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.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.     

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.     

Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin

The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.     

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins.The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.     

Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.     

Evidence that subcellular flagellin pools in Caulobacter crescentus are precursors in flagellum assembly

To study the assembly of the Caulobacter crescentus flagellar filament, we have devised a fractionation protocol that separates the cellular flagellin into three compartments: soluble, membrane, and assembled. Radioactive labeling in pulse-chase and pulse-labeling experiments has demonstrated for the first time that both soluble and membrane-associated flagellin pools are precursors in the assembly of the flagellar filament. The results of these experiments also indicate that flagellar filament assembly occurs via the translocation of newly synthesized flagellins from the soluble pool to the membrane pool to the assembled flagellar filaments. It is not possible to conclude whether the soluble flagellin fraction is synthesized cytoplasmically or as a loosely associated membrane intermediate which is released during lysis. It is clear, however, that the soluble and membrane flagellins are in physically and functionally distinct pools. The implications of these findings for the study of protein secretion from cells and the invariant targeting of flagellar proteins to the stalk-distal pole of the dividing cell during flagellum morphogenesis are discussed.     

Variation in bacterial flagellins: from sequence to structure

Bacterial motility relies chiefly on the rotation of a molecular propeller, the flagellar filament, which is constructed from the protein flagellin. Here, flagellin sequence conservation and diversity is examined in the light of the recently determined flagellar filament structure. As expected, the surface-exposed domains are not conserved. However, the sequences that mediate filament assembly show remarkable conservation, which indicates that all bacterial flagellins are likely to pack into filaments in a similar manner. Flagellins provide a striking illustration of the twin evolutionary themes of conservation and variability.     

The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins

In order to circumvent problems associated with direct chemical analysis of the phase-1 flagellar filament protein (flagellin) of Salmonella typhimurium, the covalent structure was determined by recombinant DNA procedures. The corresponding structural gene (H-1i) was cloned into plasmid pBR322 in a 4.3-kilobase fragment produced by EcoRI digestion of chromosomal DNA, and the nucleotide sequence of the region specifying the flagellar protein was determined. Comparison of the data obtained with the limited information available for other salmonellar flagellins supported the concept that both ends of the molecule are conserved in this genus. Additionally, a conservation of base sequence in the region of H-1 genes coding for the N-terminal end of flagellins was apparent, suggesting that this area may have an additional regulatory role. The i flagellin was found to be unrelated to proteins in the NBRF data base with the exception of other flagellins. The three flagellins which have been sequenced to date (those produced by Bacillus subtilis, Caulobacter crescentis, and phase-1 S. typhimurium) show homologies in amino acid sequence at both the N-terminal and C-terminal ends despite large differences in their total molecular weight, and comparison suggests that B. subtilis and Salmonella are more closely related to each other than either is to Caulobacter.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

The structure of the helically perturbed flagellar filament of Pseudomonas rhodos: implications for the absence of the outer domain in other complex flagellins and for the flexibility of the radial spokes

Bacterial flagella, the organelles of motility, are commonly divided into two classes: 'plain' and 'complex'. The complex filaments are pairwise, helically perturbed forms of the plain filaments and have been reported to occur only in Rhizobium and Pseudomonas. Previously, we reconstructed and analysed the structure of the complex filaments of Rhizobium lupini H13-3 and determined their unique symmetry and origin of the perturbations (Trachtenberg et al., 1986, J Mol Biol 190: 569-576; 1987, 195: 603-620; 1998, 276: 759-773; Cohen-Krausz and Trachtenberg, 1998, J Struct Biol 122: 267-282). Here, we analyse the structure of the flagellar filament of the other known complex filament, that of Pseudomonas rhodos, as reconstructed from electron microscope images. Compared with the filament of R. lupini, the filament of P. rhodos is more flexible, as implied from high-intensity darkfield light microscopy and, although constructed from flagellins of higher molecular weights (59 versus 41 kDa), has similar symmetry. Using cryonegative stained specimens and low-dose, field emission electron microscopy, we reconstructed and averaged 158 filaments each containing 170 statistically significant layer lines. The three-dimensional density maps of P. rhodos clearly suggest, when compared with those of R. lupini and the right-handed Salmonella typhimurium SJW1655, that R. lupini is missing the outer flagellin domain (D3), that the interior of the complex filament is rather similar to that of the plain filament and that the radial spokes (connecting domains D0 and D1), present in individual density maps, average out because of their variability and implied flexibility. Extending the three-start grooves and ridges on the propeller's surface, in the form of an Archimedean screw, may further improve the motility of the cell in viscous environments.     

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.     

Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

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.     

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.     

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.     

Conserved N-terminal sequences in the flagellins of archaebacteria

Methanococcus voltae produces two flagellins of molecular weight 31,000 and 33,000. Amino acid analysis as well as peptide mapping with cyanogen bromide, chymotrypsin and Staphylococcus aureus V-8 protease indicates that the two flagellins are distinct. N-terminal sequencing of the 31,000 Mc. voltae flagellin as well as the 24,000 and 25,000 molecular weight flagellins of Methanospirillum hungatei GP1 shows an extensive homology with the reported N-terminus of the flagellins from Halobacterium halobium, deduced from the nucleotide sequence of the cloned genes. However, the N-termini of all three sequenced methanogen flagellins lack a terminal methionine and start at position 13 from the N-terminus of H. halobium flagellins. This initial 12 amino acid stretch may be a leader peptide which is subsequently cleaved to generate the mature flagellin, which could suggest flagellar assembly in archaebacteria occurs by a mechanism distinct from that in eubacteria. The high degree of conservation of the N-terminus of the flagellins from Mc. voltae, Msp. hungatei and H. halobium suggests an important role for this sequence, and that the archaebacteria share a common mechanism for flagellar biosynthesis.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998