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The basal hook structure of the flagellar organelle Caulobacter crescentus was isolated from release flagella. Hook preparations contained a single major proteins species of 73,000 molecular weight and proteins in smaller amounts that may be minor hook components. Hooks isolated from C. crescents CB13B1a and CB15 were immunologically cross-reactive.
Bacterial motility relies chiefly on the rotation of a molecular propeller, the flagellar filament, which is constructed from the protein flagellin. Here, flagellin sequence conservation and diversity is examined in the light of the recently determined flagellar filament structure. As expected, the surface-exposed domains are not conserved. However, the sequences that mediate filament assembly show remarkable conservation, which indicates that all bacterial flagellins are likely to pack into filaments in a similar manner. Flagellins provide a striking illustration of the twin evolutionary themes of conservation and variability.
Bacterial flagella, the organelles of motility, are commonly divided into two classes: 'plain' and 'complex'. The complex filaments are pairwise, helically perturbed forms of the plain filaments and have been reported to occur only in Rhizobium and Pseudomonas. Previously, we reconstructed and analysed the structure of the complex filaments of Rhizobium lupini H13-3 and determined their unique symmetry and origin of the perturbations (Trachtenberg et al., 1986, J Mol Biol 190: 569-576; 1987, 195: 603-620; 1998, 276: 759-773; Cohen-Krausz and Trachtenberg, 1998, J Struct Biol 122: 267-282). Here, we analyse the structure of the flagellar filament of the other known complex filament, that of Pseudomonas rhodos, as reconstructed from electron microscope images. Compared with the filament of R. lupini, the filament of P. rhodos is more flexible, as implied from high-intensity darkfield light microscopy and, although constructed from flagellins of higher molecular weights (59 versus 41 kDa), has similar symmetry. Using cryonegative stained specimens and low-dose, field emission electron microscopy, we reconstructed and averaged 158 filaments each containing 170 statistically significant layer lines. The three-dimensional density maps of P. rhodos clearly suggest, when compared with those of R. lupini and the right-handed Salmonella typhimurium SJW1655, that R. lupini is missing the outer flagellin domain (D3), that the interior of the complex filament is rather similar to that of the plain filament and that the radial spokes (connecting domains D0 and D1), present in individual density maps, average out because of their variability and implied flexibility. Extending the three-start grooves and ridges on the propeller's surface, in the form of an Archimedean screw, may further improve the motility of the cell in viscous environments.
The membrane-like flagellar sheath of Helicobacter pylori is of unknown function and little is known of its composition. A murine monoclonal antibody to H. pylori, designated GF6, which reacts by immunoblot with a polypeptide with an apparent molecular mass of 29 kDa was shown by immunogold-electron microscopy to label specifically the flagellar sheath structure. The antigen was detected by immunoblot using the monoclonal antibody in all 11 strains, of diverse geographic origin, so far tested. The antibody also reacted weakly with polypeptides with apparent molecular masses of 65 kDa in Vibrio cholerae and Vibrio parahaemolyticus. The antigen was shown by one- and two-dimensional electrophoretic analysis and immunoblotting to be distinct from the abundant urease subunit UreA, of similar molecular mass. Identification of this flagellar sheath polypeptide will facilitate investigation of the structure and function of the flagellar sheath of this important gastric pathogen.
The spermatozoa from four infertile patients showing a flagellar dyskinesia due to abnormal flagellar wave development have been studied by light and transmission electron microscopy (TEM) for flagellar morphology. No axonemal anomalies were found but modification of the periaxonemal structures was observed. The results of a stereological analysis revealed abnormal extension of the individual dense fibres along the axoneme in the four cases as compared with a control group. The order of termination of those structures was therefore altered. However, the overall fibre extension was the same in both groups (ie, 60% of the principal piece). The number and the location of the longitudinal columns were also modified, the predominant anomaly being the presence of a single column. The possible influence of those structural anomalies on the pattern of sperm movement is discussed. Our observations seem to agree with a previous hypothesis of the literature, that the dense fibres might play a role in flagellar flexibility. More particularly, we suggest that the abnormal extension of dense fibres No. 2, 3, and 4 and the symmetric distribution of the dense fibres on both sides of the plane of beating may alter the flagellar curvature amplitude and the cell rotation frequency.
The three-dimensional architecture of a quartet of microtubules (QM) of Trypanosoma evansi and its related structures was examined with a transmission electron microscope (TEM). The initiation of the QM as a successive bundle of microtubules was confirmed at the juxtaposition of the second barren body (SBB). After connecting with the distal part of the basal body (BB), the QM ascended to the level of the terminal plate (TP) and gained access most closely to the inner membrane of the flagellar pocket (FP) and formed the special structure together with macula adherens (MA) and the electron dense linear substance (EDLS); then it follows its way around the FP from the posterior to the anterior and fills the subpellicular space at the underneath of the flagellum. This special structure disappeared when the associated-flagellum became the extracellular flagellum (EF), where the QM intervened into the row of the subpellicular microtubules (SM). This EDLS is suggested to represent a demarcating line (DL). In the deep cytoplasm of the flagellar root, connections via several microtubules between the BB and the SBB and via electron zonal substance (EDZS) between the BB and the mitochondrion (M) were observed. As a result, a connecting system among the flagellar apparatus and its accessory structures seems probable.
The proximal hooks of plain and complex flagella produced by a strain of Pseudomonas rhodos have been analyzed by electron microscopy and optical diffraction and filtering. Plain flagellar hooks are cone-shaped, 70 nm long, and 13 to 21.5 nm wide, and consist of helically arranged subunits. Complex flagellar hooks are cylinders, 180 to 190 nm long, and 15 to 16 nm wide, and are composed of globular subunits. The structure comprises four small-scale helical rows of subunits intersecting bewteen 10 and 11 large-scale helices of pitch angle 80 degrees. The axial and lateral dimensions of the unit cell, which define the surface lattice, are 4.9 and 4.7 nm, respectively. In addition, a core structure, approximately 5 nm wide, has been demonstrated inside the hook cylinder. Complex flagellar hooks were isolated and purified by gradient centrifugation after acid degradation of the attached filaments. Isolated hook particles have an average sedimentation constant of 130S and consist of a protein of molecular weight 43,000. A model of the complex flagellar hook is presented, and its possible role in flagellar assembly and rotation is discussed.
Bacterial motility involves switching between the left and right supercoiled states of the flagellar filament. The polymorphism of this assembly of identical flagellin molecules has presented a structural puzzle. Supercoiling has been attributed to coexistence of two conformational states of the 11 nearly axially aligned protofilament strands of subunits. The helical parameters of straight filaments in the left (L) and right (R) lattice states have now been accurately determined by X-ray fiber diffraction. The 9 A resolution electron density map of the R-type filament, refined from the X-ray data, reveals the interlocked alpha-helical segments of the core portion, which constitute the inner and outer tubes. While the inner-tube domain interactions remain invariant, the strand joints in the outer tube can switch between the L- and R-state by 2-3 A axial shifts, which change the strand periodicity of approximately 50 A by 0.8 A. This bi-stable quaternary switching results in supercoiling. Based on the measured helical parameters of the L and R lattices and the switching model, the twist and curvature calculated for the ten possible supercoils are in quantitative accord with observed supercoiled forms of flagellar filaments.
A method for preparing bacterial flagellar hook structures is described. The method involves isolating intact flagella from a mutant which makes thermally labile flagellar filaments and heat-treating them to disaggregate the filament preferentially. The resulting hook preparation can be separated and purified by velocity and isopycnic centrifugation. The purified hooks sediment at a relative S value of 77. On acrylamide gel electrophoresis in sodium dodecyl sulfate, they show one major and a number of minor protein bands. The purified hooks can be used to immunize rabbits, and the resulting antiserum is hook-specific. These results support the notion that hooks are composed of a protein that differs from flagellin.
Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.
The Escherichia coli flaA gene product (also called cheC) plays a crucial role in switching flagellar rotational direction during chemotactic responses. Wild-type and mutant alleles have been cloned onto plasmid vectors, and the gene product has been identified as a 37,000-dalton protein. The flaA product appeared as a soluble protein in the cytoplasm when overproduced in minicells and maxicells. The protein could not be detected in flagellar basal structures purified from a wild-type strain. To assess the effects of altered flaA expression, the gene was fused to a synthetic tac promoter that could be regulated by the addition of an inducer. Overproduction resulted in strong counterclockwise flagellar rotational bias and partial paralysis of flagellar motors. These results suggest that the flaA protein provides the interface between the flagellar machinery and the chemotaxis signaling system in a motor structure external to the basal body.
Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Delta1-18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Delta1-18) crystals grew in the monoclinic space group P2(1), with unit-cell parameters a = 48, b = 73, c = 126 A, beta = 94 degrees, and diffracted to 2.4 A resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination.
The FliF ring complex, which consists of the M-S ring and a proximal portion of the rod of the flagellar basal body, is the base structure for the bacterial flagellar assembly. The FliF ring is also thought to be part of the export apparatus for flagellar proteins from its amino acid sequence homology to proteins involved in type III protein export systems. We established a new purification procedure for the FliF ring particles and carried out electron microscopic image analyses in their two distinct forms: well-dispersed single particles in the presence of salt and ordered monolayer arrays of hexagonal packing formed in the absence of salt. In both cases, the axial projection maps showed a common feature, a pair of concentric rings: the inner ring corresponds to the proximal rod; the outer ring represents the thick, edge portion of the M-S ring. However, the central channel of the FliF ring, the putative pathway for the flagellar protein export, appeared to show distinct structural features in the two forms. This suggests that a domain of FliF partially occupies the central channel to be involved in the export and gate mechanism, and the domain changes its conformation depending on the ionic strength.
The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.
Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes.
Chlamydiae are obligate intracellular bacterial pathogens whose entry into mucosal epithelial cells is required for intracellular survival and subsequent growth. After a seemingly stealthy entry, chlamydiae quickly modify their vacuole (i) for exit from the endosomal pathway to the exocytic pathway and (ii) to permit fusion with intercepted endoplasmic reticulum- and Golgi-derived vesicles carrying glycerophospholipids and sphingolipids for chlamydiae-containing vacuole membrane expansion. Chlamydiae possess novel hollow proteinaceous structures, termed projections, which they use to pierce the inclusion membrane, possibly to acquire from the epithelial cytoplasm nutrients they cannot synthesize; whether or not these truncated flagellar-like structures serve a dual exchange function for secretion of molecules to programme host cell signalling is unknown. Despite the accumulation of some 500-1000 progeny in the enormously enlarged inclusion, host cell function is surprisingly little disrupted, and progeny escape can be unobtrusive. This elegant adaptive pathogen strategy, which leads to silent, chronic human infection, is fascinating from a cellular microbiology perspective.
Chemotactic receptors at the bacterial cell surface communicate with flagellar basal structures to elicit appropriate motor behavior in response to extracellular stimuli. Genetic and physiological studies indicate that the product of the cheY gene interacts directly with components of the flagellar motor to control swimming behavior. We have purified and characterized the Salmonella typhimurium CheY protein and have determined the nucleotide sequence of the cheY gene. Amino acid sequence comparisons showed CheY to be homologous over its entire length (129 residues) to the N-terminal regulatory domain of another protein involved in chemotaxis, the CheB methyl esterase. The entire CheY protein and the regulatory domain of CheB also homologous to the N-terminal portions of the Escherichia coli OmpR and Dye proteins and the Bacillus subtilis Spo0A protein. These homologies suggest an evolutionary and functional relationship between the chemotaxis system and systems that are thought to regulate gene expression in response to changing environmental conditions.
The terminal regions of monomeric hook protein from Salmonella typhimurium are known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far-UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles. Secondary structure prediction, together with the analysis of far-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain beta-sheeted structures without significant alpha-helical content. The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly alpha-helical domain. Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments. Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies. The formation of alpha-helical bundles is commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella. To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerization behavior was investigated. We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filaments in vitro under appropriately high salt concentrations. Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.