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PubMed1987

DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor.

Biosynthesis of bacterial flagella involves the coordinated expression of 30 or more genes in several separate operons. We have recently shown that in Bacillus subtilis, the sigma 28 factor is essential for flagellar synthesis, suggesting that transcription of these genes is directly under the control of this alternative sigma factor. In enteric bacteria structural genes for flagellar, chemotaxis, and motility operons appear to be under coordinate control, however, the nature of the regulatory factors has not been determined. Sequence analysis of many such genes has failed to reveal plausible promoter sequences for the predominant bacterial RNA polymerase, and several such genes are not transcribed effectively in vitro by the Escherichia coli sigma 70 RNA polymerase. However, all of the sequenced flagellar, chemotaxis, and motility operons from the enteric bacteria are preceded by DNA sequences highly homologous to B. subtilis sigma 28 promoters. We propose that an alternative sigma factor controls expression of the flagellar regulon in both B. subtilis and in the enteric bacteria.

Bacterial Proteins↗

PubMed1999

Assessment of inner dynein arm structure and possible function in ciliary and flagellar axonemes.

The construction and assessment of a three-dimensional computer-generated model of inner dynein arms on a 96-nm repeat unit of an axonemal doublet is described. The model is based on published electron micrographs of axonemes from Tetrahymena cilia and eel sperm, which were prepared using several different techniques: negative stain, freeze etch, and thin section. The inner arm structure is represented as three inner dynein arm complexes containing four inner dynein arms (IDAs), three dyads, and one single-headed arm, each capable of bridging the interdoublet gap. The IDA structures in the model have been correlated with the domains containing dynein heavy-chain isoforms mapped by several authors using genetic analyses of Chlamydomonas mutants. The model is consistent with micrographic evidence from axonemes of cilia and flagella from other organisms that led previously to conflicting structural interpretations. In this reconciling interpretation, the different alignments of the IDAs relative to the corresponding outer dynein arms observed in micrographs of differently prepared samples, result from the IDAs being arrested at different stages of their cycles of activity in each preparation. By interpolating between these positions of arrest, cycles of activity are proposed for each of the IDAs during which the arms attach to the neighbouring doublet microtubule and drive it tipwards.

Animals↗

PubMed2005

Centrin scaffold in Chlamydomonas reinhardtii revealed by immunoelectron microscopy.

In the flagellate green alga Chlamydomonas reinhardtii the Ca(2+)-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.

Animals↗

PubMed2005

Building the atomic model for the bacterial flagellar filament by electron cryomicroscopy and image analysis.

The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a well-ordered helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments, each in either of the two distinct conformations, L- and R-type, for supercoiling. We have been studying the three-dimensional structures of the flagellar filaments by electron cryomicroscopy and recently obtained a density map of the R-type filament up to 4 angstroms resolution from an image data set containing only about 41,000 molecular images. The density map showed the features of the alpha-helical backbone and some large side chains, which allowed us to build the complete atomic model as one of the first atomic models of macromolecules obtained solely by electron microscopy image analysis (Yonekura et al., 2003a). We briefly review the structure and the structure analysis, and point out essential techniques that have made this analysis possible.

Cryoelectron Microscopy↗

PubMed1980

Structures linking the tips of ciliary and flagellar microtubules to the membrane.

The distal tips of both central pair and outer doublet microtubules are linked to flagellar membranes of Chlamydomonas and to ciliary membranes of both Tetrahymena and Aequipecten by the central microtubule cap and by distal filaments, respectively. The distal filaments are linked to ciliary membranes at their distal ends and, at their proximal ends, form a carrot-shaped plug which inserts into the lumen of the A-tubule of each outer doublet. Both types of tip structures are present in regenerating cilia and flagella. At least 2 different types of distal filaments are present in Tetrahymena. Certain cilia in Tetrahymena oral apparatuses and in certain molluscan gills contain distal filaments which join with the central microtubule cap and with the ciliary membrane to form a cap-filament-membrane complex.

Animals↗

PubMed2004

Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats.

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.

Animals↗

PubMed1992

The cytoplasmic component of the bacterial flagellar motor.

We have used electron microscopy to examine freshly isolated Salmonella typhimurium and Escherichia coli basal flagellar fragments, purified without resort to extremes of pH or ionic strength. Such fragments contain the large bell-like basal structures visualized recently in freeze-substituted or fixed preparations. We have found mot (non-motile) mutants produced by lesions in fli genes (G, M, N) in which the bell structures do not coisolate with the flagellar basal body. The coisolation of the bell with the flagellar basal body was unaffected in strains lacking the genes for the motility-associated Mot proteins or for the Che family of proteins, which are necessary for chemotaxis. Proper assembly and interaction of the cytoplasmically located bell with the membrane-associated flagellar basal structures appears to be necessary for motor function. The FliG, FliM, and FliN proteins are thought to form a structural complex responsible for energization and switching of the flagellar motor. Our findings are consistent with the existence of such a complex and imply that it forms part of the flagellar bell.

Cell Fractionation↗

PubMed2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus.

The transcription factor FlbD regulates the temporal and spatial transcription of flagellar genes in the bacterium Caulobacter crescentus. Activation of FlbD requires cell cycle progression and the assembly of an early (class II) flagellum structure. In this report, we identify 20 independent gain-of-function mutations in flbD that relieve regulation by flagellar assembly. One of these, flbD-1204, contained a mutation in the receiver domain (V17M) and another, flbD-1231, in the DNA binding domain (V451G). Both of these mutations resulted in an aberrant pattern of cell cycle transcription. The presence of the FlbD-1204 allele also resulted in a loss of swarmer-pole-specific transcription. These results indicate that temporal and spatial transcription is influenced by the assembly of the nascent flagellar structure. The trans-acting positive and negative regulatory factor, FliX, couples flagellar assembly to the activation of FlbD and, as we show here, also influences temporal transcription. Furthermore, we show that FliX can suppress the activity of FlbD mutants that cannot be phosphorylated, and that FliX is required for FlbD stability, and vice versa. These results indicate that FliX may interact directly with FlbD to regulate its activity.

Amino Acid Sequence↗

PubMed1995

Conserved machinery of the bacterial flagellar motor.

Novel periplasmic and cytoplasmic structural modules of the bases of bacterial flagella have been observed in situ and isolated using new biochemical protocols. Flagellar rotation may depend upon interactions of these modules with the intramembrane particle rings, a ubiquitous feature of flagellar bases necessary for torque generation. The outer membrane-associated basal disk of the Wolinella succinogenes polar flagellum has architecture well suited for interaction with the ring particles. However, antibody against the main W. succinogenes basal disk protein did not cross-react with flagella-enriched fractions from Salmonella typhimurium and Bacillus firmus; nor have such structures been observed in these species thus far. Antibodies against two S. typhimurium proteins, FliG and FliM, known to be involved in motor function and part of the cytoplasmic module in this species cross-reacted with flagella-enriched fractions from both W. succinogenes and B. firmus. In addition, flagellar cytoplasmic structure could be isolated from B. firmus. The basal disk may anchor the flagellar motor to the cell wall in some polar bacteria, but this does not seem to be a unique strategy. In contrast, the data indicate that the cytoplasmic module is conserved.

Antigens, Bacterial↗

PubMed2002

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.

Animals↗

PubMed1995

Torque generation by the flagellar rotary motor.

A review is given of the structure and dynamics of the flagellar rotary motor. Force-generating elements in a motor driving a tethered bacterium (a cell fixed to the substratum by a single flagellum) exert forces of order 20 pN while moving at speeds of order 1 micron/s. Force-generating elements in a motor driving a flagellar filament in a bundle exert forces some 10-fold lower but move at speeds more than 10-fold higher. The motor torque-speed relationship has been measured over a wide dynamic range. Motors strongly resist being driven backwards and are easily broken.

Bacterial Proteins↗

PubMed1986

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures.

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.

Base Sequence↗

PubMed1998

Structure and expression of the FlaA periplasmic flagellar protein of Borrelia burgdorferi.

The spirochete which causes Lyme disease, Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of the B. burgdorferi flagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that a flaA homolog was expressed in B. burgdorferi and that it mapped in a fla/che operon. These results led us to analyze the PFs and FlaA of B. burgdorferi in detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purify B. burgdorferi PFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes, B. burgdorferi FlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart from Serpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant of B. burgdorferi (HB19Fla-). Although this mutant still synthesized flaA message in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis using Treponema pallidum anti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs of B. burgdorferi and that they are regulated differently from flagellin proteins of other bacteria.

Borrelia↗

PubMed1975

Observations on the ultrastructure of the choanoflagellate Codosiga botrytis (Ehr.) Saville-Kent with special reference to the flagellar apparatus.

The ultrastructure of the choanoflagellate Codosiga botrytis is described with particular reference to the flagellar appendages, the flagellar rootlet system, the transition zone, the basal body and accessory centrioles, and the stalk. The controversial early reports of flagellar appendages in this species have been confirmed and they have been detected in 2 further species, Salpingoeca frequentissima and Monosiga sp. The appendages consist of a delicate bilateral vane 2 mum wide on either side of the axis, composed of extremely fine overlapping or interwoven fibrils. The flagellar root system consists of a large number of radiating microtubules associated with bands of electron-dense material near the basal body; striated roots are absent. The microtubules extend from several separate foci, those in any one group originating near a composite electron-dense band, and for a distance of 300 nm from the basal body they are separated by blocks of interstitial material. The flagellar basal body forms one of a diplosome pair of centrioles. The triplet microtubules of the accessory centriole are embedded in amorphous electron-dense material and the whole is enveloped in a sheath of similar appearance. The existence of a third centriole close to the diplosome pair is also reported. The relatively complex structure of the flagellar transitional zone is described. The stalk is composed of a core of circular lacunae, which may or may not contain finger-like protoplasmic extensions of the posterior end of the cell, surrounded by a continuation of the sheath material which encloses the remainder of the protoplast. In the stalk only there is a further closely sheathing layer about 15 nm thick which is regularly striated, the spacing of the striations in shadowcast material and sections being about 3 times that measured by negative staining. The structure of choanoflagellates differs widely from that of the algal class Chrysophyceae, the group in which they are included in some classifications, and from the remainder of the algae; they do not appear to have a place in either the algae or the plant kingdom. The structure of Codosiga botrytis is briefly compared with that of sponge choanocytes and collared cells in the Metazoa and some of the possible phylogenetic implications of this are indicated.

Animals↗

PubMed1999

FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly.

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.

Bacterial Proteins↗

PubMed1975

Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei.

The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50 percent of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the acitvity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.

Acid Phosphatase↗

PubMed2005

FlhB regulates ordered export of flagellar components via autocleavage mechanism.

The bacterial flagellum is a predominantly cell-external super-macromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB(C)), which is consistently and specifically cleaved into two subdomains (FlhB(CN) and FlhB(CC)) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB(C)(P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB(C) and FlhB(C)(P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.

Bacterial Proteins↗

PubMed1988

The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution.

Chondrichthyes possess an evolved type of spermatozoa. Their flagellar apparatus is characterized by the presence of flagellar roots which form the axis of the midpiece, and the existence of one or two lateral elements associated with the axoneme. Osteichthyes, mainly teleosteans, show a great diversity of spermatic forms. The primitive spermatozoon with a 9 + 2 pattern flagellum is common. The primitive spermatozoon has evolved along different lines. The spermatic diversity which results from this is mainly evident in the structure of the flagellar apparatus. In the animal kingdom the primitive spermatozoon with a 9 + 2 pattern flagellum, present in primitive metazoa, is retained in phyla where external fertilization is maintained. The main evolutionary tendencies--elongation, aflagellarity or biflagellarity--are generally connected with the acquisition of internal fertilization. These evolutionary tendencies are found in teleosteans. It is not possible to link aflagellarity or biflagellarity of the gamete in certain fishes to this method of fertilization. Only the elongation of the spermatozoon is connected, in certain cases, with internal fertilization, but this cannot be taken as general.

Animals↗