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PubMed1992

Endocytosed transferrin in African trypanosomes is delivered to lysosomes and may not be recycled.

It has been shown in mammalian systems that the passage of transferrin-colloidal gold (Tf-Au) through the endocytic system is influenced by the size of the gold colloid (Neutra, M. R. et al., J. Histochem. Cytochem. 33, 1134-1144 (1985); Woods, J. W. et al., Eur. J. Cell Biol. 50, 132-143 (1989)). However, in both Trypanosoma brucei brucei and Trypanosoma congolense, widely varying sizes of Tf-Au (Tf-Au5 and Tf-Au15) have been shown to proceed to lysosomes (Webster, P., Eur. J. Cell Biol. 49, 295-302 (1989); Webster, P., D. Grab, J. Cell Biol. 106, 279-288 (1988)). Using an affinity-purified anti-bovine transferrin IgG we have demonstrated that, in both T. brucei and T. congolense, native transferrin, like Tf-Au, is found in the flagellar pocket, coated vesicles, tubular structures, and lysosome-like organelles where it appears to be concentrated. The presence of Tf in the lysosomes was confirmed in colocalization experiments using T. congolense, where native bovine transferrin colocalized with a trypanosome lysosomal marker, a cysteine protease. The data suggest that, unlike the situation in mammalian cells where most transferrin is recycled to the cell surface, in African trypanosomes transferrin is routed into lysosomes and may not, therefore, be recycled.

Animals↗

PubMed1986

Cytochemical localization of acid phosphatase in Leishmania mexicana amazonensis.

Acid phosphatase was cytochemically detected at the ultrastructural level in infective and non-infective promastigotes and in amastigotes of the parasitic protozoan Leishmania mexicana amazonensis. Cerium chloride was used as the capture agent of the phosphate liberated during the hydrolysis of the substrate (Na-beta-glycerophosphate). Reaction product, indicative of enzyme activity, was seen in the outer face of the plasma membrane of many, but not all, infective and noninfective promastigote forms. No reaction product was seen in the plasma membrane of amastigote forms. Reaction product was seen in the endoplasmic reticulum, in the Golgi complex, in vesicles located close to the flagellar pocket and in cytoplasmic structures which may represent lysosomes. No reaction product was seen when the substrate was omitted from or sodium fluoride was added to the incubation medium. The possible role played by the acid phosphatase present in the plasma membrane of Leishmania parasites is discussed.

Acid Phosphatase↗

PubMed1993

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.

Amino Acid Sequence↗

PubMed2006

Structure of FliM provides insight into assembly of the switch complex in the bacterial flagella motor.

Bacteria switch the direction their flagella rotate to control movement. FliM, along with FliN and FliG, compose a complex in the motor that, upon binding phosphorylated CheY, reverses the sense of flagellar rotation. The 2.0-A resolution structure of the FliM middle domain (FliM(M)) from Thermotoga maritima reveals a pseudo-2-fold symmetric topology similar to the CheY phosphatases CheC and CheX. A variable structural element, which, in CheC, mediates binding to CheD (alpha2') and, in CheX, mediates dimerization (beta'(x)), has a truncated structure unique to FliM (alpha2'). An exposed helix of FliM(M) (alpha1) does not contain the catalytic residues of CheC and CheX but does include positions conserved in FliM sequences. Cross-linking experiments with site-directed cysteine mutants show that FliM self-associates through residues on alpha1 and alpha2'. CheY activated by BeF(3)(-) binds to FliM with approximately 40-fold higher affinity than CheY (K(d) = 0.04 microM vs. 2 microM). Mapping residue conservation, suppressor mutation sites, binding data, and deletion analysis onto the FliM(M) surface defines regions important for contacts with the stator-interacting protein FliG and for either counterclockwise or clockwise rotation. Association of 33-35 FliM subunits would generate a 44- to 45-nm-diameter disk, consistent with the known dimensions of the C-ring. The localization of counterclockwise- and clockwise-biasing mutations to distinct surfaces suggests that the binding of phosphorylated CheY cooperatively realigns FliM around the ring.

Amino Acid Sequence↗

PubMed1966

Attachment and structural features of flagella of certain bacilli.

Abram, Dinah (Purdue University, Lafayette, Ind.), A. E. Vatter, and Henry Koffler. Attachment and structural features of flagella of certain bacilli. J. Bacteriol. 91:2045-2068. 1966.-The attachment of flagella to cells of various mesophilic and thermophilic strains of Bacillus was studied electron microscopically. Studies of ghost cells and membrane fragments indicate that flagella are connected to the cytoplasmic membrane. Flagella removed from cells mechanically, during autolysis, or by phage lysis, have attached to the base of their proximal hooks material that is heterogeneous in character. In part, this material consists of cytoplasmic membrane; its varied shape appears to be caused by the folding of the membrane around the proximal end of the flagellum at the site of attachment. It is uncertain whether this material represents a real structure or an artifact. Highresolution microscopy reveals differences in the fine structure of intact flagella of the various strains studied. The proximal hook and the flagellar filament are distinct in morphology and fine structure. A specialized structure is associated with the hook of flagella of B. brevis and B. circulans. The filament of flagella of B. stearothermophilus 2184 has two regions that show marked differences in the manner in which the subunits appear to be organized. No correlation was found between the site of origin of flagella and the location of reduced tellurite when the reduction of potassium tellurite was used to indicate the loci of enzymatic respiratory activities.

Bacillus↗

PubMed2006

Flagellar glycosylation - a new component of the motility repertoire?

The biosynthesis, assembly and regulation of the flagellar apparatus has been the subject of extensive studies over many decades, with considerable attention devoted to the peritrichous flagella of Escherichia coli and Salmonella enterica. The characterization of flagellar systems from many other bacterial species has revealed subtle yet distinct differences in composition, regulation and mode of assembly of this important subcellular structure. Glycosylation of the major structural protein, the flagellin, has been shown most recently to be an important component of numerous flagellar systems in both Archaea and Bacteria, playing either an integral role in assembly or for a number of bacterial pathogens a role in virulence. This review focuses on the structural diversity in flagellar glycosylation systems and demonstrates that as a consequence of the unique assembly processes, the type of glycosidic linkage found on archaeal and bacterial flagellins is distinctive.

Archaea↗

PubMed1993

A motility in the eukaryotic flagellum unrelated to flagellar beating.

We report a motility in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flagellar beating. This motility, referred to as intraflagellar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the flagella. Intraflagellar transport could be experimentally separated from other, previously reported, nonbeat flagellar motilities. EM of flagella showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport.

Animals↗

PubMed2000

Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli.

How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor sigma(28). FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of sigma(28)-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.

Amino Acid Sequence↗

PubMed1977

Normal-to-curly flagellar transitions and their role in bacterial tumbling. Stabilization of an alternative quaternary structure by mechanical force.

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Flagella↗

PubMed2006

sigma28-dependent transcription in Salmonella enterica is independent of flagellar shearing.

The FlgM anti-sigma28 factor is secreted in response to flagellar hook-basal body completion to allow sigma28-dependent transcription of genes needed late in flagellar assembly, such as the flagellin structural gene, fliC. A long-standing hypothesis was that one role of FlgM secretion was to allow rapid expression of flagellin in response to shearing. We tested this hypothesis by following FlgM secretion and fliC transcription in response to flagellar shearing. Experiments showed that the level of FlgM inside the cell was unchanged after shearing whereas the extracellular FlgM levels increased in the growth medium as time passed. Identical results were obtained with cells that were not exposed to shear forces: internal FlgM levels remained constant while external FlgM levels rose with time at rates similar to those for the sheared culture. Consistent with this find, FlgM/sigma28-dependent class 3 gene expression was unaffected by flagellar shearing but was affected by the growth phase of the cell. Regardless of exposure to shear forces, flagellar class 3 transcription rose sharply and then declined. These results demonstrate that flagellar regrowth following shearing is independent of FlgM secretion.

Agglutination Tests↗

PubMed1985

The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique.

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.

Cell Wall↗

PubMed1989

Structure of the core and central channel of bacterial flagella.

X-ray fibre diffraction analysis of bacterial flagellar filaments has allowed the subunit packing and secondary structure arrangement in the filament core to be determined. The central hole, presumably a channel for flagellin transport, is large enough to accommodate the folded elongated flagellin molecules during their transport to the distal end for filament growth.

Amino Acid Sequence↗

PubMed2001

Extensive alanine scanning reveals protein-protein and protein-DNA interaction surfaces in the global regulator FlhD from Escherichia coli.

FlhD and FlhC are the transcriptional activators of the flagellar regulon. The heterotetrameric complex formed by these two proteins activates the transcription of the class II flagellar genes. The flagellar regulon consists not only of flagellar genes, but also of the chemotactic genes and some receptor proteins. Recently, a connection between the flagellar regulon and some virulence genes has been found in some species. Furthermore, FlhD, but not FlhC, regulates another non-flagellar target. As a first attempt to understand the mechanism of the flagellar transcriptional activation by FlhD and FlhC, the structure of FlhD has been solved. In order to understand the mechanism of the action of FlhD when it regulates the flagellar genes, we conducted site-directed mutagenesis based on its three-dimensional structure. Six interaction surfaces in the FlhD dimer were mapped by alanine scanning mutagenesis. Two of them are surface clusters formed by residues His-2, Asp-28, Arg-35, Phe-34 and Asn-61 located at each side of the dimer core. The other four are located in the flexible arms of the dimer. The residues Ser-82, Arg-83, Val-84, His-91, Thr-92, Ile-94 and Leu-96 are located at this region. All these residues are involved in the FlhD/FlhC interaction with the exception of Ser-82, Arg-83 and Val-84. These three residues affect the DNA-binding ability of the complex. The three-dimensional topology of FlhD and the site-directed mutagenesis results support the hypothesis of FlhC as an allosteric effector that activates FlhD for the recognition of the DNA.

Alanine↗

PubMed1998

Type III protein secretion systems in plant and animal pathogenic bacteria.

Among many interesting and sophisticated mechanisms used by bacterial pathogens to subvert eukaryotic hosts is a class of specialized protein secretion systems (known as type III protein secretion systems) that deliver bacterial virulence proteins directly into the host cell. Recent studies have revealed four important features of these secretion systems. First, they are widespread among plant and animal bacterial pathogens, and mutations affecting type III protein secretion often eliminate bacterial virulence completely. Second, at least eight type III secretion components share sequence similarities with those of the flagellar assembly machinery and flagellum-like structures are associated with type III secretion, raising the possibility that these secretion systems are derived from the presumably more ancient flagellar assembly apparatus. Third, type III secretion is activated in vivo upon contact with host cells. Fourth, the type III secretion mechanism is Sec-independent and the effector proteins may possess mRNA-based targeting signals. This review highlights the similarities and differences among type III secretion systems of selected model plant and animal pathogenic bacteria.

Journal Article↗

PubMed1978

Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli.

Of the more than 30 genes required for flagellar function, 6 are located between pyrC and ptsG on the Escherichia coli genetic man. This cluster of genes is called flagellar region I. Four-point transductional crosses were used to establish the position and order of the region I flagellar genes with respect to the outside markers ptsG and pyrC. Bacteriophage lambda-E. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. The properties of specific hybrids that carried the region I fla genes 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 lambda hybrids demonstrated the existence of at least six flagellar-specific cistrons. These directed the synthesis of polypeptides with the following apparent molecular weights: flaV, 11,000; flaK, 42,000; flaL, 30,000 and 27,000; flaM, 38,000; flS, 60,000; and flaT, 35,000. Plasmid ColE1-E. coli hybrids with region I flagellar genes were also used to program the synthesis of polypeptides in minicell-producing strains. The polypeptides synthesized in these experiments were identical to polypeptides of the hook-basal body structure and helped to confirm the assignment of genes to specific polypeptides. The synthesis of all of these polypeptides was regulated by the same mechanism that regulates the synthesis of other flagellar-related structural components.

Bacterial Proteins↗

PubMed2000

The flagellar hook protein, FlgE, of Salmonella enterica serovar typhimurium is posttranscriptionally regulated in response to the stage of flagellar assembly.

We investigated the posttranscriptional regulation of flgE, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the flgE gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook-basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly.

Bacterial Proteins↗

PubMed2001

Crystal structure of an activated response regulator bound to its target.

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Amino Acid Sequence↗

PubMed1996

The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles.

We previously reported that the FLA10 locus on the uni linkage group of Chlamydomonas encodes a kinesin homologous protein, KHP1. The fla10 phenotype, which is a temperature-sensitive defect for flagellar assembly and maintenance, is rescued by transformation with the wild-type KHP1 gene. In the present study we identify the molecular defect associated with the fla10 mutation and examine the subcellular localization of KHP1 throughout the cell cycle. The mutation in the fla10-1 allele consists of a C to A transversion, which alters amino acid 329 in the motor domain of KHP1. This residue and the sequence of the carboxy-terminal third of the motor domain in which it is located are highly conserved throughout eukaryotic evolution in a subfamily of kinesin-related proteins from mouse (KIF3), sea urchin (KRP85/95), Xenopus (XKLP3), and Drosophila (KLP68D). These data suggest a conserved function for this family of proteins. Immunofluorescence studies reveal that: (1) in interphase cells KHP1 is associated with basal bodies and with the proximal portion of the flagella; (2) in cells undergoing flagellar regeneration KHP1 occurs in punctate structures that extend to the tip of the developing axoneme; and (3) in dividing cells KHP1 remains associated with centrioles throughout mitosis and localizes to the mitotic spindle. KHP1 is the first kinesin homologous protein to be found in association with basal bodies and centrioles throughout the cell cycle. These observations provide evidence for a direct role of basal bodies in the process of flagellar development, which we propose is based on KHP1 acting as a transporter of flagellar components from the basal bodies out to the distal site of assembly. The localization of KHP1 in mitosis suggests that this protein may play an analogous role in the centriole-based assembly of the mitotic spindle.

Alleles↗