Interactions between chemotaxis genes and flagellar genes in Escherichia coli

Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli.     

Cell cycle regulation of flagellar genes.

The expression of the flagellar master operon, flhDC, peaked in the middle of three consecutive cell cycles. The level of expression was lowest at the time of cell division. The expression of the second-level operon, flhB, peaked at cell division. The swimming speed of individual cells was also highest at the time of cell division.     

A family of six flagellin genes contributes to the Caulobacter crescentus flagellar filament

The Caulobacter crescentus flagellar filament is assembled from multiple flagellin proteins that are encoded by six genes. The amino acid sequences of the FljJ and FljL flagellins are divergent from those of the other four flagellins. Since these flagellins are the first to be assembled in the flagellar filament, one or both might have specialized to facilitate the initiation of filament assembly.     

Separation of temporal control and trans-acting modulation of flagellin and chemotaxis genes in Caulobacter

Summary The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. We have found that the trans regulation that modulates the amounts of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. This conclusion is based on two observations: (1) the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and (2) complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control.     

Isolation and characterization of Caulobacter crescentus flagellar hooks.

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.     

The organization of the Caulobacter crescentus flagellar filament

The structural organization of the flagellar filament of Caulobacter crescentus, as revealed by immunoelectron microscopy, shows five antigenically distinct regions within the hook-filament complex. The first region is the hook. The second region is adjacent to the hook and is approximately 10 nm in length. On the basis of its location in the hook-filament complex, this region may contain hook-associated proteins. Next to this is the third region, which is approximately 60 nm in length. Antibody decoration experiments using mutant strains with deletions of the structural gene for the 29 x 10(3) Mr flagellin (flgJ) showed that the presence of this region is correlated with the expression of the 29 x 10(3) Mr flagellin gene. The next region (region IV), of length approximately 1 to 2 microns, appears to contain the 27.5 x 10(3) Mr flagellin, but at its distal end includes, in gradually increasing amounts, the 25 x 10(3) Mr flagellin. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Except for the hook, there are no morphological features that would otherwise distinguish these regions. A functional flagellum, having the wild-type length and morphology, is assembled by mutant strains deficient in the 29 x 10(3) Mr flagellin and 27.5 x 10(3) Mr flagellin.     

Reconstitution and purification of flagellar filaments from Caulobacter crescentus.

Filaments from isolated flagella of Caulobacter crescentus have been purified by successive dissociation and reconstitution. After the second and third reconstitutions from subunits in 0.8 M sodium citrate, filament preparations contained only two proteins, flagellin A (26,000 daltons) and flagellin B (28,000 daltons). There was some enrichment for flagellin A during reconstitution by this procedure, since isolated flagella contained flagellin A and flagellin B in a ratio of approximately 3.8:1 and filaments after the third reconstitution contained the two proteins in a ratio of 5.0:1.     

Three-dimensional reconstruction of the flagellar hook from Caulobacter crescentus

The structure of the bacterial flagellar hook produced by a mutant of Caulobacter crescentus was studied by electron microscopy, optical diffraction, and digital image processing techniques. The helical surface lattice of the hook is defined by a single, right-handed genetic helix having a pitch of about 23 Å, an axial rise per subunit of 4 Å and an azimuthal angle between subunits of 64·5 °. The lattice is also characterized by intersecting families of 5-start, 6-start and long-pitch 11-start helices. These helical parameters are remarkably similar to those determined for the flagellar filaments from several strains of gram-negative bacteria. The technique of three-dimensional image reconstruction (DeRosier & Klug, 1968) was applied to nine of the better preserved specimens and the diffraction data from five of these were correlated and averaged and used to generate an average three-dimensional model of the hook. The pattern of density modulations in the three-dimensional model is suggestive of an elongated, curved shape for the hook subunit (100 Å × 25 Å × 25 Å). The subunits are situated in the lattice of the polyhook such that their long axes are tilted about 45 ° with respect to the hook axis. The subunits appear to make contact with each other along the 6-start helices at a radius of 80 Å and also along the 11-start helices at a radius of 65 Å. Few structural features are revealed at radii between 15 å and 45 Å and, therefore, we are unable to decide to what extent the hook subunits extend into this region. The most striking characteristic of the model is the presence of deep, broad, continuous 6-start helical grooves extending from an inner radius of about 50 Å to the perimeter of the particle at 105 Å radius. Normal hooks usually appear curved in electron micrographs and sometimes so are the mutant hooks; the prominent 6-start grooves appear to allow for bending with minimal distortion of matter in the outer regions of the hook. A round stain-filled channel about 25 Å in diameter runs down the center of the polyhook. Such a channel supports a model for flagellar assembly in which flagellin subunits travel through the interior of the flagellum to the growing distal end of the filament.     

Caulobacter crescentus flagellar filament has a right-handed helical form

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.     

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.     

Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants

Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.