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.     

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.     

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.     

A three-start helical sheath on the flagellar filament of Caulobacter crescentus.

An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some filaments are surrounded by a set of three windings that form a sheath. We provide evidence that the sheath is composed of subunits having a molecular mass of 24,000 Da. We suggest that the sheath could be composed of protofilaments of flagellin wound around the filament.     

Flagellin redundancy in Caulobacter crescentus and its implications for flagellar filament assembly

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.     

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.     

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.     

Flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking part of the outer domain of flagellin.

We have isolated spontaneous mutants of Salmonella typhimurium which can swim in the presence of antifilament antibodies. The molecular masses of flagellins isolated from these mutants were smaller than that (52 kDa) of wild-type flagellin. Two mutants which produced the smallest flagellins (42 and 41 kDa) were selected, and the domain structures of the flagellins were analyzed by trypsin digestion and then subjected to amino acid sequencing. The two flagellins have deletions at Ala-204 to Lys-292 and Thr-183 to Lys-279, respectively. These deleted parts belong to the outer domain (D3) of flagellin, which is believed to be at the surface of the filament. These mutant filaments aggregated side by side in the presence of salt, resulting in disordered motility.     

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.     

Caulobacter crescentus synthesizes an S-layer-editing metalloprotease possessing a domain sharing sequence similarity with its paracrystalline S-layer protein

Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to "scan" for modifications.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

N-terminal amino acid sequence of the Borrelia burgdorferi flagellin

Abstract The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44–55%), in contrast to the high degree (67–95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.     

Three-dimensional structure of the frozen-hydrated flagellar filament. The left-handed filament of Salmonella typhimurium

Electron micrographs of frozen-hydrated preparations of flagellar filaments of Salmonella typhimurium were used to obtain a three-dimensional reconstruction of the structure. The filaments were obtained from the mutant SJW1660, which produces straight, left-handed filaments. The subunits in this filament are thought to be all in the L-state. The structure consists of a set of 11 longitudinal segmented rods of density that lie at a radius of 70 A. The outermost feature of the filament is a set of knobs of density that project outward from the rods. The interior of the filaments consists of arms that extend inward radially from the segmented rods. The 11 segmented rods and their interconnections are noteworthy because current theories regarding filament structure involve switching of subunits between the L and R states co-operatively along the directions of the rods.     

Refolding and biophysical characterization of the Caulobacter crescentus copper resistance protein,PcoB: An outer membrane protein containing an intrinsically disordered domain

Copper cations play fundamental roles in biological systems, such as protein folding and stabilization, or enzymatic reactions. Although copper is essential to the cell, it can become cytotoxic if present in too high concentration. Organisms have therefore developed specific regulation mechanisms towards copper. This is the case of the Pco system present in the bacterium Caulobacter crescentus, which is composed of two proteins: a soluble periplasmic protein PcoA and an outer membrane protein PcoB. PcoA oxidizes Cu⁺ to Cu²⁺, whereas PcoB is thought to be an efflux pump for Cu²⁺. While the PcoA protein has already been studied, very little is known about the structure and function of PcoB. In the present work, PcoB has been overexpressed in high yield in E. coli strains and successfully refolded by the SDS-cosolvent method. Binding to divalent cations has also been studied using several spectroscopic techniques. In addition, a three-dimensional structure model of PcoB, experimentally supported by circular dichroism, has been constructed, showing a β-barrel conformation with a N-terminal disordered chain. This peculiar intrinsic disorder property has also been confirmed by various bioinformatic tools.     

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.