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PubMed2005

Role of motility and flagellin glycosylation in the pathogenesis of Pseudomonas aeruginosa burn wound infections.

In this study, we tested the contribution of flagellar motility, flagellin structure, and its glycosylation in Pseudomonas aeruginosa using genetically defined flagellar mutants. All mutants and their parent strains were tested in a burned-mouse model of infection. Motility and glycosylation of the flagellum appear to be important determinants of flagellar-mediated virulence in this model. This is the first report where genetically defined flagellar variants of P. aeruginosa were tested in the burned-mouse model of infection.

Animals↗
PubMed2004

Self-assembly and type III protein export of the bacterial flagellum.

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.

Bacterial Proteins↗
PubMed1985

Electron microscopic observations of structures associated with the flagella of Spirillum volutans.

Electron microscopy of thin-sectioned Spirillum volutans (ATCC 19554) showed that at the insertion site of the flagellum there was a cylindrical structure with a diameter of ca. 36 nm which extended ca. 19 nm into the cytoplasm. This structure, termed a cytoplasmic flagellar base, enclosed a central rod which was continuous with the hook. There was a continuation of the flagellar base into the peptidoglycan layer, enclosing ringlike structures and the central rod. The flagellar hook and proximal part of the flagellar filament contained a central channel which was large enough to accommodate the flagellin subunit. The flagella of fixed cells may project perpendicularly from the outer membrane in a position corresponding to a trailing, swimming orientation or may bend almost parallel to the membrane in a leading orientation. Maximum bending occurred in the hook region, which may be the structure responsible for executing changes in swimming direction.

Flagella↗
PubMed2000

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio.

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.

Amino Acid Sequence↗
PubMed2001

Regulation of late flagellar gene transcription and cell division by flagellum assembly in Caulobacter crescentus.

Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a trans-acting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a trans-acting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.

Amino Acid Sequence↗
PubMed1994

Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium.

The proximal end of the flagellar basal body consists of a structure called the MS ring complex: two (M and S) rings with different thicknesses closely apposed and a rod extending from the center of the S ring. It has been shown that the MS ring complex consists of multiple copies of single protein FliF (molecular mass 61 kDa). We analyzed the domains of FliF to elucidate how a single protein can be used to construct a complicated particle with several distinct sub-structures. Tryptic digestion of the MS ring complex gave rise to a structure which lacked most of the M ring portion by electron microscopy and showed a major band at 25 kDa by SDS/gel electrophoresis. Amino acid sequence analysis of this band showed that both terminal regions of FliF have been digested, leaving a semi-stable peptide starting from Phe120 and ending at around 400. In addition, we constructed a truncated fliF gene which encodes a FliF lacking 103 amino acid residues from the C terminus. Amplification of the truncated FliF gave rise to a ring complex lacking the rim of the M ring. From these results we assign both terminal regions of FliF to the M ring. Possible domain structures of FliF corresponding to the S ring and the rod are also discussed.

Amino Acid Sequence↗
PubMed2004

Type III flagellar protein export and flagellar assembly.

Bacterial flagella, unlike eukaryotic flagella, are largely external to the cell and therefore many of their subunits have to be exported. Export is ATP-driven. In Salmonella, the bacterium on which this chapter largely focuses, the apparatus responsible for flagellar protein export consists of six membrane components, three soluble components and several substrate-specific chaperones. Other flagellated eubacteria have similar systems. The membrane components of the export apparatus are housed within the flagellar basal body and deliver their substrates into a channel or lumen in the nascent structure from which point they diffuse to the far end and assemble. Both on the basis of sequence similarities of several components and structural similarities, the flagellar protein export systems clearly belong to the type III superfamily, whose other members are responsible for secretion of virulence factors by many species of pathogenic bacteria.

Bacteria↗
PubMed1997

The flk gene of Salmonella typhimurium couples flagellar P- and L-ring assembly to flagellar morphogenesis.

The flagellum of Salmonella typhimurium is assembled in stages, and the negative regulatory protein, FlgM, is able to sense the completion of an intermediate stage of assembly, the basal body-hook (BBH) structure. Mutations in steps leading to the formation of the BBH structure do not express the flagellar filament structural genes, fliC and fljB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regulatory gene, flk, which appears to sense the completion of another assembly stage in the flagellar morphogenic pathway just prior to BBH formation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural genes. Mutations by insertional inactivation in either the flk or flgM locus allow expression of the fljB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fljB gene in strains defective in all of the steps leading to BBH formation. The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk+ allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein, B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene product of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independent of the FlhDC and sigma28 flagellar transcription factors. The Flk protein observed in a T7 RNA polymerase-mediated expression system showed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly hydrophilic protein with a very C-terminal membrane-spanning segment preceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG.

The FliG protein is essential for assembly, rotation and clockwise/counter-clockwise (CW/CCW) switching of the bacterial flagellum. About 25 copies of FliG are present in a large rotor-mounted assembly termed the 'switch complex', which also contains the proteins FliM and FliN. Mutational studies have identified the segments of FliG most crucial for flagellar assembly, rotation and switching. The structure of the C-terminal domain, which functions specifically in rotation, was reported previously. Here, we describe the crystal structure of a larger fragment of the FliG protein from Thermotoga maritima, which encompasses the middle and C-terminal parts of the protein (termed FliG-MC). The FliG-MC molecule consists of two compact globular domains, linked by an alpha-helix and an extended segment that contains a well-conserved Gly-Gly motif. Mutational studies indicate that FliM binds to both of the globular domains, and given the flexibility of the linking segment, FliM is likely to determine the relative orientation of the domains in the flagellum. We propose a model for the organization of FliG-MC molecules in the flagellum, and suggest that CW/CCW switching might occur by movement of the C-terminal domain relative to other parts of FliG, under the control of FliM.

Amino Acid Motifs↗
PubMed1985

ATPase and cytochrome oxidase activities at the polar organelle in swarm cells of Sphaerotilus natans: an ultrastructural study.

The polar organelle of bacteria presumably is part of the flagellar apparatus. In order to characterize this structure, cytochemical studies on Sphaerotilus natans have been performed. Marked ATPase activity is associated with the inner boundary layer and central layer of this organelle. The spaces between the boundary layers and the central layer of the polar organelle which are traversed by fine fibrilles are positive for reactions with diaminobenzidine. This indicates cytochrome oxidase activity. S. natans possesses a ribbon-like, helically shaped polar organelle which is divided concomitantly with cell fission, possibly explaining inheritance of this structure and of the flagellar apparatus.

Adenosine Triphosphatases↗
PubMed2003

Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion.

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.

Bacteria↗
PubMed1978

Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera.

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.

Agglutination↗
PubMed1992

Structure of striated microtubule-associated fibers of flagellar roots. Comparison of native and reconstituted states.

Several fiber systems are associated with the flagella and basal bodies of eukaryotic cells. Apart from the contractile and Ca(2+)-sensitive system-II fibers, these include the noncontractile system-I fibers that run parallel to flagellar root microtubules. Using electron microscopy and image reconstruction, we have investigated the structure of the system-I fibers of the flagellate green alga Spermatozopsis similis. The fibers were observed in three different states: (1) in situ, (2) after isolation of the intact fibers, (3) after disassembly and reconstitution of fibers in vitro from their 34 kDa subunit protein. The fibers are highly ordered; they show a constant repeat of 28 nm, they are polar, and they contain several transverse and longitudinal striations. A model is discussed showing the system-I fiber to be built from rod-like molecules with a staggered arrangement and identical polarities.

Chlorophyta↗
PubMed1977

Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis.

The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its (35)S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with (35)S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.

Alleles↗
PubMed1996

Flagellar assembly in Salmonella typhimurium.

The bacterial flagellum is a motility apparatus in which a long helical filament--the propeller--is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.

Bacterial Proteins↗