Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes.     

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.     

Role of gene flaFV on flagellar hook formation in Salmonella typhimurium.

Nine temperature-sensitive nonflagellate mutants defective in flaFV were isolated from a strain of Salmonella typhimurium. Among them three mutants were found to produce flagella with abnormally shaped (either straight or irregularly curved) hooks at the permissive temperature. Two mutations that rendered hooks straight were located in one of the eight segments of flaFV defined by deletion mapping. The mutation that rendered hooks irregularly curved was located in a different segment. An flaR mutation was introduced into the latter mutant. At the permissive temperature, the resulting double mutant produced polyhooks whose wavelength and amplitude were both exceedingly reduced. These polyhook structures were more thermolabile than those of the flaFV+ strain. Hook protein of the former strain was shown to have a slightly positive electric charge compared with that of the latter. From these results and other available information, it is inferred that flaFV is the structural gene for the hook protein in Salmonella.     

Caulobacter flagellar organelle: synthesis, compartmentation, and assembly.

In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Length Control of the Flagellar Hook in a Temperature-Sensitive flgE Mutant of Salmonella enterica Serovar Typhimurium

The flagellar hook is a short, curved, extracellular structure located between the basal body and the filament. The hook is composed of the FlgE protein. In this study, we analyzed flagellum assembly in a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium. When the mutant cells were grown at 30°C, they produced flagella of a normal length (71% of the population) and short hooks without filaments (26%). At 37°C, 70% of the basal bodies lacked hooks, and intact flagella made up only 6% of the population. Mutant cells secreted monomeric FlgE in abundance at 37°C, suggesting that the mutant FlgE protein might be defective in polymerization at higher temperatures. The average length of the hooks in intact filaments was 55 nm, whereas after acid treatment, it was 45 nm. SDS-PAGE analysis of the hook-basal body showed that HAP1 was missing in the mutant but not in the wild type. We concluded that hook length in the mutant is controlled in the same way as in the wild type, but the hook appeared short after acid treatment due to the lack of HAP1. We also learned that the true length of the hook is possibly 45 nm, not 55 nm, as has been believed.     

Flagellar hook and basal complex of Caulobacter crescentus.

Intact bacterial flagella possessing a membrane-free hook and basal complex were purified from Caulobacter crescentus CB15, as well as from mutants which synthesize incomplete flagella. The basal body consisted of five rings mounted on a rod. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. The lower rings were all approximately 21 nm in diameter, although they varied significantly in width. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component.     

Differentiation Within the Bacterial Flagellum and Isolation of the Proximal Hook

Purified and crude flagellar isolates from cells of Bacillus pumilus NRS 236 were treated with acid, alcohol, acid-alcohol, or heat, and were examined electron microscopically in negatively stained and shadow-cast preparations. Under certain conditions, each of these agents causes the flagella to break between the proximal hooks and the spiral filaments. In such preparations, filaments are seen in various stages of disintegration, whereas hooks of fairly constant length retain their integrity and morphological identity. When crude isolates of flagella are treated under these conditions, the hooks remain attached to membrane fragments or bear basal material. These findings substantiate previous structural observations that led to the view that the proximal hook is a distinct part of the bacterial flagellum and further confirm that the hook is tightly associated with basal material and the cytoplasmic membrane. It appears that the hook is a polarly oriented structure, and that the interactions between the hook and the basal material or the cytoplasmic membrane are different from those between the hook and the filamentous portion of the organelle. Moreover, both types of interaction apparently differ still from those by which the flagellin subunits are held together in the flagellar filament. Hooks were isolated by exploiting the differences in relative stability shown by the various morphological regions of the bacterial flagellum.     

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.     

Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia.

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.     

Structure of plain and complex flagellar hooks of Pseudomonas rhodos.

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.     

Image reconstruction of the flagellar basal body of Caulobacter crescentus

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.     

Flagellar assembly mutants in Escherichia coli

Genetic and biochemical analysis of mutants defective in the synthesis of flagella in Escherichia coli revealed an unusual class of mutants. These mutants were found to produce short, curly, flagella-like filaments with low amplitude ( approximately 0.06 mum). The filaments were connected to characteristic flagellar basal caps and extended for 1 to 2 mum from the bacterial surface. The mutations in these strains were all members of one complementation group, group E, which is located between his and uvrC. The structural, serological, and chemical properties of the filament derived from the mutants closely resemble those of the flagellar hook structure. On the basis of these properties, it is suggested that these filaments are "polyhooks", i.e., repeated end-to-end polymers of the hook portion of the flagellum. Polyhooks are presumed to be the result of a defective cistron which normally functions to control the length of the hook region of the flagellum.     

Compliance of bacterial polyhooks measured with optical tweezers.

In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.     

FLAGELLAR REGENERATION IN SPERMATOZOPSIS SIMILIS (CHLOROPHYTA)

The unicellular green alga Spermatozopsis similis Preisig et Melkonian bears two flagella of unequal length. After deflagellation, cells first regenerated the longer flagellum to about one third of its original length, before the shorter flagellum started to develop. Growth rates were similar for both flagella. Thus, the length difference between both flagella was restored by a lag-phase during regeneration of the shorter flagellum. To explain the lag-phase, we have considered a gating mechanism near the flagellar base that controls the entry of precursors into the flagellum. This would allow cells to restrict the time of effective flagellar growth and thereby control flagellar length. Our data indicated that cells are capable of individually regulating flagellar assembly onto basal bodies. We discuss a recent model of flagellar length regulation based on a balance of assembly and disassembly and conclude that flagellar length is controlled by additional factors, including the availability of flagellar proteins and the developmental status of basal bodies.     

Purification and Partial Characterization of Bacillus subtilis Flagellar Hooks

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.     

Functional Activation of the Flagellar Type III Secretion Export Apparatus

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.     

The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock

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.