle ring structures in the cytoplasmic membranes of peritrichously flagellated bacteria by means of freeze-fracture electron microscopy. These structures were identified as flagellar motor components because their distribution matched that of flagella, and because they were absent in non-flagellated mutants of Escherichia coli. Particle rings were present in both the Gram-positive Streptococcus and the Gram-negative E. coli. In E. coli, a non-functional mocha operon produced flagellated but immotile cells lacking the particle rings. Simultaneous introduction of the motA and motB genes, led to recovery of both motility and the ring structures but neither gene alone was sufficient. The concomitant loss of the rings and motility is consistent with the ring particles having a central role in the flagellar motor.

Escherichia coli↗

PubMed1996

Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum.

A locus encoding polypeptides associated with flagellar structure and function was identified, sequenced, and characterized in Treponema phagedenis and Treponema pallidum. This locus includes homologs of the FlgD, FlgE, MotA, MOB, FliL, and FliM polypeptides found in Salmonella typhimurium and Bacillus subtilis. These polypeptides are extensively conserved between the two treponemes. Several additional polypeptides or unknown function, including Tapl, located upstream of FlgD, and ORF4, located between FlgE and MotA, were also identified. Transcription analysis using RNA PCR indicated that these genes are likely transcribed as part of a single operon and comprise the 5' region of the treponemal fla operon. Primer extension analysis identified a putative promoter, preceding T. phagedenis tap1 in a region of divergent transcription. Pfla resembles the class II or class III motility-related promoters of S. typhimurium. FlgE and Tap1 were further characterized. Western blotting (immunoblotting) indicated that T. pallidum FlgE exhibited an unusual polypeptide ladder that was similar but not identical to that of T. phagedenis. Triton X-114 phase partitioning of T. phagedenis cells coupled with Western blotting revealed that Tap1 was located in the aqueous phase. Computer analysis indicated that Tap1 had no significant membrane spanning regions, suggesting that it resides primarily in the cytoplasm. The organization and expression of this operon are similar in both treponemes but different from those of previously described motility-related operons. These results indicate that despite extensive amino acid sequence conservation, the expression of spirochete flagellar polypeptides is different from that in other bacteria.

Amino Acid Sequence↗

PubMed2004

Direct measurement of the passive stiffness of rat sperm and implications to the mechanism of the calcium response.

Glass microprobes were used to measure the stiffness of the flagella of Triton X-100-extracted rat sperm models. The sperm models were treated with 50 microM sodium vanadate and 0.1 mM Mg-ATP to evaluate the stiffness of the passive flagellar structure without the influence of the dynein motor proteins. The passive stiffness was determined to be 4.6 (+/- 1.1) x 10(-19) N x m(2). Rat sperm models exposed to greater than 10(-5) M calcium ions exhibit a strong bend in the basal 40 microm of the flagellum, resulting in a fishhook-like appearance. The torque required to bend a passive rat sperm flagellum into the fishhook-like configuration was determined. The result was compared to the previously published measurement of the torque required to straighten the flagella of rat sperm in the Ca(2+)-induced fishhook configuration [Moritz et al., 2001: Cell Motil. Cytoskeleton 49:33-40]. The torque required to induce a fishhook in a passive flagellum was 2.7 (+/- 0.7) x 10(-14) N x m and the torque to straighten an active Ca(2+)-induced fishhook was 2.6 (+/- 1.4) x 10(-14) N x m. These values are identical within the limit of error of the measurement technique. This finding suggests that the fishhook configuration observed in the Ca(2+) response of rat sperm is the result of a Newtonian equilibrium, where active torque produced by dynein is counterbalanced by an equal and opposite passive torque that results from bending the flagellum. Consistent with this mechanism, the Ca(2+)-induced fishhook configuration is progressively relaxed by incremental increases in sodium vanadate concentration. This supports an active role of the dynein motors in producing the torque for the response. When rat sperm respond to Ca(2+), the bend in the flagellum always forms in the direction opposite the curvature of the asymmetric sperm head. Based on this polarity, the bending torque for the Ca(2+) response must result from the action of the dyneins on outer doublets 1 through 4.

Animals↗

PubMed1989

External mechanical control of the timing of bend initiation in sea urchin sperm flagella.

The movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351-352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth-like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 microns of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid- and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.

Animals↗

PubMed2003

Spermatid nuclear and sperm periaxonemal anomalies in the mouse Ube2b null mutant.

Ube2b (yeast Ubc2b/Rad6 homolog) null mice were described previously. Ube2b encodes the murine ubiquitin conjugating enzyme mHR6B. Ube2b(-/-) mice were shown to present male infertility and their sperm head shape anomalies suggested that Ube2b may be involved in the replacement of nuclear proteins during spermatid chromatin condensation. Apoptosis of spermatocytes suggested additional targets of Ube2b during spermatogenesis. Consistently, we found Ube2b transcription in both meiotic and postmeiotic stages by in situ hybridization. Immuno-electron microscopy revealed that transition proteins 1 and 2, protamines 1 and 2, and actin appear normally distributed during morphogenesis of Ube2b(-/-) spermatid heads. Surprisingly, electron microscopy revealed a particular sperm flagellum phenotype characterized by an abnormal distribution of periaxonemal structures. Flagellar anomalies of Ube2b null mice were previously described in infertile men indicating a possible genetic pathway for flagellar periaxonemal assembly in human.

Animals↗

PubMed2004

Tektin3 encodes an evolutionarily conserved putative testicular microtubules-related protein expressed preferentially in male germ cells.

Tektins are microtubule-associated cytoskeletal proteins that are expressed primarily in the male germ cell-lineage in centrioles and basal bodies and within ciliary and flagellar doublet microtubules. They are proposed to be important for axonemal architecture and microtubule stability in the sperm tail and in other ciliated and flagellar structures. Using an in silico (electronic database) subtractive approach to identify germ cell-specific genes in vertebrates, we isolated a new member of the Tektin gene family from mice, Tektin3. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses confirm that Tektin3 encodes a 1.7 kb transcript detectable preferentially in the testes of adult mice. In situ hybridization analysis in the testes revealed Tektin3 mRNA expression exclusively in late pachytene spermatocytes and early round spermatids. The putative TEKTIN3 protein shares 83.5% overall sequence identity with the human ortholog and includes a fully conserved carboxy terminal nonapeptide signature sequence present in all TEKTIN family members. In addition, using database mining, highly conserved TEKTIN3 orthologs were identified in puffer fish (Fugu rubripes) and rats (Rattus norvegicus) which shared 60.9 and 91.4% identity, respectively, with mouse TEKTIN3. The Tekt3 gene maps to murine chromosome 11 in a region that is syntenic to the human 17p12 chromosomal region containing the human TEKTIN3 gene. Our studies demonstrate that TEKTIN3 is a novel evolutionarily conserved male germ cell-enriched protein and suggest that it might perform important roles in male reproductive development and physiology.

Amino Acid Sequence↗

PubMed1987

Purification, reconstitution and polymorphic transition of halobacterial flagella.

Flagellar filaments of Halobacterium halobium have been purified by dissociation and reconstitution. Three different protein bands (23,500, 26,500 and 31,500 apparent molecular weight) are seen on sodium dodecyl sulphate/polyacrylamide gels, thus confirming that all three proteins are intrinsic to the flagellar structure. We designate them as flagellin Fla I (23,500), Fla II (26,500) and Fla III (31,500). Polymorphic transitions from normal to a curly, a ring and a straight form are induced by different pH values and heat treatments.

Flagella↗

PubMed1994

Sequence analysis of the flgA gene and its adjacent region in Salmonella typhimurium, and identification of another flagellar gene, flgN.

The flagellar genes flgA and flgM are located at the terminus of the region-I flagellar gene cluster on the chromosome of Salmonella typhimurium. The flgA gene is involved in P-ring formation of the flagellar basal body, whereas flgM encodes the anti-sigma factor which acts as a negative regulator of the flagellar regulon. The nucleotide sequence of the DNA fragment containing these flagellar genes and the adjacent region was determined. The flgA gene was found to encode a 219-amino-acid (aa) protein of 23,556 Da. The N-terminal region of FlgA has the characteristics of a typical signal sequence, suggesting that FlgA may function in the periplasmic space where P-ring assembly takes place. The flgM gene was found to constitute an operon together with an ORF which encodes a 140-aa protein of 15,899 Da. A gene disruption mutant was constructed by inserting a cat gene cartridge into the ORF on the chromosome. This mutant showed only weak motility, indicating that the product of the ORF is involved in flagellar formation. Therefore, this ORF was designated as flgN. Electron microscopic observation revealed that most of the flagellar structures produced by the flgN mutant are hook-basal body complexes lacking the filament portions. Based on these results, we concluded that the flgN product is required for the efficient initiation of filament assembly.

Amino Acid Sequence↗

PubMedDate not supplied

Electron microscopy of frozen hydrated eukaryotic flagella.

Resting and active sea urchin sperm flagella have been examined by low-dose electron microscopy of frozen hydrated specimens. The flagella are unfixed, unstained, completely intact, and able to swim vigorously after going through the entire preparative procedure. The most prominent features of the image arise from the edges of the axonemal doublets and central-pair microtubules seen in projection. By comparison with these longitudinal markings, transverse features are less easy to discern, being camouflaged by superposition. However, Fourier transforms of digitized micrographs reveal a remarkable degree of crystalline order in quiescent flagella. Filtered images derived from these Fourier transforms show clearly features arising from the central-pair complex and radial spokes that were obscured in the original data. Potentially complicating effects of specimen thickness are shown to be quantitatively insignificant in the formation of images of unstained frozen hydrated flagella. Determination of native flagellar structure by 3-D reconstruction from multiple-tilted views appears to be feasible.

Animals↗

PubMed2003

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process.

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.

Animals↗

PubMed1999

Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis.

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.

Alleles↗

PubMed2000

Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions.

We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild-type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl-accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co-localization of CheY and CheZ with MCPs was CheA dependent, and co-localization of CheA with MCPs was CheW dependent, as expected. Co-localization with MCPs was confirmed by immunofluorescence using an anti-MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild-type cells and in a fliN mutant, but not in flhC or fliG mutants. Co-localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co-localization of CheY with motors, even under conditions in which cells tumbled.

Bacterial Proteins↗

PubMed2000

FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase activity.

Both FliH and the ATPase FliI are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N-terminus of FliI interacts with FliH and that this interaction is important for the ATPase function of the C-terminal domain of FliI. N-terminally histidine-tagged, wild-type FliI retarded untagged FliH in a Ni-NTA affinity chromatography assay, as did N-His-tagged versions of FliI carrying catalytic mutations. In contrast, N-His-tagged FliI carrying the double mutation R7C/L12P did not, further indicating that the N-terminus of FliI is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and FliI form a complex. Analytical gel filtration with in-line multiangle light scattering indicated that FliH alone forms a dimer, FliI alone remains as a monomer, and FliH and FliI together form a (FliH)2FliI complex. Ni-NTA affinity chromatography using N-His-tagged FliH and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH-FliI complex was about 10-fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.

Adenosine Triphosphate↗

PubMed2003

Regulatory and functional co-operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease.

Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.

Bacterial Adhesion↗

PubMed1993

Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5-trisphosphate and requires Ca2+ entry.

C. reinhardtii sheds its flagella in response to acidification. Previously, we showed correlations between pH shock, deflagellation, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, but 100% of cells deflagellated by 5 s, which was the earliest that Ins(1,4,5)P3 accumulation could be accurately measured by techniques available to us at that time (Quarmby, L. M., Y. G. Yueh, J. L. Cheshire, L. R. Keller, W. J. Snell, and R. C. Crain. J. Cell Biol. 1992. 116:737-744). To learn about the causal relationship between Ins(1,4,5)P3 accumulation and deflagellation, we extended these studies to early times using a continuous-flow rapid-quench device. Within 1 s of acidification to pH 4.3-4.5, 100% of cells deflagellated. A transient peak of Ins(1,4,5)P3 was observed 250-350 ms after pH shock, preceding deflagellation. Preincubation with 10 microM neomycin, which prevents hydrolysis of phosphatidylinositol 4,5-bisphosphate, inhibited both the transient production of Ins(1,4,5)P3 and the subsequent deflagellation. The nonspecific Ca2+ channel blockers La3+ and Cd2+ prevented flagellar excision induced by mastoparan without inhibiting rapid Ins(1,4,5)P3 production. Likewise, the Ins(1,4,5)P3-gated channel inhibitors ruthenium red and heparin blocked deflagellation in response to mastoparan. These studies were extended to mutants defective in flagellar excision. Fa-1, a mutant defective in flagellar structure, produced Ins(1,4,5)P3 but failed to deflagellate. These results support a model in which acid pH activates a putative cellular receptor leading to G-protein dependent activation of phospholipase C and accumulation of Ins(1,4,5)P3. These events are upstream of Ins(1,4,5)P3-dependent Ca2+ entry from the medium, and of deflagellation.

Animals↗