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PubMed1984

Isolation of the flagellum and characterization of the paraxial structure of Herpetomonas megaseliae.

It was observed in thin sections that the flagellum from Herpetomonas megaseliae, a protozoan of the Trypanosomatidae family, has, besides the axoneme, a filamentous lattice-like structure, the paraxial rod. Crithidia deanei, a protozoan of the same family, does not possess a paraxial rod in its flagellum. The flagella of these two trypanosomatids were isolated. Cultures were sonified and fractionated thus resulting the following fractions: total homogenates, deflagellated cell bodies, crude flagellar fraction and purified flagellar fraction from both H. megaseliae and C. deanei. The flagellar fractions are very pure and the flagella have their structures well preserved: axonemes and paraxial structures, where they exist, are intact, associated with one another and surrounded by the flagellar membrane. A comparative analysis of the purified flagellar fractions from both trypanosomatids was made using polyacrylamide gel electrophoresis. Two protein bands, with molecular weights of 78,000 and 73,000 daltons, which were enriched along the process of purification of the flagella from H. megaseliae, were found to correspond to the main components of the paraxial structure. These two bands were not observed in the purified flagellar fraction from C. deanei.

Animals↗

PubMed2006

Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori.

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

Amino Acid Sequence↗

PubMed1985

"Cap" on the tip of Salmonella flagella.

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.

Flagella↗

PubMed2000

FlbT, the post-transcriptional regulator of flagellin synthesis in Caulobacter crescentus, interacts with the 5' untranslated region of flagellin mRNA.

Flagellar gene expression' in Caulobacter crescentus is regulated by a complex trans-acting hierarchy, in which the assembly of early structural proteins is required for the expression of later structural proteins. The flagellins that comprise the filament are regulated at both the transcriptional and the post-transcriptional levels. Post-transcriptional regulation is sensitive to the assembly of the flagellar basal body and hook structures. In mutant strains lacking these structures, flagellin genes are transcribed, but not translated. Mutations in the flagellar regulatory gene, flbT, restore flagellin translation in the absence of flagellar assembly. In this report, we investigate the mechanism of FlbT-mediated post-transcriptional regulation. We show that FlbT is associated with the 5' untranslated region (UTR) of fljK (25 kDa flagellin) mRNA and that this association requires a predicted loop structure in the transcript. Mutations within this loop abolished FlbT association and resulted in increased mRNA stability, indicating that FlbT promotes the degradation of flagellin mRNA by associating with the 5' UTR. We also assayed the effects on gene expression using mutant transcripts fused to lacZ. Interestingly, the mutant transcript that failed to associate with FlbT in vitro was still repressed in mutants defective in flagellum assembly, suggesting that other factors in addition to FlbT couple assembly to translation.

5' Untranslated Regions↗

PubMed2006

The flagellar cytoskeleton of the spirochetes.

The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells.

Borrelia burgdorferi↗

PubMed1983

Mutational disruption of the 9 + 2 structure of the axoneme of Chlamydomonas flagella.

A mutant of Chlamydomonas, gam-5, which has short but variable-length, paralysed flagella has been characterized using electron microscopy and genetics. The flagellar axoneme shows varying degrees of disorganization of its 9 + 2 structure, and the structures that attach to the microtubule pairs may be wholly or partly missing or, if present, abnormal. Flagellar length is inversely related to temperature, and gametes of this mutant are generally incapable of mating at higher temperatures. This pleiotropic mutation shows Mendelian (2:2) segregation and is unlinked to the mating-type locus. It is suggested that gam-5 leads to the production of a thermal-sensitive flagellar component, which results in increased assembly and/or maintenance difficulties as temperature is increased.

Chlamydomonas↗

PubMed1994

Structural and antigenic characteristics of Campylobacter coli FlaA flagellin.

The polar flagellar filament of Campylobacter coli VC167 is composed of two highly related (98%) flagellin subunit proteins, FlaA and FlaB, whose antigenic specificities result from posttranslational modification. FlaA is the predominant flagellin species, and mutants expressing only FlaA form a full-length flagellar filament. Although the deduced M(r) of type 2 (T2) FlaA is 58,884 and the apparent M(r) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 59,500, the solution weight-average M(r) by sedimentation analysis was 63,000. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate or 50% trifluorethanol showed that the secondary structure of T2 FlaA flagellin was altered, with alpha-helix structure being increased to 25% in the nonpolar environment. The molecule also contained 35 to 48% beta-sheet and 11 to 29% beta-turn structure. Mimeotope analysis of octapeptides representing the sequence of FlaA together with immunoelectron microscopy and enzyme-linked immunosorbent assay with a panel of antisera indicated that many residues in presumed linear epitopes were inaccessible or nonepitopic in the assembled filament, with the majority being in the N-terminal 337 residues of the 572-residue flagellin. Residues at the carboxy-terminal end of the T2 FlaA subunit also become inaccessible upon assembly. Digestion with trypsin, chymotrypsin, and endoproteinase Glu-C revealed a protease-resistant domain with an approximate M(r) of 18,700 between residues 193 and 375. Digestion with endoproteinase Arg-C and endoproteinase Lys-C allowed the mapping of a segment of surface-exposed FlaA sequence which contributes serospecificity to the VC167 T2 flagellar filament at residues between 421 and 480.

Amino Acid Sequence↗

PubMed1979

Rhizoplast and rootlet system of the flagellar apparatus of Chlamydomonas moewusii.

The ultrastructure of the flagellar apparatus of Chlamydomonas moewusii was examined in detail. Two rhizoplasts were found associated with the basal bodies of this biflagellate and were observed to extend to the central area of the cell. A segment of smooth endoplasmic reticulum ran parallel to each rhizoplast. These 2 segments anastomose beneath the basal bodies and the tubule proceeds over the top of the distal connecting fibre. A functional role for the smooth endoplasmic reticulum in these locations is discussed. Four sets of rootlet microtubules emanate from a region between the 2 basal bodies and the distal connecting fibre. Two sets have a three-over-one arrangement and the other 2 sets are doublets. Cytoplasmic microtubules were seen associated with possible nucleating sites on the rootlet microtubules. The association of the observed structures are discussed and compared to the flagellar apparatus of C. reinhardtii.

Chlamydomonas↗

PubMed2005

Hypothesis: chemotaxis in Escherichia coli results from hyper-structure dynamics.

Large assemblies of different molecules and macromolecules,termed hyper structures, have been proposed to be the units of an intermediate level of organisation in bacteria. Here we propose a model for chemotaxis in Escherichia coli in which (1) the size and functioning of the chemo-signalling hyper-structure depends not only on the protein constituents but also on cardiolipin and calcium,(2) the coupled transcription, translation and insertion of nascent proteins (transertion) is a potentially powerful influence in determining the size of the chemo-signalling hyper-structure and therefore in affecting its function, and (3) a single transertional hyper-structure is jointly responsible for the synthesis of chemo-signalling and flagellar proteins so as to divorce the size of the chemo-signalling hyper-structure from the transertion of its constituents.

Cardiolipins↗

PubMed2001

Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus.

The MS ring of the flagellar basal body of Salmonella is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliF mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in flhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the fliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.

Amino Acid Substitution↗

PubMed2001

Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex.

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

Bacterial Proteins↗

PubMed1978

Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.

The temperature-sensitive gametogenesis-defective mutant, gam-1 is sex-limited, expressed only in mating type minus (mt-), and can sexually agglutinate but not fuse at the restrictive temperature (35 degrees C) with gametes of wild type (wt) mt+. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gam-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35 degrees C, each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gam-1 flagella. The two mutant phenotypes displayed by gam-1 have been denoted gam-1-I and gam-1-II. An agglutination reaction involving gam-1-I cells does not result in activation of the wt mt+ mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-1-II cells, but because the gam-1 mt- mating sturcture still fails to activate, cell fusion is precluded. We conclude that the gam-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.

Agglutination↗

PubMed1997

The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.

Amino Acid Sequence↗

PubMed2000

Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro.

Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.

Calcium-Binding Proteins↗

PubMed2000

Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa.

Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.

Amino Acid Sequence↗

PubMed1995

Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins.

Amino Acid Sequence↗

PubMed1989

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.

Amino Acid Sequence↗

PubMed1984

Genetic and biochemical dissection of the eucaryotic flagellum.

The axoneme is the basic functional unit of the eucaryotic flagellum. Periodic structures appended to its 9+2 microtubule core are responsible for generation of flagellar bending. An account of biochemical and genetic studies of flagellar-defective mutants of Chlamydomonas reinhardtii is presented. These studies provide insights into the complex molecular composition of the appended structures, their mode of assembly, and the way in which they interact to modulate flagellar function.

Cell Movement↗