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1.
Basal-body-associated disks are additional structural elements of the flagellar apparatus isolated from Wolinella succinogenes. 总被引:5,自引:5,他引:0
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The intact flagella of Wolinella succinogenes, a gram-negative, anaerobic bacterium with a single polar flagellum, were obtained by an improved procedure, introduced recently by Aizawa et al. (S.-J. Aizawa, G. E. Dean, C. J. Jones, R. M. Macnab, and S. Yamaguchi, J. Bacteriol. 161:836-849, 1985) for the flagellum of Salmonella typhimurium. Disks with a diameter of 130 +/- 30 nm, which were attached to the basal body of the isolated intact flagella, could be identified by electron microscopy as additional structural elements of the bacterial flagellar apparatus. In freeze-dried and metal-shadowed samples, two rings of the basal body were detected on one side and a terminal knob was located on the other side of the disks. Suspension of the flagellar apparatus in acidic solution dissociated the flagellar filaments, yielding hook-basal body complexes with and without the associated disks. If whole cells were subjected to low pH, double disks of the same diameter and with a central hole of about 13 nm could be isolated. Similar parallel disks could be seen also in negatively stained whole cells. When uranyl acetate was used for negative staining of the intact flagella, concentric rings were detected on the disks, similar to the concentric membrane rings found by Coulton and Murray (J. W. Coulton and R. G. E. Murray, J. Bacteriol. 136:1037-1049, 1978) on platelike arrays of proteins in outer membrane preparations of Aquaspirillum serpens. Because the disks of W. succinogenes can be isolated together with the flagellar hook-basal body complex, they appear to be basal-body-rather than secondary membrane-associated structures. It is possible that these disks are the bearing or stator of this rotary device. 相似文献
2.
G E Sosinsky N R Francis M J Stallmeyer D J DeRosier 《Journal of molecular biology》1992,223(1):171-184
The Salmonella typhimurium basal body, a part of the flagellar rotary motor, consists of four rings (denoted M, S, P and L) and a coaxial rod. Using low-dose electron microscopy and image averaging methods on negatively stained and frozen-hydrated preparations, we examined whole basal body complexes and subcomplexes obtained by dissociation in acid. Dissociation occurs in steps, allowing us to obtain images of substructures lacking the M ring, lacking the M and S rings, and lacking the M and S rings and the proximal portion of the rod. We obtained images of the L and P ring subcomplex. The existence of a subcomplex missing only the M ring suggests either that the S and M rings derive from two different proteins, or that the M ring is a labile domain of a single protein, which makes up both rings. At the 25 to 30 A resolution of our averaged images, the L, P and S rings appear cylindrically symmetric. Images of the M ring show variability that may be due to differences in angular orientation of the grid, but equally could be due to structural variations. Three-dimensional reconstructions of these structures from the averaged images reveal the internal structure and spatial organization of these components. 相似文献
3.
The basal body is thought to be a part of the rotary motor of the bacterial flagellum. It consists of a central rod coaxial with a set of four rings, which are associated with the cell envelope. We used single-particle averaging methods to analyze images of negatively stained basal bodies of Salmonella typhimurium. Several averages were computed, so that the reliability of features could be assessed. We carried out the same analysis on electron micrographs of isolated, negatively stained L-P rings. In order to interpret the averages in terms of a three-dimensional structure, we carried out image reconstruction on them. The resulting three-dimensional map corresponds to the cylindrically averaged structure of the basal body. To show that the reconstruction procedure is legitimate, we demonstrate it on model data. The results of the modelling show that features very near to the axis of the reconstruction are not reliable but that broader, off-axis features are represented faithfully. The L ring is L-shaped, with the long arm of the L parallel to the axis of the rod, and the short arm pointing away from the rod. The P ring, on the other hand, appears to be a ring or disk. The position of the L-P ring complex on the rod seems to vary somewhat, consistent with its putative role as a bushing. The cross-sectional shape of the S ring is that of a frustum rather than a disk. The M ring, which is oval in cross section, sits atop the S ring, making contact with it at an outer radius. The S ring appears to make contact with the rod, whereas the M ring does not. This situation, if true, is difficult to reconcile with the common notion that the S ring is stationary and the M ring rotates. It seems more likely that the S ring and rod rotate as a unit. 相似文献
4.
Specific regions of the cell envelope associated with the flagellar basal complex of the gram-negative bacterium Aquaspirillum (Spirillum) serpens were identified by studying each of the envelope layers: outer membrane, mucopeptide, and plasma membrane. The outer membrane around the flagella insertion site was differentiated by concentric membrane rings and central perforations surrounded by a closely set collar. The perforations in both the outer membrane and the isolated mucopeptide layer were of a size accomodating the central rod of the basal complex but smaller than either the L or the P disks. The P disk of the complex may lie between the mucopeptide and the outer membrane. Electron microscopy of intact, spheroplasted, or autolyzed preparations did not adequately resolve the location of the inner pair of disks of the basal complex. Freeze-etching, however, revealed differentiation within the plasma membrane that appeared to be related to the basal complex. The convex fracture face showed depressions which are interpreted as impressions of a disk surrounded by a set of evenly spaced macromolecular studs and containing a central "plug" interpreted as the central rod. In thin sections, blebs, which appear to be associated with the flagellar apparatus, were seen on the cytoplasmic side of the plasma membrane. Superimposing the dimensions of the flagellar basal complex and the spacings of the cell envelope layers and using the position of the L disk within the outer membrane for reference, showed that the S disk might be within and the M disk beneath the plasma membrane. A tentative model was developed for comparison with that based on the structure of the Escherichia coli basal complex. 相似文献
5.
Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia. 总被引:19,自引:13,他引:6
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The periplasmic flagella of Spirochaeta aurantia were isolated and were found to be ultrastructurally and biochemically complex. Generally, flagellar filaments were 18 to 20 nm in diameter and appeared to consist of an 11 to 13-nm-wide inner region and an outer layer. The hook-basal body region consisted of two closely apposed disks connected to a hook by a rod. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella together with a Western blot analysis of a motility mutant that produces hooks and basal bodies but not flagellar filaments revealed that the filaments were composed of three major polypeptides of 37,500, 34,000, and 31,500 apparent molecular weight (37.5K, 34K, and 31.5K polypeptides) and three minor polypeptides of 36,000, 33,000, and 32,000 apparent molecular weight (36K, 33K, and 32K polypeptides). Purified hook-basal body preparations were greatly enriched in three polypeptides in the range of 62,000 to 66,000 apparent molecular weight. Immunogold labeling experiments with a monoclonal antibody specific for the 37.5K flagellin and one that reacts with an epitope common to the 36K, 34K, 33K, 32K, and 31.5K flagellins revealed that the 37.5K major polypeptide was a component of the outer layer, whereas one or more of the other polypeptides constituted the core. 相似文献
6.
The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings. When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane. Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings. Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body. We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body. 相似文献
7.
M J Stallmeyer K M Hahnenberger G E Sosinsky L Shapiro D J DeRosier 《Journal of molecular biology》1989,205(3):511-518
The bacterium Caulobacter crescentus has a single polar flagellum, which is present for only a portion of its cell cycle. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. The flagellum is composed of a transmembrane basal body, a hook and a filament. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. These basal bodies have five rings threaded on a rod. The L and P rings are connected by a bridge of material at their outer radii. The E ring is a thin, flat disk. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. Averages of the rod-hook-filament subassembly ejected by swarmer cells reveal that the rod consists of two parts with the E ring marking the approximate position of the break. The structures of basal bodies from two mutants defective in the hook assembly were found to be indistinguishable from wild-type basal bodies, suggesting that the assembly of the basal body is independent of the hook or filament assembly. 相似文献
8.
Fine Structure and Isolation of the Hook-Basal Body Complex of Flagella from Escherichia coli and Bacillus subtilis 总被引:53,自引:43,他引:10
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The hook-basal body complex comprising the basal end of purified intact flagella from Escherichia coli and Bacillus subtilis was studied in detail with an electron microscope. The E. coli hook can be described as having five or six concentric helical coils. The basal body from E. coli is 27 nm in length and consists of four rings, 22.5 nm in diameter, arranged in two pairs and mounted on a rod. The top pair of rings is connected near their periphery, resembling a closed cylinder. In B. subtilis the basal body looks like that from E. coli, except that the top pair of rings is missing. Hook-basal body complexes from both organisms could be isolated by dissociating the filaments with either urea or acid. Based on our results, two types of basal body structures are proposed, as exemplified by E. coli and B. subtilis, which directly reflect the structure of the gram-negative and gram-positive cell envelopes. 相似文献
9.
Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium. 总被引:46,自引:33,他引:13
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The hook-basal body complex of Salmonella typhimurium, a major component of its flagellar apparatus, was subjected to detailed analysis by electron microscopy and gel electrophoresis. The study was facilitated by the development of an improved protocol for isolation of the complexes in high yield and purity. Nine proteins were identified with the structure. These proteins had apparent molecular weights of 65,000 (65K), 60K, 42K, 38K, 32K, 30K, 27K, 16K, and 14K. Small but reproducible shifts in the apparent molecular weights of specific proteins from conditionally nonflagellate mutants indicated the following gene-polypeptide correspondences: flaFV, 42K; flaFVI, 32K; flaFVII, 30K; flaFIX, 38K; flaAII.1, 65K. Several new morphological features of hook-basal body complexes were recognized, including a clawlike structure on the cytoplasm-proximal M ring and additional material at the cytoplasmic face of the M ring. Based on this study and the work of others, we suggest that the morphological features of the hook-basal body complex correspond to the following proteins: hook-filament junction, 60K; hook, 42K; rod, 30K and 32K; L ring and outer cylinder wall, 27K; P ring, 38K; S ring, unknown; M ring 65K. 相似文献
10.
Naoki Hosogi Hideki Shigematsu Hiroyuki Terashima Michio Homma Kuniaki Nagayama 《Journal of structural biology》2011,173(1):67-76
Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm. 相似文献
11.
Identification of proteins of the outer (L and P) rings of the flagellar basal body of Escherichia coli 总被引:14,自引:9,他引:5
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Synthesis of the Salmonella typhimurium hook protein from the gene cloned on a multicopy plasmid results in partial suppression of the flagellar assembly defects of certain classes of Escherichia coli mutants (K. Ohnishi, M. Homma, K. Kutsukake, and T. Iino, J. Bacteriol, 169:1485-1488, 1987). This phenomenon allowed hook-basal body complexes from such mutants to be purified and analyzed by electron microscopy and gel electrophoresis. The absence of the P and L rings in such structures was found to correlate with the absence of proteins of apparent molecular weight 39,000 and 26,000, respectively. Gene-polypeptide correlations from other studies enabled us to complete gene-polypeptide-structure correspondences for these two proteins as flaM----39-kilodalton protein----P ring and flaY----26-kilodalton protein----L ring. 相似文献
12.
13.
Attachment of Flagellar Basal Bodies to the Cell Envelope: Specific Attachment to the Outer, Lipopolysaccharide Membrane and the Cytoplasmic Membrane 总被引:2,自引:1,他引:1
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A procedure is described for the purification of the Escherichia coli outer membrane (lipopolysaccharide or L membrane) with flagella still attached. The resulting lipopolysaccharide membrane was in the form of vesicles that had a trilaminar structure in thin section and contained about 55% lipopolysaccharide and 45% protein. T2 or T4 phage preadsorbed to E. coli were found attached to the purified lipopolysaccharide membrane. Flagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring). The cytoplasmic membrane in preparations from osmotically lysed E. coli spheroplasts or Bacillus subtilis protoplasts was specifically attached to flagella at the basal body ring farthest from the hook (the M ring). In the E. coli preparation, lipopolysaccharide membrane was also present and was attached to the L ring. From these data and a knowledge of the structure and dimensions of the E. coli flagellar basal body and cell envelope, a model for flagellar attachment is deduced. 相似文献
14.
Suzuki H Yonekura K Murata K Hirai T Oosawa K Namba K 《Journal of structural biology》1998,124(2-3):104-114
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. 相似文献
15.
The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor. We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively. Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages. 相似文献
16.
The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σD-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. 相似文献
17.
New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy. 总被引:6,自引:4,他引:2
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The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella. 相似文献
18.
Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants. 总被引:25,自引:18,他引:7
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The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression. 相似文献
19.
Structure and cell envelope associations of flagellar basal complexes of Vibrio cholerae and Campylobacter fetus 总被引:11,自引:0,他引:11
F G Ferris T J Beveridge M L Marceau-Day A D Larson 《Canadian journal of microbiology》1984,30(3):322-333
To isolate intact flagella with basal complexes from Vibrio cholerae, a rhamnolipid hemolysin from Pseudomonas aeruginosa was used to disrupt the cell envelope and flagellar sheath. The nonionic detergent, Triton X-100, provided similar results for Campylobacter fetus. Each of these basal complexes possessed, in addition to the four classical rings, concentric membrane rings (CMR's) similar to those found in Aquaspirillum serpens. Through the use of stereo imaging (which allows structures to be visualized in three dimensions) of thin sections of cells which had been sequentially treated with a number of envelope perturbants (i.e., ethylenediaminetetraacetate, lysozyme, Triton X-100, rhamnolipid hemolysin, and sodium dodecyl sulfate), we have progressively exposed the component parts of the basal organelles in V. cholerae and C. fetus. Since the action of these envelope perturbants has been well documented, we have been able to determine the associations of the exposed portions of the flagellar basal complex and the layer of the cell envelope in which they would normally reside. From our observations we have concluded that in both V. cholerae and C. fetus the L ring is embedded in the outer membrane and the P ring is associated with the peptidoglycan. The CMR's are bracketed by the L and P rings and are sandwiched between the outer membrane and the peptidoglycan. Elements of both the S and M rings appear to be associated with the plasma membrane. 相似文献
20.
Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age 总被引:1,自引:0,他引:1
Kathleen Boesze-Battaglia Arlene D. Albert James S. Frye Philip L. Yeagle 《Bioscience reports》1996,16(4):289-297
The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning31P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity. 相似文献