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1.
In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ∼22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis.  相似文献   

2.
In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.  相似文献   

3.
We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild-type cells and inhibition of the export of substrates such as the hook protein FlgE. Co-overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His-FLAG-tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook-type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament-type export substrates FlgK and FlgL (hook-filament junction proteins) and FliC (flagellin). We tested for protein-protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook-type substrates were strong, whereas those with filament-type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI.  相似文献   

4.
Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.  相似文献   

5.
The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.  相似文献   

6.
fliL is the first gene in a flagellar operon that specifies members of the switch complex and type III export system in Salmonella enterica and Escherichia coli , but no function has been ascribed to this gene thus far. Here we report that a fliL mutant is slightly impaired for swimming but completely defective in swarming in both organisms, and have studied this phenotype further in S. enterica . We have found that on swarm agar, mutant cells release or 'eject' their flagellar filaments. The released filaments are attached to the hook and part of the rod structure; we have identified the distal rod protein FlgG but not the proximal rod protein FlgF in these filaments. Rod fracture was not observed if flagellar rotation was prevented by removal of proteins that supply proton flow through the motor. Based on these and other results, we propose that motors experience a higher torque on swarm agar owing to an increased proton motive force, and that FliL allows the rod to withstand the increased torsional stress. The flagella-release phenotype of the S. enterica fliL mutant has a bearing on FliL-dependent flagellar ejection during the swimmer- to stalk-cell transition in the developmental cycle of Caulobacter crescentus .  相似文献   

7.
Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.  相似文献   

8.
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.  相似文献   

9.
The stoichiometries of components within the flagellar hook-(basal-body) complex of Salmonella typhimurium have been determined. The hook protein (FlgE), the most abundant protein in the complex, is present at approximately 130 subunits. Hook-associated protein 1 (FlgK) is present at approximately 12 subunits. The distal rod protein (FlgG) is present at approximately 26 subunits, while the proximal rod proteins (FlgB, FlgC and FlgF) are present at only approximately six subunits each. The stoichiometries of the proximal rod proteins and hook-associated protein 1 are, within experimental error, consistent with values of 5 or 6, and 11, respectively. Such values would correspond to either one or two turns of a helical structure with a basic helix of approximately 5.5 subunits per turn, which is the geometry of both the hook and the filament and, one supposes, the rod and hook-associated proteins. These stoichiometries may derive from rules for the heterologous interactions that occur when a helical structure consists of successive segments constructed from different proteins; the stoichiometries within the hook and the distal portion of the rod must, however, be set by different mechanisms. The stoichiometries for the ring proteins are approximately 26 subunits each for the M-ring protein (FliF), the P-ring protein (FlgI), and the L-ring protein (FlgH); the protein responsible for the S-ring feature is not known. The rings presumably have rotational rather than helical symmetry, in which case the stoichiometries would be directly constrained by the intersubunit bonding angle. The ring stoichiometries are discussed in light of other information concerning flagellar structure and function.  相似文献   

10.
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.  相似文献   

11.
The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk from 70 to 200 nm in diameter, and a terminal knob. In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring. The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk. The diameter of the disk is smaller in the presence of the P ring. The L-P ring complex is therefore assumed to be a bushing for the rod. Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane. At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts. A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations. Its diameter (60 nm) is larger than that of the M ring. A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units.  相似文献   

12.
Thin section electron micrographs of rapidly fixed Chlamydomonas cells were used to establish a relationship between flagellar bends and orientation of the central pair microtubule complex. Using conditions that preserve flagellar waveforms during both forward swimming (asymmetric bends) and backward swimming (symmetric bends), we found that central pair orientation differs in bent regions and straight regions. During forward swimming, a plane through the two central pair microtubules is parallel to the bend plane throughout principal bends, in both effective stroke and recovery stroke phases of the beat cycle. In these curved segments, the C1 microtubule always faces the outer edge of the curve. This parallel orientation twists in straight regions both proximal and distal to bends. During backward swimming episodes induced by photoshock, when Chlamydomonas flagella beat with principal and reverse bends of similar magnitude, the central pair twists by 180 degrees between successive bends. These observations support a model in which central pair orientation in Chlamydomonas is linked to doublet-specific dynein activation, and bend propagation is linked to rotation of the central pair complex.  相似文献   

13.
Morphological pathway of flagellar assembly in Salmonella typhimurium.   总被引:14,自引:0,他引:14  
The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.  相似文献   

14.
The C-terminal half of the Salmonella flagellar protein FlgJ has peptidoglycan hydrolyzing activity and it has been suggested that it is a flagellum-specific muramidase which locally digests the peptidoglycan layer to permit assembly of the rod structure to proceed through the periplasmic space. It was also suggested that FlgJ might be involved in rod formation itself, although there was no direct evidence for this. We purified basal body structures from SJW1437(flgJ) transformed with plasmids encoding various mutant FlgJ proteins and found that these basal bodies possessed the periplasmic P ring but lacked the outer membrane L ring; they also lacked a hook at their distal end. All of these mutant FlgJ proteins had an altered or missing C-terminal domain but had at least the first 151 amino acid residues of the N-terminal domain. Immunoblotting analysis of fractionated cell extracts revealed that a rod/hook export class protein, FlgD, was exported to the periplasm but not to the culture supernatant in these mutants. FlgJ was shown to physically interact with several proteins, and especially FliE and FlgB, which are believed to reside at the cell-proximal end of the rod. On the basis of these results, we conclude that the N-terminal 151 amino acid residues of FlgJ are directly involved in rod formation and that the muramidase activity of FlgJ, though needed for formation of the L ring and subsequent events such as hook formation, is not essential for rod or P ring formation. In contrast, muramidase activity alone does not support rod assembly.  相似文献   

15.
Cell envelope associations of Aquaspirillum serpens flagella.   总被引:12,自引:9,他引:3       下载免费PDF全文
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   

16.
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.  相似文献   

17.
A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.  相似文献   

18.
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.
Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.  相似文献   

20.
Salmonella flagellar hook length is controlled at the level of export substrate specificity of the FlhB component of the type III flagellar export apparatus. FliK is believed to be the hook length sensor and interacts with FlhB to change its export specificity upon hook completion. To find properties of FliK expected of such a molecular ruler, we assayed binding of FliK to the hook and found that the N-terminal domain of FliK (FliK(N)) bound to the hook-capping protein FlgD with high affinity and to the hook protein FlgE with low affinity. To investigate a possible role of FlgE in hook length control, flgE mutants with partially impaired motility were isolated and analyzed. Eight flgE mutants obtained all formed flagellar filaments. The mutants produced significantly shorter hooks while the hook-type substrates such as FlgE, FliK and FlgD were secreted in large amounts, suggesting defective hook assembly with the mutant FlgE proteins. Upon overexpression, mutant FlgEs produced hooks of normal length and wild-type FlgE produced longer hooks. These results suggest that hook length is dependent on the hook polymerization rate and that the start of hook polymerization initiates a "time countdown" for the specificity switch to occur or for significant slow down of rod/hook-type export after hook length reaches around 55 nm for later infrequent FliK(C)-FlhB(C) interaction. We propose that FliK(N) acts as a flexible tape measure, but that hook length is also dependent on the hook elongation rate and a switch timing mechanism.  相似文献   

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