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PubMed1976

Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

Cell Membrane↗
PubMed2006

In situ structure of the complete Treponema primitia flagellar motor.

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.

Cryoelectron Microscopy↗
PubMed1999

Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium.

The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium. Here we report the molecular structure and in vivo and in vitro expression of the flhD operon. Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site. Primer extension analysis demonstrated six possible transcription start sites for this operon. They include CRP-dependent and CRP-repressible transcription start sites. The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the sigma 70-dependent promoter. The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site. The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site. These two start sites were confirmed by in vitro transcription experiments using sigma 70-RNA polymerase with or without cAMP-CRP.

Amino Acid Sequence↗
PubMed2006

Flk prevents premature secretion of the anti-sigma factor FlgM into the periplasm.

The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l-ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-sigma28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-sigma28 factor FlgM was increased in flk mutant strains. Using FlgM-beta-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes.

Bacterial Proteins↗
PubMed1977

Micromorphology of Gram-negative hydrogen bacteria. I. Cell morphology and flagellation.

The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P.pseudoflava, P.palleronii, and P.facilis.

Alcaligenes↗
PubMed1994

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed2000

Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching.

We have investigated the properties of the cytoplasmic domain (FlhB(C)) of the 383-amino-acid Salmonella membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB(C) was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB(CN) and FlhB(CC), which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB(delta CC) (i.e., the N-terminal transmembrane domain FlhB(TM) plus FlhB(CN)) and FlhB(CC), resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB(C), existing as a FlhB(CN)-FlhB(CC) complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB(C) (mutant or wild type) or the FlhB(CC) polypeptide alone bound FlgD and FliC to about the same extent. FlhB(CN) by itself did not bind substrates appreciably. We propose that FlhB(C) has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB(CN) and FlhB(CC), is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB(C) resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.

Amino Acids↗
PubMed1999

Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium.

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.

Amino Acid Substitution↗
PubMed1985

Basal bodies and associated structures are not required for normal flagellar motion or phototaxis in the green alga Chlorogonium elongatum.

The interphase flagellar apparatus of the green alga Chlorogonium elongatum resembles that of Chlamydomonas reinhardtii in the possession of microtubular rootlets and striated fibers. However, Chlorogonium, unlike Chlamydomonas, retains functional flagella during cell division. In dividing cells, the basal bodies and associated structures are no longer present at the flagellar bases, but have apparently detached and migrated towards the cell equator before the first mitosis. The transition regions remain with the flagella, which are now attached to a large apical mitochondrion by cross-striated filamentous components. Both dividing and nondividing cells of Chlorogonium propagate asymmetrical ciliary-type waveforms during forward swimming and symmetrical flagellar-type waveforms during reverse swimming. High-speed cinephotomicrographic analysis indicates that waveforms, beat frequency, and flagellar coordination are similar in both cell types. This indicates that basal bodies, striated fibers, and microtubular rootlets are not required for the initiation of flagellar beat, coordination of the two flagella, or determination of flagellar waveform. Dividing cells display a strong net negative phototaxis comparable to that of nondividing cells; hence, none of these structures are required for the transmission or processing of the signals involved in phototaxis, or for the changes in flagellar beat that lead to phototactic turning. Therefore, all of the machinery directly involved in the control of flagellar motion is contained within the axoneme and/or transition region. The timing of formation and the positioning of the newly formed basal structures in each of the daughter cells suggests that they play a significant role in cellular morphogenesis.

Chlamydomonas↗
PubMed1996

Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images.

The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy. In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes of Salmonella typhimurium overproduced in the cytoplasmic membrane of Escherichia coli. The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm. On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible. We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm. It was identified as the C ring, since it was located at the base of the polyhook. Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photogrammetry, and a three-dimensional model of the total basal structure is presented.

Cell Membrane↗
PubMed2000

Overproduced Salmonella typhimurium flagellar motor switch complexes.

Three Salmonella typhimurium flagellar motor proteins, FliG, FliM and FliN, are required for the switching of rotation sense. The proteins have been localized to the cytoplasmic module of the flagellar base. Structures, which were morphologically indistinguishable from the native transmembrane MS-ring and cytoplasmic C-ring basal body modules, formed in Escherichia coli upon plasmid-encoded synthesis of these proteins together with FliF. The structures localized to the cell membrane and contained all three motor proteins, as determined by immuno-electron microscopy. This result supports the deduction, based on earlier biochemical analysis, that the C-ring is composed entirely of these proteins and, therefore, functions as a dedicated motor component. In addition, it demonstrates that the morphologically correct assembly of the C-ring onto the MS-ring proceeds independently of other structural components of these modules.

Bacterial Proteins↗
PubMed1995

Identification and classification of Listeria by two-dimensional protein mapping.

The cellular proteins of 29 Listeria strains belonging to different species and serotypes were analysed by two-dimensional (2-D) electrophoresis with the help of a computerized 2-D gel analysis system. The protein patterns were similar among strains within a Listeria species, but were different from one species to another. The comparative analysis of these protein maps enabled us to find specific proteins and to determine the genetic relatedness among Listeria spp. strains. The cluster analysis based on protein mapping showed a division between species and a clear separation of L. monocytogenes from other Listeria species. Inside L. monocytogenes the strains were divided into two main clusters in correlation with flagellar antigenic structures; this is in concordance with the results that have been found on the basis of multilocus enzyme electromorphs or DNA-restriction patterns. This technique enabled us to subtype the strains sharing the same serovar or the same lysovar. Because of its independence and high discriminating capacity, the 2-D protein mapping technique might provide a powerful tool for the identification and classification of Listeria strains.

Autoradiography↗
PubMed2003

Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi.

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding. The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1). Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.

Amino Acid Sequence↗
PubMed2004

Flagellar sensilla of Quadraspidiotus perniciosus Comstock (Rhynchota: Diaspididae) male.

The flagellar sensory structures of Quadraspidiotus perniciosus Comstock (Rhynchota: Diaspididae) male have been investigated, using scanning and transmission electron microscopy. Five categories of sensilla have been identified: three types of sensilla trichodea, a sensillum coeloconicum and a sensillum basiconicum. The possible function of sensilla is discussed, considering their location, morphology and ultrastructure. No gustatory sensilla have been detected. All the setae (sensilla trichodea) present on the flagellomeres, except for the three 'knobbed' ones that are mechanosensilla, are cuticular components of olfactory structures. These olfactory sensilla are of two distinct types, having fluted or smooth hairs; furthermore, the fluted ones present different typologies for a varied number of dendrites. They are conceivably used for perceiving female sexual pheromone. It might be that each of the different components of the female sexual pheromone is perceived by a specific sensillum. Two types of supposed thermo-hygroreceptors have been found, one sensillum coeloconicum and two sensilla basiconica.

Animals↗
PubMed1977

Bacterial flagella rotating in bundles: a study in helical geometry.

Bacterial flagella are semi-rigid helices that undergo true rotation. In peritrichously flagellated bacteria (e.g., Escherichia and Salmonella) there are many flagella on each cell; during translational cell movement these operate as a coordinated bundle that actively disperses upon reversal of the rotation sense. The dynamic behavior of a set of helices originating on separate rotational axes is explored by a working model, geometrical analysis, and hydrodynamic calculations. A critical relationship exists between the interaxial separation and phase difference of parallel helices with overlapping domains; in the subcritical case the filaments are not intertwisted, whereas in the supercritical case they are intertwisted in the same sense (left-handed) as the helices, with one twist per helical turn. During counter-clockwise rotation (the sense operative in forward swimming) any preexisting twists of this kind are automatically cancelled and the helices brought progressively into phase. Hydrodynamic calculations suggest that some wrapping then occurs in a right-handed sense, opposite to that of the helices; this necessitates a distortion from true helical geometry which is minimized by maintaining a coaxial in-phase relationship. A highly coordinated helical bundle results that is capable of operating smoothly for an indefinite period, in agreement with the observed behavior of swimming bacteria. During reverse rotation, the supercritical case develops to cause jamming of the bundle, as has been observed with bacteria in high-viscosity medium. The explosive dispersal of the bundle during reversal in low-viscosity medium is a consequence of a complicating phenomenon, namely, a drastic change in flagellar quaternary structure. The overall conclusion is that bundle formation and function are perfectly compatible with a rotational mechanism for the individual flagella.

Bacterial Physiological Phenomena↗
PubMed1987

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei.

alpha-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.

Animals↗
PubMed1977

Identification of polypeptides necessary for chemotaxis in Escherichia coli.

Molecular cloning techniques were used to construct Escherichia coli-lambda hybrids that contained many of the genes necessary for flagellar rotation and chemotaxis. The properties of specific hybrids that carried the classical "cheA" and "cheB" loci were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the hybrids redefined the "cheA" and "cheB" regions. Six genes were resolved: cheA, cheW, cheX, cheB, cheY, and cheZ. They directed the synthesis of specific polypeptides with the following apparent molecular weights: cheA, 76,000 and 66,000; cheW, 12,000; cheX, 28,000; cheB, 38,000; cheY, 8,000; and cheZ, 24,000. The presence of another gene, cheM, was inferred from the protein synthesis experiments. The cheM gene directed the synthesis of polypeptides with apparent molecular weights of 63,000, 61,000, and 60,000. The synthesis of all of these polypeptides is regulated by the same mechanisms that regulate the synthesis of flagellar-related structural components.

Bacterial Proteins↗