MotD of Sinorhizobium meliloti and Related -Proteobacteria Is the Flagellar-Hook-Length Regulator and Therefore Reassigned as FliK
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《细菌学杂志》
Institut fur Biochemie, Genetik und Mikrobiologie, Universitt Regensburg, D-93040 Regensburg, Germany,Department Chemie, Technische Universitt Munchen, D-85747 Garching, Germany
ABSTRACT
The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.
INTRODUCTION
Free-swimming bacteria modulate their swimming patterns in response to environmental changes. They are propelled by helical flagellar filaments connected to the basal body by a flexible hook and driven by a flagellar rotary motor. The basal body consists of a central rod and four coaxial structures, the L ring in the outer membrane, the P ring in the peptidoglycan layer, the MS ring in the cytoplasmic membrane, and the bell-shaped C ring in the cytoplasm. In enterobacterial species like Escherichia coli and Salmonella enterica serovar Typhimurium, temporal and structural assembly of the flagellar apparatus is strictly regulated by a hierarchy of transcriptional controls (1, 2, 22, 23, 27, 59). The MS ring complex appears at the very beginning of flagellar assembly. Then the C ring and the export apparatus, composed of six transmembrane proteins and two cytoplasmic proteins, are assembled on the cytoplasmic side of the M ring. The active and selective export of flagellar proteins starts with five proteins comprising the rod, followed by the hook, which is composed of FlgE subunits (13, 21, 34). Upon completion of the basal structure and the hook, genes necessary for the flagellar filament and the energizing motility complexes are expressed. At this assembly point, the flagellar protein export apparatus switches specificity from rod/hook-type substrates to filament-type substrates. Whereas the length of the flagellar filament is not strictly regulated, the length of the hook is well defined by the hook length control protein, FliK (19). The accurately defined length of the flexible hook is essential for proper formation of bundles of the flagellar filaments and therefore for efficient propulsion of the cell (14). Once the hook reaches its mature length (55 nm), the flagellar export protein FlhB, together with FliK, mediates the switching of export specificity (32, 59). Defects in fliK or flhB prevent this switch, resulting in abnormally long hooks, called polyhooks (15).
The behavioral scheme of the nitrogen-fixing plant symbiont Sinorhizobium meliloti, a member of the -subgroup of the Proteobacteria (39), differs from the enterobacterial (-subgroup) behavioral scheme in the filament structure, the mode of flagellar rotation, signal processing, and gene regulation (52). The rigid "complex" flagellar filaments consist of four related flagellin subunits, and interflagellin bonds lock the filaments in right-handedness (6, 11, 50). Hence, S. meliloti cells are propelled by exclusively clockwise rotating flagella, and swimming cells respond to tactic stimuli by modulating their rotary speed (3, 49). This mode of motility control has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, which are present in addition to the ubiquitous MotA/MotB energizing proton channel. MotC binds to the periplasmic portion of MotB and requires a specific chaperone, MotE, for proper folding and stability (7). Platzer et al. (42) described the presence of an additional motility protein, MotD. These authors determined that the proper assignment was Mot on the basis of an in-frame deletion introduced into motD that resulted in paralyzed cells with intact flagella. The arrangement of chemotaxis (che), flagellar (fla, flg, flh, and fli), motility (mot), and regulatory (visN, visR, and rem) genes differs from the enterobacterial pattern in that all known 51 genes are clustered in one contiguous 56-kb chromosomal region, the flagellar regulon (9, 45, 57). Most flagellar gene homologues were identified in the original S. meliloti genome annotation. However, a small number of genes remained unassigned due to sequence divergence from the E. coli-Salmonella paradigm. Indeed, homology searches revealed great variability in the regulatory flagellar genes, as well as the absence of a few specific flagellar chaperones in the genomes of a representative set of phylogenetically diverse bacterial species (40). In S. meliloti, the global enterobacterial transcriptional activator, FlhD2C2, is functionally replaced by the LuxR- and OmpR-like transcriptional regulators VisNR and Rem (9, 45, 57). In addition, several specific flagellar chaperones and export proteins have not been identified (or are missing) in S. meliloti. Among these unrecognized components are (i) FliD, the filament cap protein; (ii) FliS, the chaperone for flagellins; and (iii) FliK, the hook length control protein. Based on homology of a conserved motif in the otherwise unrelated sequence, we identified the gene coding for the hook length regulator, FliK, in the S. meliloti genome. Here we provide bioinformatic, genetic, physiological, biochemical, and structural proof for reassignment of MotD, a protein previously thought to function as a cytoplasmic motility protein (42), as a hook length regulator, FliK. In conformity with the nomenclature, we proposed that MotD from S. meliloti and other related -proteobacteria should be renamed FliK.
MATERIALS AND METHODS
Bacterial strains and plasmids. Derivatives of E. coli K-12, S. enterica serovar Typhimurium, and S. meliloti MV II-1 (18) and the plasmids used are listed in Table 1.
Media and growth conditions. E. coli strains were grown in Luria broth (26) at 37°C. S. meliloti strains were grown in TYC (0.5% tryptone, 0.3% yeast extract, 0.13% CaCl2 · 6H2O [pH 7.0]) at 30°C (42). Motile cells prepared for Western blot analysis were grown for 2 days in TYC, diluted 1:100 in 10 ml RB minimal medium, layered on Bromfield agar plates (55), and incubated at 30°C for 14 h to obtain an optical density at 600 nm (OD600) of 0.2 to 0.5. Motile cells prepared for -galactosidase assays were grown for 2 days in TYC, diluted in 15 ml of RB minimal medium to obtain an OD600 of 0.05, layered on Bromfield agar plates, and incubated on a slowly rotating platform at 30°C for 16 h to obtain an OD600 of 0.2 to 0.5. Antibiotics were used at the following final concentrations: for E. coli, 100 mg/liter ampicillin, 50 mg/liter kanamycin, and 10 mg/liter tetracycline; and for S. meliloti, 120 mg/liter neomycin, 600 mg/liter streptomycin, and 10 mg/liter tetracycline.
Motility assay. Swarm plates containing Bromfield medium and 0.3% Bacto agar were inoculated with 3-μl droplets of the test culture and incubated at 30°C for 3 days. Second-site suppressors (pseudorevertants) of the fliK deletion were isolated on swarm plates incubated at 30°C for 10 to 15 days in a humidity chamber. Free-swimming tracks were analyzed by computerized motion analysis using a Hobson BacTracker (Hobson Tracking Systems, Ltd., Sheffield, England) as previously described (56).
Genetic manipulations. Mutants of S. meliloti (Table 1) were generated in vitro by overlap extension PCR as described by Higuchi (12). Constructs containing the mutation were cloned into the mobilizable suicide vector pK18mobsacB, used to transform E. coli S17-1, and conjugally transferred to S. meliloti by filter mating using the method of Simon et al. (53). Allelic replacement was achieved by sequential selection with neomycin and 10% sucrose as described previously (55). Confirmation of allelic replacement and elimination of the vector was obtained by performing gene-specific primer PCR, Southern blotting, and DNA sequencing.
DNA methods. S. meliloti DNA was isolated and purified as described previously (55). Plasmid DNA was purified with NucleoSpin (Macherey Nagel, Duren, Germany), and DNA fragments or PCR products were purified from agarose gels using a QiaEx DNA purification kit (QIAGEN, Hilden, Germany). PCR amplification of chromosomal DNA and Southern blotting were carried out by using previously described protocols (42, 57).
Flagellar filament isolation. Flagella were prepared as described previously (50). Briefly, flagella were detached from motile cells (from 250-ml cultures) by agitation in a Braun MX32 mixer (Braun, Melsungen, Germany) at maximum power for 20 s, separated from cells by centrifugation at 8,000 x g for 8 min, and further purified by serial centrifugation at 15,000 x g for 15 min and at 25,000 x g for 1 h. Purified flagella that were sedimented by centrifugation at 87,000 x g for 2 h were resuspended in 100 μl of RB minimal medium.
Expression and purification of FliK. Recombinant FliK protein was overproduced from plasmid pRU2315 in E. coli ER2566 (Table 1). Cells were grown at 37°C in Luria broth containing 100 mg/liter ampicillin to an OD600 of 0.7, and gene expression was induced with 0.3 mM isopropyl--D-thiogalactopyranoside (IPTG). Cultivation was continued for 16 h at 16°C until harvest. Cells from 2-liter cultures were resuspended in 20 ml column buffer (20 mM HEPES [pH 8.0], 500 mM NaCl, 1 mM EDTA) containing 10 μg/ml DNase I and were lysed by two passages through a French press at 20,000 lb/in2. Cell membranes and debris were removed by centrifugation at 48,000 x g and 4°C for 30 min. The supernatant was loaded onto a chitin agarose (New England Biolabs, Beverly, MA) column (2.6 by 5.0 cm) equilibrated with column buffer and washed thoroughly with 5 bed volumes of column buffer at 4°C. Intein-mediated cleavage at the intein cleavage site was elicited by equilibration of the column with 2 bed volumes of cleavage buffer (50 mM dithiothreitol in column buffer) and further incubation at 4°C for 16 h. FliK was eluted during equilibration with cleavage buffer and after incubation for 16 h. Protein was eluted with column buffer, and pooled fractions were dialyzed against 1 mM EDTA-20 mM Tris-HCl (pH 7.5) and concentrated by ultrafiltration on a regenerated cellulose membrane (30-kDa cutoff). Samples were subjected to fast protein liquid ion-exchange chromatography (MonoQ HR5/5; Amersham Pharmacia, Freiburg, Germany) equilibrated with 20 mM Tris-HCl-1 mM EDTA (pH 7.5) (buffer A) at a flow rate of 0.5 ml/min. FliK eluted with a linear gradient of buffer A and buffer A containing 1 M NaCl at an NaCl concentration of 0.17 M.
Analytical size exclusion chromatography. Size exclusion high-performance liquid chromatography was performed using a Phenomenex BioSep-SEC-S3000 column (Phenomenex, Aschaffenburg, Germany) with a separation range of 10 to 1,500 kDa. FliK was subjected to 50-μl portions at a concentration of 0.5 mg/ml. The column was equilibrated and developed with 150 mM KCl-40 mM HEPES-KOH (pH 7.4) at a flow rate of 0.5 ml/min. Protein was detected by fluorescence at 330 nm after excitation at 280 nm using an FP 920 fluorescence detector (Jasco, Groumstadt, Germany). For calibration, thyroglobulin (669 kDa), catalase (232 kDa), ovalbumin (48 kDa) and chymotrypsin (25 kDa) were used as molecular mass markers (Sigma, St. Louis, Mo.).
Analytical ultracentrifugation. The native molecular weight of FliK was determined using a Beckman XL-I analytical ultracentrifuge and a Beckman Ti-60 rotor equipped with a UV/Vis and interference detection unit. Centrifugation was performed at 4°C and 13,000 rpm until the sedimentation equilibrium was reached. Complete protease inhibitors (Roche Applied Science) were added to avoid proteolytic degradation. The protein concentration was 0.4 mg/ml in a buffer containing 20 mM Tris-HCl and 1 mM EDTA (pH 7.5). For data analysis we used the one-species model included in the Origin software package available from Beckman.
Limited proteolysis and mass spectrometry. Limited proteolysis of FliK by trypsin (Promega, Madison, WI) was carried out in buffer containing 20 mM Tris-HCl (pH 7.5) at 25°C. Protein was incubated with trypsin at a protein-to-protease ratio of 600:1 (wt/wt), and samples were collected at 0.5, 1, 2, 5, and 10 min. The digestion products were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) or were subsequently purified using C4 Zip Tips (QIAGEN, Hilden, Germany) and analyzed with a Biflex-II mass spectrometer (Bruker Daltonik, Bremen, Germany) operated in the linear mode to determine the masses of the digestion products. To correlate the fragment masses determined with the different parts of the protein, the digestion products were incubated with 0.01% sodium desoxycholate on ice for 20 min and precipitated on ice for 30 min in the presence of 8% trichloroacetic acid. After centrifugation at 4°C and 18,000 x g for 30 min, proteins were analyzed on 15% acrylamide gels stained with Coomassie brilliant blue. Single bands were excised, and total in-gel digestion of the protein with trypsin was performed by using the protocol of Schfer et al. (48). Samples used for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry fingerprinting were prepared using C18 ZipTips (QIAGEN, Hilden, Germany) following the manufacturer's protocol. Data analysis was performed using Mascot (Matrix Science, London, United Kingdom).
Immunoblotting. Polyclonal antibodies raised against purified S. meliloti FliK and flagellar filaments were isolated from whole serum by affinity purification using 100 μg of protein and previously described protocols (50). Culture supernatants (filtered through a 0.2-μm cellulose acetate syringe filter) and whole-cell extracts were separated in 10% acrylamide gels, transferred to nitrocellulose, and probed using purified anti-S. meliloti FliK polyclonal antibody at a 1:100 dilution and anti-S. meliloti filament polyclonal antibody at a 1:500 dilution (50). Quantitative immunoblotting was performed and immunoblots were analyzed as described previously (7, 51). One-milliliter aliquots of RU11/001 (wild type) were harvested by centrifugation, resuspended in 30 μl SDS sample buffer, and heated to 100°C for 10 min. Samples of this mixture were stored at –20°C. Control samples were prepared by adding 0.5 to 10 ng of purified FliK to an extract prepared from a 1-ml aliquot of strain RU11/212 (fliK). Two samples and six standards (at six different concentrations) were applied to each gel. Gel electrophoresis, blotting, and probing were done as described above. Hyperfilm ECL (Amersham Pharmacia) was used for detection with a series of exposures. Films were scanned in the presence of a calibrated Step Tablet (Eastman Kodak) by using an Epson Perfection 1640SU and Corel Photo-Paint 10 software. Scans were analyzed using NIH Image 1.60 and Origin 5.0. Flagellin expression was semiquantified by comparing the flagellin band intensities for mutant strains with the wild-type band intensity using NIH Image 1.60.
Protein and -galactosidase assays. Protein concentrations were determined by the Bradford microassay (Bio-Rad, Munich, Germany). For quantitative immunoblotting, the concentration of purified FliK was determined from the Ala and Phe content by amino acid analysis performed by the Analytical Research and Services, University of Bern (Switzerland). Cultures of S. meliloti containing lacZ fusions were sampled, diluted in Z buffer (30) to obtain an OD600 of 0.2, permeabilized with 1 drop of toluene, and assayed for -galactosidase activity by the method of Miller (30).
Electron microscopy. Samples were applied to carbon-coated copper grids, negatively stained with aqueous 3% uranyl acetate (pH 4.5), and examined with a CM12 transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at 120 keV. The magnification was calibrated using catalase crystals negatively stained with uranyl acetate (44). The images were digitally recorded using a slow-scan charge-coupled device camera connected to a PC running TVIPS software (TVIPS GmbH, Gauting, Germany).
RESULTS
S. meliloti FliK (formerly MotD) contains a conserved Flg hook motif in the carboxy-terminal moiety. The fliK (motD, Smc03044) (9) gene maps in the motility (mot) operon of S. meliloti downstream of motB (encoding a channel protein) and motC (encoding a periplasmic motility protein) (42). This genetic linkage, together with the paralyzed phenotype of a knockout mutant, led to its assignment as a novel motility gene in S. meliloti (42). Here, we performed a bioinformatic analysis of the derived 475-residue polypeptide sequence (49 kDa) and of its orthologues from Agrobacterium tumefaciens, Brucella melitensis, Mesorhizobium loti, and Rhizobium lupini. These orthologues are positioned in an identical genetic linkage and in a very similar operon arrangement. The derived polypeptide sequences of the four orthologues exhibit only low levels of homology with the S. meliloti FliK sequence (ca. 37% similar residues and 29% identical residues). All five proteins contain a Flg hook motif (PF02120) in the carboxy-terminal region, as revealed by Pfam (protein families database of alignments) analysis (Fig. 1) (5, 29). The 124-residue motif is typical for flagellar-hook-length control proteins and is designated FliK, and it has been assigned to more than 90 bacterial species, including 11 species belonging to the -subgroup of the Proteobacteria. The homology in this C-terminal region is significantly higher (58% similar residues and 45% identical residues on average) than that in the N-terminal two-thirds of the sequence. According to the significant homology with the Flg hook motif, the aligned proteins can be newly assigned as FliK. Below, we provide experimental evidence for the function of S. meliloti FliK in controlling the length of the flagellar hook.
Deletion of fliK causes the formation of polyhooks. If FliK controls the length of the S. meliloti flagellar hook, mutations in fliK are expected to result in hooks whose length is uncontrolled, so-called polyhooks (15). We therefore isolated flagellar structures of an fliK knockout mutant (RU11/212, having a 649-bp deletion in fliK) generated by allelic exchange (47) and the S. meliloti wild-type strain (RU11/001) and examined the negatively stained samples by electron microscopy (Fig. 2). In contrast to the wild type, which produced normal flagella and a curved hook that was ca. 60 nm long (Fig. 2D), cells of the fliK deletion mutant lacked typical flagellar filaments. The lack of flagellar filaments occurred concurrently with the nonmotile phenotype of mutant strain RU11/212, as observed on swarm plates (Fig. 3A) (42). Instead, cells of the mutant strain produced hooks which were four to eight times longer than wild-type hooks (Fig. 2A to C). Hence, deletion of the fliK gene resulted in a failure to terminate hook assembly and initiate filament assembly.
Mutations in flhB suppress the fliK polyhook phenotype. To our surprise, a closer examination of the fliK deletion strain by phase-contrast microscopy revealed that a small fraction of cells (less than 0.1%) were motile. As described previously for Salmonella (58), these cells could be divided in two classes: (i) "leaky" mutants producing swimming cells with normal hooks and (ii) suppressor mutants having a mutation which permitted the formation of functional flagellar filaments in the absence of an fliK gene product. We isolated pseudorevertants from a fliK strain (RU11/212) after prolonged incubation on swarm plates which could swim but swam with less efficiency than the wild type (Fig. 3A). As described previously for enterobacterial fliK polyhook mutants, pseudorevertants fall into two classes: intragenic (mutations in fliK) and extragenic (mutations in flhB) (15, 23). The 649-bp deletion in strain RU11/212 resulted in the loss of amino acid residues 2 to 218 of FliK; 257 codons were still present in the genome. Gain-of-function mutations that produced frame-shifted sequences have been described for the truncated Salmonella gene (59). We therefore assayed the isolated pseudorevertants for expression of FliK in immunoblots using anti-FliK antibody. None of the nine strains expressed FliK at a detectable level, which ruled out the possible occurrence of such intragenic suppressors (data not shown). All known extragenic suppressor mutations of Salmonella fliK polyhook mutants map to the cytoplasmic domain of the integral membrane protein FlhB (15, 59). We amplified this part of the flhB gene from the nine mutant strains by PCR, and the amplified fragments were sequenced using internal primers. All suppressing mutations indeed mapped in a 68-amino-acid region of the cytoplasmic FlhB domain and were caused by single-base substitutions (Table 1). One residue, Ala at position 300, represented a hot spot of mutation, with four independently isolated changes to Thr or Val. Ala-300 and Gly-295 are highly conserved residues encoded by the flhB gene (23, 59). The other five amino acid substitutions identified (A278V, G295E, L320F, S323P, and L345P) (Tables 1 and 2) are novel suppressor mutations for the S. meliloti polyhook mutant (RU11/212).
Flagellar structures were isolated from these strains and inspected by electron microscopy (Fig. 2E and F). For all strains, as illustrated for RU13/202 (A300T), the hook structures of the flagella were polyhooks but had filaments at the distal end, known as polyhook filament (phf) structures (15, 41). Hence, the suppressor mutations can suppress the fliK deletion for filament formation but not for determination of the hook length. In addition, immunoblots comparing the levels of flagellin expression in the wild type and the suppressor mutants revealed that the levels in the phf strains were 30 to 60% of the wild-type levels (Table 2). When the swarming proficiencies were assessed, the phf mutants formed swarms that were much smaller than those of the wild-type strain, with swarm sizes ranging from 17% to 32% of the wild-type sizes (Table 2), as shown for RU13/202 (A300T) as a typical example in Fig. 3A. There was a moderate positive correlation between the efficiency of swarming and the amount of flagellin protein expressed. An additional motility assay was used, which monitored and averaged the free-swimming speeds of cell populations. All mutants exhibited comparable swimming speeds that were 60% of the wild-type speed (Table 2). We therefore concluded that extragenic suppressor mutants of the fliK deletion strain restored filament assembly and hence swimming proficiency, albeit in the presence of polyhook structures.
Defined hook length is essential for proper motility. Rhizobial cells do not exhibit a strong relationship between flagellar filament length and swimming speed (49). In contrast, in phf mutants there was a remarkable reduction in the swimming speed despite significant expression of flagellin protein (Table 2 and Fig. 2E and F). We analyzed the swimming behavior of phf mutants in greater detail. The "smooth-swimming rate" is defined as the ratio of the straight-line velocity (linear distance traveled per second between endpoints of a given track) to the instantaneous velocity (velocity measured at every point in the track and averaged for every swimming track). This ratio reflects the suppression of directional changes in response to positive stimulation and is a corollary of the suppression of tumbles in the chemotaxis of enterobacteria (55). Interestingly, all phf mutants exhibited a much lower smooth-swimming rate (average, 0.54) than nonstimulated S. meliloti wild-type cells exhibited (0.84) and changed their swimming directions more frequently than the wild type (Table 2). This behavior was nicely illustrated by tracking the swimming path over time. While the S. meliloti wild type (RU11/001) (Fig. 3B) swam in wide, slightly curved tracks, a phf mutant strain (RU13/202) (Fig. 3D) was distinguished by swimming in tiny spirals. The extended polyhooks of the mutants (Fig. 2E and F) apparently interfered with proper formation of a flagellar bundle which pushed the cells straight forward. Regulation of the appropriate hook length is therefore necessary for maximal motility.
FliK is necessary for flagellin secretion. Electron micrographs of the fliK deletion mutant revealed the presence of polyhooks concurrent with the lack of typical flagellar filaments (Fig. 2E and F). The lack of filament genesis was also reflected by the low (<20%) expression of flagellins, as analyzed by Western blotting of whole-cell extracts (Fig. 4 and Table 2). We asked whether the failure of filament assembly in the fliK deletion strain is consistent with a lack of flagellin monomer secretion through the export apparatus. Cell culture supernatants of the wild type and the fliK mutant strain were isolated, sheared flagellar filaments were removed by filtration, and samples were analyzed by Western blotting. Supernatants of wild-type cultures contained considerable amounts of flagellins, whereas no flagellin proteins were present in supernatants of the mutant strain. In the absence of FliK, export of flagellin monomers through the flagellar export apparatus was prevented.
We have shown previously that transcription of flaA, the gene encoding the principal flagellin of the S. meliloti complex filament, is down-regulated in abortive fla mutants (50). Is the blockage of filament assembly in an fliK deletion mutant passed on to transcriptional control of the flagellin genes We compared the transcriptional activity of all four fla genes (flaA to flaD) in fliK (RU11/212) with that in the wild type (Table 3). Strong transcriptional control was exerted on the flaA promoter activity, which was reduced by more than 70%, when filament assembly could not be initiated. The transcription of the three secondary flagellin genes, flaB, flaC, and flaD, was weakly affected, with an average reduction of 30%. Hence, transcription of the principal flagellin gene, flaA, was effectively controlled at the control switch between hook and filament assembly. Flagellin genes belong to the late (class III) genes in the regulatory cascade, together with genes for the chemotaxis machinery (54). We wondered whether FliK also exerts control over the members of the class III gene group, all of which are in the chemotaxis operon. Indeed, the activity of the che promoter was reduced by more than 80% (Table 3). In conclusion, fliK can be integrated as a class IIA gene in the hierarchical cascade of flagellar gene expression, regulating the expression of all class III genes.
FliK is secreted into the culture medium. The enterobacterial counterpart of FliK contains an export signal in its N-terminal region (amino acids 1 to 40) (31). To investigate whether S. meliloti FliK is restricted to the cytoplasm, we analyzed supernatants of wild-type cultures for the presence of FliK. Using anti-FliK antibody, FliK could be detected in whole-cell extracts and the culture supernatant equally well (Fig. 5), showing that there was secretion of FliK into the culture medium. A quantitative Western blot analysis showed that there were 74 ± 28 FliK molecules per cell. The question of whether FliK is actually secreted through the flagellar export apparatus was approached by analyzing a mutant defective in flagellar export. Deletion of fliF, the gene coding for the MS ring, blocks assembly of the export apparatus at an early stage. An FliK band was detected in whole-cell extracts of an fliF deletion strain (fliF, RU11/807), and the intensity was identical to that in wild-type extracts. However, this band was missing in supernatants of the fliF cell culture. We concluded that FliK is secreted through the flagellar export system into the culture medium. Therefore, similar to Salmonella FliK (31), it is an export substrate recognized by the flagellum-specific export apparatus.
FliK is an elongated monomeric molecule. Most properties of FliK determined so far are analogous to those of its enterobacterial counterpart. We examined the question of whether the two proteins share any structural properties. FliK was overexpressed in E. coli and purified using affinity and anion-exchange chromatography. To determine the molecular mass of FliK independent of its shape, we carried out sedimentation equilibrium measurements at a protein concentration of 0.4 mg ml–1. Using the manufacturer's software, a single-species model produced the best fit and calculated a native molecular mass of 50.0 ± 1.5 kDa, which agrees well with the deduced molecular mass, 48.8 kDa (Fig. 6). Hence, FliK exists as a monomer in solution.
To analyze the hydrodynamic properties of FliK, we performed size exclusion chromatography. Purified FliK eluted after 19 min from a BioSep-SEC-S3000 column, at an apparent molecular mass of 150 kDa (Fig. 7). The observation that FliK eluted from the size exclusion column much earlier than expected for a 50-kDa (Fig. 6) monomer suggests that FliK is an elongated molecule. A similar behavior was described for Salmonella FliK (33).
Flg hook motif is a compactly folded domain. To study the possible domain organization of FliK, we carried out limited proteolysis of FliK by using the protease trypsin, which cleaved C terminal of Arg and Lys residues. The time course of proteolysis, as monitored by SDS-PAGE, is shown in Fig. 8A. Seven metastable fragments (FliKT1 to FliKT7) and two stable fragments (FliKT8 and FliKT9) were produced. The cleaved products were analyzed by MALDI-TOF mass spectrometry, and the identities of the fragments were as follows: FliKT1 was FliK24-467, FliKT2 was FliK105-467, FliKT3 was FliK105-429, FliKT4 was FliK146-467, FliKT5 was FliK105-391, FliKT6 wasFliK217-467, FliKT7 was FliK217-429, FliKT8 was FliK217-391, andFliKT9 was FliK245-391 (Fig. 8B). All the calculated masses were in excellent agreement with the experimental values. This indicated that five trypsin-sensitive sites are located in the N-terminal region of FliK and three trypsin-sensitive sites are located in the C-terminal region of FliK. The only two stable fragments maintained after limited proteolysis contained the Flg hook motif (amino acids 297 to 402). The Flg hook region apparently comprises a compactly folded, globular domain, rendering it insensitive to protease digestion.
DISCUSSION
Here we describe reassignment of a gene, motD, previously described as a gene encoding a cytoplasmic motility protein having an unknown function, as fliK, the gene coding for the hook length regulator. The physiological significance of hook length control is obvious in mutants with extraordinarily long hooks (polyhooks). In this case, the presence of a filament is not sufficient for proper propulsion. Instead, such mutants swim in small, aberrant spirals due to (i) their inability to form a functional flagellar bundle and (ii) their inability to produce enough thrust to propel the cell forward (Fig. 3D). Hence, a hook length control mechanism is expected to be present in all bacteria that have a flagellar hook filament structure. The primary function of FliK, together with a transmembrane protein of the export apparatus, FlhB, is to regulate the length of the flagellar hook. However, the occurrence of polyhook mutants has been reported for only a small number of bacterial species, including the soil bacterium Rhizobium lupini (10, 25, 35, 36, 46).
In the past, reliable annotation of fliK in the flagellar loci of distantly related bacteria was hampered by the low level of similarity to the enterobacterial counterpart (40). The wealth of sequence data, together with refinement of search engines, led to identification of S. meliloti FliK. Despite significant divergence (24% overall identity), an Flg hook domain in the C-terminal region, centered on a highly conserved Pro-X-X-Leu-Gly motif (Fig. 1) (5, 29), was identified in the former motility protein MotD. Our results corroborated the bioinformatic approach results by experimentally defining the function of the protein as a flagellar-hook-length regulator, FliK, as follows: (i) deletion of fliK resulted in a failure to terminate hook assembly and initiate filament assembly; (ii) FliK was secreted through the flagellar export apparatus; (iii) the number of FliK molecules per cell (74 ± 28) correlated with the number reported for Salmonella FliK (37); and (iv) the fliK defect could be suppressed by extragenic mutations in the cytoplasmic domain of FlhB. The frequent occurrence of extragenic suppressor mutations resulting in a polyhook filament phenotype might explain the previous misassignment of FliK as a new motility protein. Platzer et al. (42) described fliK knockout mutant cells as cells that were paralyzed and nonswarming but had normal flagellation as determined by electron microscopy. The second observation is obviously not true. We can only assume that the analysis was not performed under appropriate conditions and/or that the data were misinterpreted due to the high frequency of leaky mutants and suppressor mutants. These authors apparently failed to recognize the concurrent presence of polyhooks, since elongated hooks are very difficult to discern in whole-cell preparations. They named the new gene motD.
The C-terminal domain of Salmonella FliK is directly involved in substrate specificity switching of the flagellar export apparatus (59). Data reported by Minamino and colleagues (33) suggest that the N-terminal domain of FliK receives the length information from the growing hook structure and activates the C-terminal domain of FliK to bind to FlhB. A conformational change in FlhB then triggers the substrate switch of the flagellar export machinery (8, 32, 33). In contrast to the two-domain structure (receiver and activator) of Salmonella FliK, we identified only one stable, compactly folded region comprising the Flg hook motif of S. meliloti FliK (Fig. 8). The C-terminal region of Salmonella FliK is rich in glutamine residues (16 mol%) (19). Accumulation of glutamine residues has also been noted for S. meliloti FliK, although it is less pronounced (9 mol%). In contrast, the S. meliloti homologue lacks the characteristic accumulation of proline residues in the central part of the protein (residues 180 to 205) (19, 33), underscoring the differences between the two proteins. Minamino and colleagues (33) suggested that these proline residues ensure flexibility of the hinge region connecting the two compactly folded FliK domains. Since S. meliloti FliK consists of only one domain, there is no requirement for such a flexible hinge region. Therefore, accumulation of proline residues in the central part of the protein is pointless.
The complex flagellar filaments of S. meliloti and R. lupini are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their stability, which prevents a switch of handedness (6, 50). It is conceivable that filaments composed of heterodimers formed by four and three different flagellin subunits, respectively, place different demands on the flagellar export channel than single-subunit filaments place. This possibility is supported by the fact that S. meliloti FliK, when expressed from plasmid pRU2930 (a derivative of pQE60) (Table 1) and introduced into E. coli HCB7 and Salmonella strain SJW880, is not able to complement the fliK polyhook phenotype. Furthermore, S. meliloti FliK is not secreted by either of the two enterobacterial strains (data not shown).
There are two hypothetical models for the function of FliK after completion of the hook structure, the quantized measuring cup model proposed by Aizawa and colleagues (28) and the molecular ruler model (17, 33). In the first model, the hook proteins accumulate to fill the C ring and are then secreted to form a hook having a precise length. In the ruler model, the N- and C-terminal ends of FliK are anchored to the tip of the hook (strictly speaking, to the hook cap FlgD) and to the basal body, respectively. When FliK is fully stretched because the hook has grown to its full length, it transmits information to FlhB, switching the secretion specificity. Preliminary binding studies using affinity chromatography and Far Western blot analysis revealed an interaction of S. meliloti FliK with the C-ring component FliM, which would be more in agreement with the measuring cup model but would not contradict the ruler model (data not shown). Interaction of FliK with the C-ring proteins might shed light on the mechanism of hook length control and will be the topic of future studies.
ACKNOWLEDGMENTS
We thank Helmut Krause and Titus Franzmann for excellent practical assistance and Howard C. Berg and Karen Fahrner for providing strains HCB7 and SJW880.
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Scha914/1-3) and Fonds der chemischen Industrie to M.H.
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ABSTRACT
The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.
INTRODUCTION
Free-swimming bacteria modulate their swimming patterns in response to environmental changes. They are propelled by helical flagellar filaments connected to the basal body by a flexible hook and driven by a flagellar rotary motor. The basal body consists of a central rod and four coaxial structures, the L ring in the outer membrane, the P ring in the peptidoglycan layer, the MS ring in the cytoplasmic membrane, and the bell-shaped C ring in the cytoplasm. In enterobacterial species like Escherichia coli and Salmonella enterica serovar Typhimurium, temporal and structural assembly of the flagellar apparatus is strictly regulated by a hierarchy of transcriptional controls (1, 2, 22, 23, 27, 59). The MS ring complex appears at the very beginning of flagellar assembly. Then the C ring and the export apparatus, composed of six transmembrane proteins and two cytoplasmic proteins, are assembled on the cytoplasmic side of the M ring. The active and selective export of flagellar proteins starts with five proteins comprising the rod, followed by the hook, which is composed of FlgE subunits (13, 21, 34). Upon completion of the basal structure and the hook, genes necessary for the flagellar filament and the energizing motility complexes are expressed. At this assembly point, the flagellar protein export apparatus switches specificity from rod/hook-type substrates to filament-type substrates. Whereas the length of the flagellar filament is not strictly regulated, the length of the hook is well defined by the hook length control protein, FliK (19). The accurately defined length of the flexible hook is essential for proper formation of bundles of the flagellar filaments and therefore for efficient propulsion of the cell (14). Once the hook reaches its mature length (55 nm), the flagellar export protein FlhB, together with FliK, mediates the switching of export specificity (32, 59). Defects in fliK or flhB prevent this switch, resulting in abnormally long hooks, called polyhooks (15).
The behavioral scheme of the nitrogen-fixing plant symbiont Sinorhizobium meliloti, a member of the -subgroup of the Proteobacteria (39), differs from the enterobacterial (-subgroup) behavioral scheme in the filament structure, the mode of flagellar rotation, signal processing, and gene regulation (52). The rigid "complex" flagellar filaments consist of four related flagellin subunits, and interflagellin bonds lock the filaments in right-handedness (6, 11, 50). Hence, S. meliloti cells are propelled by exclusively clockwise rotating flagella, and swimming cells respond to tactic stimuli by modulating their rotary speed (3, 49). This mode of motility control has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, which are present in addition to the ubiquitous MotA/MotB energizing proton channel. MotC binds to the periplasmic portion of MotB and requires a specific chaperone, MotE, for proper folding and stability (7). Platzer et al. (42) described the presence of an additional motility protein, MotD. These authors determined that the proper assignment was Mot on the basis of an in-frame deletion introduced into motD that resulted in paralyzed cells with intact flagella. The arrangement of chemotaxis (che), flagellar (fla, flg, flh, and fli), motility (mot), and regulatory (visN, visR, and rem) genes differs from the enterobacterial pattern in that all known 51 genes are clustered in one contiguous 56-kb chromosomal region, the flagellar regulon (9, 45, 57). Most flagellar gene homologues were identified in the original S. meliloti genome annotation. However, a small number of genes remained unassigned due to sequence divergence from the E. coli-Salmonella paradigm. Indeed, homology searches revealed great variability in the regulatory flagellar genes, as well as the absence of a few specific flagellar chaperones in the genomes of a representative set of phylogenetically diverse bacterial species (40). In S. meliloti, the global enterobacterial transcriptional activator, FlhD2C2, is functionally replaced by the LuxR- and OmpR-like transcriptional regulators VisNR and Rem (9, 45, 57). In addition, several specific flagellar chaperones and export proteins have not been identified (or are missing) in S. meliloti. Among these unrecognized components are (i) FliD, the filament cap protein; (ii) FliS, the chaperone for flagellins; and (iii) FliK, the hook length control protein. Based on homology of a conserved motif in the otherwise unrelated sequence, we identified the gene coding for the hook length regulator, FliK, in the S. meliloti genome. Here we provide bioinformatic, genetic, physiological, biochemical, and structural proof for reassignment of MotD, a protein previously thought to function as a cytoplasmic motility protein (42), as a hook length regulator, FliK. In conformity with the nomenclature, we proposed that MotD from S. meliloti and other related -proteobacteria should be renamed FliK.
MATERIALS AND METHODS
Bacterial strains and plasmids. Derivatives of E. coli K-12, S. enterica serovar Typhimurium, and S. meliloti MV II-1 (18) and the plasmids used are listed in Table 1.
Media and growth conditions. E. coli strains were grown in Luria broth (26) at 37°C. S. meliloti strains were grown in TYC (0.5% tryptone, 0.3% yeast extract, 0.13% CaCl2 · 6H2O [pH 7.0]) at 30°C (42). Motile cells prepared for Western blot analysis were grown for 2 days in TYC, diluted 1:100 in 10 ml RB minimal medium, layered on Bromfield agar plates (55), and incubated at 30°C for 14 h to obtain an optical density at 600 nm (OD600) of 0.2 to 0.5. Motile cells prepared for -galactosidase assays were grown for 2 days in TYC, diluted in 15 ml of RB minimal medium to obtain an OD600 of 0.05, layered on Bromfield agar plates, and incubated on a slowly rotating platform at 30°C for 16 h to obtain an OD600 of 0.2 to 0.5. Antibiotics were used at the following final concentrations: for E. coli, 100 mg/liter ampicillin, 50 mg/liter kanamycin, and 10 mg/liter tetracycline; and for S. meliloti, 120 mg/liter neomycin, 600 mg/liter streptomycin, and 10 mg/liter tetracycline.
Motility assay. Swarm plates containing Bromfield medium and 0.3% Bacto agar were inoculated with 3-μl droplets of the test culture and incubated at 30°C for 3 days. Second-site suppressors (pseudorevertants) of the fliK deletion were isolated on swarm plates incubated at 30°C for 10 to 15 days in a humidity chamber. Free-swimming tracks were analyzed by computerized motion analysis using a Hobson BacTracker (Hobson Tracking Systems, Ltd., Sheffield, England) as previously described (56).
Genetic manipulations. Mutants of S. meliloti (Table 1) were generated in vitro by overlap extension PCR as described by Higuchi (12). Constructs containing the mutation were cloned into the mobilizable suicide vector pK18mobsacB, used to transform E. coli S17-1, and conjugally transferred to S. meliloti by filter mating using the method of Simon et al. (53). Allelic replacement was achieved by sequential selection with neomycin and 10% sucrose as described previously (55). Confirmation of allelic replacement and elimination of the vector was obtained by performing gene-specific primer PCR, Southern blotting, and DNA sequencing.
DNA methods. S. meliloti DNA was isolated and purified as described previously (55). Plasmid DNA was purified with NucleoSpin (Macherey Nagel, Duren, Germany), and DNA fragments or PCR products were purified from agarose gels using a QiaEx DNA purification kit (QIAGEN, Hilden, Germany). PCR amplification of chromosomal DNA and Southern blotting were carried out by using previously described protocols (42, 57).
Flagellar filament isolation. Flagella were prepared as described previously (50). Briefly, flagella were detached from motile cells (from 250-ml cultures) by agitation in a Braun MX32 mixer (Braun, Melsungen, Germany) at maximum power for 20 s, separated from cells by centrifugation at 8,000 x g for 8 min, and further purified by serial centrifugation at 15,000 x g for 15 min and at 25,000 x g for 1 h. Purified flagella that were sedimented by centrifugation at 87,000 x g for 2 h were resuspended in 100 μl of RB minimal medium.
Expression and purification of FliK. Recombinant FliK protein was overproduced from plasmid pRU2315 in E. coli ER2566 (Table 1). Cells were grown at 37°C in Luria broth containing 100 mg/liter ampicillin to an OD600 of 0.7, and gene expression was induced with 0.3 mM isopropyl--D-thiogalactopyranoside (IPTG). Cultivation was continued for 16 h at 16°C until harvest. Cells from 2-liter cultures were resuspended in 20 ml column buffer (20 mM HEPES [pH 8.0], 500 mM NaCl, 1 mM EDTA) containing 10 μg/ml DNase I and were lysed by two passages through a French press at 20,000 lb/in2. Cell membranes and debris were removed by centrifugation at 48,000 x g and 4°C for 30 min. The supernatant was loaded onto a chitin agarose (New England Biolabs, Beverly, MA) column (2.6 by 5.0 cm) equilibrated with column buffer and washed thoroughly with 5 bed volumes of column buffer at 4°C. Intein-mediated cleavage at the intein cleavage site was elicited by equilibration of the column with 2 bed volumes of cleavage buffer (50 mM dithiothreitol in column buffer) and further incubation at 4°C for 16 h. FliK was eluted during equilibration with cleavage buffer and after incubation for 16 h. Protein was eluted with column buffer, and pooled fractions were dialyzed against 1 mM EDTA-20 mM Tris-HCl (pH 7.5) and concentrated by ultrafiltration on a regenerated cellulose membrane (30-kDa cutoff). Samples were subjected to fast protein liquid ion-exchange chromatography (MonoQ HR5/5; Amersham Pharmacia, Freiburg, Germany) equilibrated with 20 mM Tris-HCl-1 mM EDTA (pH 7.5) (buffer A) at a flow rate of 0.5 ml/min. FliK eluted with a linear gradient of buffer A and buffer A containing 1 M NaCl at an NaCl concentration of 0.17 M.
Analytical size exclusion chromatography. Size exclusion high-performance liquid chromatography was performed using a Phenomenex BioSep-SEC-S3000 column (Phenomenex, Aschaffenburg, Germany) with a separation range of 10 to 1,500 kDa. FliK was subjected to 50-μl portions at a concentration of 0.5 mg/ml. The column was equilibrated and developed with 150 mM KCl-40 mM HEPES-KOH (pH 7.4) at a flow rate of 0.5 ml/min. Protein was detected by fluorescence at 330 nm after excitation at 280 nm using an FP 920 fluorescence detector (Jasco, Groumstadt, Germany). For calibration, thyroglobulin (669 kDa), catalase (232 kDa), ovalbumin (48 kDa) and chymotrypsin (25 kDa) were used as molecular mass markers (Sigma, St. Louis, Mo.).
Analytical ultracentrifugation. The native molecular weight of FliK was determined using a Beckman XL-I analytical ultracentrifuge and a Beckman Ti-60 rotor equipped with a UV/Vis and interference detection unit. Centrifugation was performed at 4°C and 13,000 rpm until the sedimentation equilibrium was reached. Complete protease inhibitors (Roche Applied Science) were added to avoid proteolytic degradation. The protein concentration was 0.4 mg/ml in a buffer containing 20 mM Tris-HCl and 1 mM EDTA (pH 7.5). For data analysis we used the one-species model included in the Origin software package available from Beckman.
Limited proteolysis and mass spectrometry. Limited proteolysis of FliK by trypsin (Promega, Madison, WI) was carried out in buffer containing 20 mM Tris-HCl (pH 7.5) at 25°C. Protein was incubated with trypsin at a protein-to-protease ratio of 600:1 (wt/wt), and samples were collected at 0.5, 1, 2, 5, and 10 min. The digestion products were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) or were subsequently purified using C4 Zip Tips (QIAGEN, Hilden, Germany) and analyzed with a Biflex-II mass spectrometer (Bruker Daltonik, Bremen, Germany) operated in the linear mode to determine the masses of the digestion products. To correlate the fragment masses determined with the different parts of the protein, the digestion products were incubated with 0.01% sodium desoxycholate on ice for 20 min and precipitated on ice for 30 min in the presence of 8% trichloroacetic acid. After centrifugation at 4°C and 18,000 x g for 30 min, proteins were analyzed on 15% acrylamide gels stained with Coomassie brilliant blue. Single bands were excised, and total in-gel digestion of the protein with trypsin was performed by using the protocol of Schfer et al. (48). Samples used for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry fingerprinting were prepared using C18 ZipTips (QIAGEN, Hilden, Germany) following the manufacturer's protocol. Data analysis was performed using Mascot (Matrix Science, London, United Kingdom).
Immunoblotting. Polyclonal antibodies raised against purified S. meliloti FliK and flagellar filaments were isolated from whole serum by affinity purification using 100 μg of protein and previously described protocols (50). Culture supernatants (filtered through a 0.2-μm cellulose acetate syringe filter) and whole-cell extracts were separated in 10% acrylamide gels, transferred to nitrocellulose, and probed using purified anti-S. meliloti FliK polyclonal antibody at a 1:100 dilution and anti-S. meliloti filament polyclonal antibody at a 1:500 dilution (50). Quantitative immunoblotting was performed and immunoblots were analyzed as described previously (7, 51). One-milliliter aliquots of RU11/001 (wild type) were harvested by centrifugation, resuspended in 30 μl SDS sample buffer, and heated to 100°C for 10 min. Samples of this mixture were stored at –20°C. Control samples were prepared by adding 0.5 to 10 ng of purified FliK to an extract prepared from a 1-ml aliquot of strain RU11/212 (fliK). Two samples and six standards (at six different concentrations) were applied to each gel. Gel electrophoresis, blotting, and probing were done as described above. Hyperfilm ECL (Amersham Pharmacia) was used for detection with a series of exposures. Films were scanned in the presence of a calibrated Step Tablet (Eastman Kodak) by using an Epson Perfection 1640SU and Corel Photo-Paint 10 software. Scans were analyzed using NIH Image 1.60 and Origin 5.0. Flagellin expression was semiquantified by comparing the flagellin band intensities for mutant strains with the wild-type band intensity using NIH Image 1.60.
Protein and -galactosidase assays. Protein concentrations were determined by the Bradford microassay (Bio-Rad, Munich, Germany). For quantitative immunoblotting, the concentration of purified FliK was determined from the Ala and Phe content by amino acid analysis performed by the Analytical Research and Services, University of Bern (Switzerland). Cultures of S. meliloti containing lacZ fusions were sampled, diluted in Z buffer (30) to obtain an OD600 of 0.2, permeabilized with 1 drop of toluene, and assayed for -galactosidase activity by the method of Miller (30).
Electron microscopy. Samples were applied to carbon-coated copper grids, negatively stained with aqueous 3% uranyl acetate (pH 4.5), and examined with a CM12 transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at 120 keV. The magnification was calibrated using catalase crystals negatively stained with uranyl acetate (44). The images were digitally recorded using a slow-scan charge-coupled device camera connected to a PC running TVIPS software (TVIPS GmbH, Gauting, Germany).
RESULTS
S. meliloti FliK (formerly MotD) contains a conserved Flg hook motif in the carboxy-terminal moiety. The fliK (motD, Smc03044) (9) gene maps in the motility (mot) operon of S. meliloti downstream of motB (encoding a channel protein) and motC (encoding a periplasmic motility protein) (42). This genetic linkage, together with the paralyzed phenotype of a knockout mutant, led to its assignment as a novel motility gene in S. meliloti (42). Here, we performed a bioinformatic analysis of the derived 475-residue polypeptide sequence (49 kDa) and of its orthologues from Agrobacterium tumefaciens, Brucella melitensis, Mesorhizobium loti, and Rhizobium lupini. These orthologues are positioned in an identical genetic linkage and in a very similar operon arrangement. The derived polypeptide sequences of the four orthologues exhibit only low levels of homology with the S. meliloti FliK sequence (ca. 37% similar residues and 29% identical residues). All five proteins contain a Flg hook motif (PF02120) in the carboxy-terminal region, as revealed by Pfam (protein families database of alignments) analysis (Fig. 1) (5, 29). The 124-residue motif is typical for flagellar-hook-length control proteins and is designated FliK, and it has been assigned to more than 90 bacterial species, including 11 species belonging to the -subgroup of the Proteobacteria. The homology in this C-terminal region is significantly higher (58% similar residues and 45% identical residues on average) than that in the N-terminal two-thirds of the sequence. According to the significant homology with the Flg hook motif, the aligned proteins can be newly assigned as FliK. Below, we provide experimental evidence for the function of S. meliloti FliK in controlling the length of the flagellar hook.
Deletion of fliK causes the formation of polyhooks. If FliK controls the length of the S. meliloti flagellar hook, mutations in fliK are expected to result in hooks whose length is uncontrolled, so-called polyhooks (15). We therefore isolated flagellar structures of an fliK knockout mutant (RU11/212, having a 649-bp deletion in fliK) generated by allelic exchange (47) and the S. meliloti wild-type strain (RU11/001) and examined the negatively stained samples by electron microscopy (Fig. 2). In contrast to the wild type, which produced normal flagella and a curved hook that was ca. 60 nm long (Fig. 2D), cells of the fliK deletion mutant lacked typical flagellar filaments. The lack of flagellar filaments occurred concurrently with the nonmotile phenotype of mutant strain RU11/212, as observed on swarm plates (Fig. 3A) (42). Instead, cells of the mutant strain produced hooks which were four to eight times longer than wild-type hooks (Fig. 2A to C). Hence, deletion of the fliK gene resulted in a failure to terminate hook assembly and initiate filament assembly.
Mutations in flhB suppress the fliK polyhook phenotype. To our surprise, a closer examination of the fliK deletion strain by phase-contrast microscopy revealed that a small fraction of cells (less than 0.1%) were motile. As described previously for Salmonella (58), these cells could be divided in two classes: (i) "leaky" mutants producing swimming cells with normal hooks and (ii) suppressor mutants having a mutation which permitted the formation of functional flagellar filaments in the absence of an fliK gene product. We isolated pseudorevertants from a fliK strain (RU11/212) after prolonged incubation on swarm plates which could swim but swam with less efficiency than the wild type (Fig. 3A). As described previously for enterobacterial fliK polyhook mutants, pseudorevertants fall into two classes: intragenic (mutations in fliK) and extragenic (mutations in flhB) (15, 23). The 649-bp deletion in strain RU11/212 resulted in the loss of amino acid residues 2 to 218 of FliK; 257 codons were still present in the genome. Gain-of-function mutations that produced frame-shifted sequences have been described for the truncated Salmonella gene (59). We therefore assayed the isolated pseudorevertants for expression of FliK in immunoblots using anti-FliK antibody. None of the nine strains expressed FliK at a detectable level, which ruled out the possible occurrence of such intragenic suppressors (data not shown). All known extragenic suppressor mutations of Salmonella fliK polyhook mutants map to the cytoplasmic domain of the integral membrane protein FlhB (15, 59). We amplified this part of the flhB gene from the nine mutant strains by PCR, and the amplified fragments were sequenced using internal primers. All suppressing mutations indeed mapped in a 68-amino-acid region of the cytoplasmic FlhB domain and were caused by single-base substitutions (Table 1). One residue, Ala at position 300, represented a hot spot of mutation, with four independently isolated changes to Thr or Val. Ala-300 and Gly-295 are highly conserved residues encoded by the flhB gene (23, 59). The other five amino acid substitutions identified (A278V, G295E, L320F, S323P, and L345P) (Tables 1 and 2) are novel suppressor mutations for the S. meliloti polyhook mutant (RU11/212).
Flagellar structures were isolated from these strains and inspected by electron microscopy (Fig. 2E and F). For all strains, as illustrated for RU13/202 (A300T), the hook structures of the flagella were polyhooks but had filaments at the distal end, known as polyhook filament (phf) structures (15, 41). Hence, the suppressor mutations can suppress the fliK deletion for filament formation but not for determination of the hook length. In addition, immunoblots comparing the levels of flagellin expression in the wild type and the suppressor mutants revealed that the levels in the phf strains were 30 to 60% of the wild-type levels (Table 2). When the swarming proficiencies were assessed, the phf mutants formed swarms that were much smaller than those of the wild-type strain, with swarm sizes ranging from 17% to 32% of the wild-type sizes (Table 2), as shown for RU13/202 (A300T) as a typical example in Fig. 3A. There was a moderate positive correlation between the efficiency of swarming and the amount of flagellin protein expressed. An additional motility assay was used, which monitored and averaged the free-swimming speeds of cell populations. All mutants exhibited comparable swimming speeds that were 60% of the wild-type speed (Table 2). We therefore concluded that extragenic suppressor mutants of the fliK deletion strain restored filament assembly and hence swimming proficiency, albeit in the presence of polyhook structures.
Defined hook length is essential for proper motility. Rhizobial cells do not exhibit a strong relationship between flagellar filament length and swimming speed (49). In contrast, in phf mutants there was a remarkable reduction in the swimming speed despite significant expression of flagellin protein (Table 2 and Fig. 2E and F). We analyzed the swimming behavior of phf mutants in greater detail. The "smooth-swimming rate" is defined as the ratio of the straight-line velocity (linear distance traveled per second between endpoints of a given track) to the instantaneous velocity (velocity measured at every point in the track and averaged for every swimming track). This ratio reflects the suppression of directional changes in response to positive stimulation and is a corollary of the suppression of tumbles in the chemotaxis of enterobacteria (55). Interestingly, all phf mutants exhibited a much lower smooth-swimming rate (average, 0.54) than nonstimulated S. meliloti wild-type cells exhibited (0.84) and changed their swimming directions more frequently than the wild type (Table 2). This behavior was nicely illustrated by tracking the swimming path over time. While the S. meliloti wild type (RU11/001) (Fig. 3B) swam in wide, slightly curved tracks, a phf mutant strain (RU13/202) (Fig. 3D) was distinguished by swimming in tiny spirals. The extended polyhooks of the mutants (Fig. 2E and F) apparently interfered with proper formation of a flagellar bundle which pushed the cells straight forward. Regulation of the appropriate hook length is therefore necessary for maximal motility.
FliK is necessary for flagellin secretion. Electron micrographs of the fliK deletion mutant revealed the presence of polyhooks concurrent with the lack of typical flagellar filaments (Fig. 2E and F). The lack of filament genesis was also reflected by the low (<20%) expression of flagellins, as analyzed by Western blotting of whole-cell extracts (Fig. 4 and Table 2). We asked whether the failure of filament assembly in the fliK deletion strain is consistent with a lack of flagellin monomer secretion through the export apparatus. Cell culture supernatants of the wild type and the fliK mutant strain were isolated, sheared flagellar filaments were removed by filtration, and samples were analyzed by Western blotting. Supernatants of wild-type cultures contained considerable amounts of flagellins, whereas no flagellin proteins were present in supernatants of the mutant strain. In the absence of FliK, export of flagellin monomers through the flagellar export apparatus was prevented.
We have shown previously that transcription of flaA, the gene encoding the principal flagellin of the S. meliloti complex filament, is down-regulated in abortive fla mutants (50). Is the blockage of filament assembly in an fliK deletion mutant passed on to transcriptional control of the flagellin genes We compared the transcriptional activity of all four fla genes (flaA to flaD) in fliK (RU11/212) with that in the wild type (Table 3). Strong transcriptional control was exerted on the flaA promoter activity, which was reduced by more than 70%, when filament assembly could not be initiated. The transcription of the three secondary flagellin genes, flaB, flaC, and flaD, was weakly affected, with an average reduction of 30%. Hence, transcription of the principal flagellin gene, flaA, was effectively controlled at the control switch between hook and filament assembly. Flagellin genes belong to the late (class III) genes in the regulatory cascade, together with genes for the chemotaxis machinery (54). We wondered whether FliK also exerts control over the members of the class III gene group, all of which are in the chemotaxis operon. Indeed, the activity of the che promoter was reduced by more than 80% (Table 3). In conclusion, fliK can be integrated as a class IIA gene in the hierarchical cascade of flagellar gene expression, regulating the expression of all class III genes.
FliK is secreted into the culture medium. The enterobacterial counterpart of FliK contains an export signal in its N-terminal region (amino acids 1 to 40) (31). To investigate whether S. meliloti FliK is restricted to the cytoplasm, we analyzed supernatants of wild-type cultures for the presence of FliK. Using anti-FliK antibody, FliK could be detected in whole-cell extracts and the culture supernatant equally well (Fig. 5), showing that there was secretion of FliK into the culture medium. A quantitative Western blot analysis showed that there were 74 ± 28 FliK molecules per cell. The question of whether FliK is actually secreted through the flagellar export apparatus was approached by analyzing a mutant defective in flagellar export. Deletion of fliF, the gene coding for the MS ring, blocks assembly of the export apparatus at an early stage. An FliK band was detected in whole-cell extracts of an fliF deletion strain (fliF, RU11/807), and the intensity was identical to that in wild-type extracts. However, this band was missing in supernatants of the fliF cell culture. We concluded that FliK is secreted through the flagellar export system into the culture medium. Therefore, similar to Salmonella FliK (31), it is an export substrate recognized by the flagellum-specific export apparatus.
FliK is an elongated monomeric molecule. Most properties of FliK determined so far are analogous to those of its enterobacterial counterpart. We examined the question of whether the two proteins share any structural properties. FliK was overexpressed in E. coli and purified using affinity and anion-exchange chromatography. To determine the molecular mass of FliK independent of its shape, we carried out sedimentation equilibrium measurements at a protein concentration of 0.4 mg ml–1. Using the manufacturer's software, a single-species model produced the best fit and calculated a native molecular mass of 50.0 ± 1.5 kDa, which agrees well with the deduced molecular mass, 48.8 kDa (Fig. 6). Hence, FliK exists as a monomer in solution.
To analyze the hydrodynamic properties of FliK, we performed size exclusion chromatography. Purified FliK eluted after 19 min from a BioSep-SEC-S3000 column, at an apparent molecular mass of 150 kDa (Fig. 7). The observation that FliK eluted from the size exclusion column much earlier than expected for a 50-kDa (Fig. 6) monomer suggests that FliK is an elongated molecule. A similar behavior was described for Salmonella FliK (33).
Flg hook motif is a compactly folded domain. To study the possible domain organization of FliK, we carried out limited proteolysis of FliK by using the protease trypsin, which cleaved C terminal of Arg and Lys residues. The time course of proteolysis, as monitored by SDS-PAGE, is shown in Fig. 8A. Seven metastable fragments (FliKT1 to FliKT7) and two stable fragments (FliKT8 and FliKT9) were produced. The cleaved products were analyzed by MALDI-TOF mass spectrometry, and the identities of the fragments were as follows: FliKT1 was FliK24-467, FliKT2 was FliK105-467, FliKT3 was FliK105-429, FliKT4 was FliK146-467, FliKT5 was FliK105-391, FliKT6 wasFliK217-467, FliKT7 was FliK217-429, FliKT8 was FliK217-391, andFliKT9 was FliK245-391 (Fig. 8B). All the calculated masses were in excellent agreement with the experimental values. This indicated that five trypsin-sensitive sites are located in the N-terminal region of FliK and three trypsin-sensitive sites are located in the C-terminal region of FliK. The only two stable fragments maintained after limited proteolysis contained the Flg hook motif (amino acids 297 to 402). The Flg hook region apparently comprises a compactly folded, globular domain, rendering it insensitive to protease digestion.
DISCUSSION
Here we describe reassignment of a gene, motD, previously described as a gene encoding a cytoplasmic motility protein having an unknown function, as fliK, the gene coding for the hook length regulator. The physiological significance of hook length control is obvious in mutants with extraordinarily long hooks (polyhooks). In this case, the presence of a filament is not sufficient for proper propulsion. Instead, such mutants swim in small, aberrant spirals due to (i) their inability to form a functional flagellar bundle and (ii) their inability to produce enough thrust to propel the cell forward (Fig. 3D). Hence, a hook length control mechanism is expected to be present in all bacteria that have a flagellar hook filament structure. The primary function of FliK, together with a transmembrane protein of the export apparatus, FlhB, is to regulate the length of the flagellar hook. However, the occurrence of polyhook mutants has been reported for only a small number of bacterial species, including the soil bacterium Rhizobium lupini (10, 25, 35, 36, 46).
In the past, reliable annotation of fliK in the flagellar loci of distantly related bacteria was hampered by the low level of similarity to the enterobacterial counterpart (40). The wealth of sequence data, together with refinement of search engines, led to identification of S. meliloti FliK. Despite significant divergence (24% overall identity), an Flg hook domain in the C-terminal region, centered on a highly conserved Pro-X-X-Leu-Gly motif (Fig. 1) (5, 29), was identified in the former motility protein MotD. Our results corroborated the bioinformatic approach results by experimentally defining the function of the protein as a flagellar-hook-length regulator, FliK, as follows: (i) deletion of fliK resulted in a failure to terminate hook assembly and initiate filament assembly; (ii) FliK was secreted through the flagellar export apparatus; (iii) the number of FliK molecules per cell (74 ± 28) correlated with the number reported for Salmonella FliK (37); and (iv) the fliK defect could be suppressed by extragenic mutations in the cytoplasmic domain of FlhB. The frequent occurrence of extragenic suppressor mutations resulting in a polyhook filament phenotype might explain the previous misassignment of FliK as a new motility protein. Platzer et al. (42) described fliK knockout mutant cells as cells that were paralyzed and nonswarming but had normal flagellation as determined by electron microscopy. The second observation is obviously not true. We can only assume that the analysis was not performed under appropriate conditions and/or that the data were misinterpreted due to the high frequency of leaky mutants and suppressor mutants. These authors apparently failed to recognize the concurrent presence of polyhooks, since elongated hooks are very difficult to discern in whole-cell preparations. They named the new gene motD.
The C-terminal domain of Salmonella FliK is directly involved in substrate specificity switching of the flagellar export apparatus (59). Data reported by Minamino and colleagues (33) suggest that the N-terminal domain of FliK receives the length information from the growing hook structure and activates the C-terminal domain of FliK to bind to FlhB. A conformational change in FlhB then triggers the substrate switch of the flagellar export machinery (8, 32, 33). In contrast to the two-domain structure (receiver and activator) of Salmonella FliK, we identified only one stable, compactly folded region comprising the Flg hook motif of S. meliloti FliK (Fig. 8). The C-terminal region of Salmonella FliK is rich in glutamine residues (16 mol%) (19). Accumulation of glutamine residues has also been noted for S. meliloti FliK, although it is less pronounced (9 mol%). In contrast, the S. meliloti homologue lacks the characteristic accumulation of proline residues in the central part of the protein (residues 180 to 205) (19, 33), underscoring the differences between the two proteins. Minamino and colleagues (33) suggested that these proline residues ensure flexibility of the hinge region connecting the two compactly folded FliK domains. Since S. meliloti FliK consists of only one domain, there is no requirement for such a flexible hinge region. Therefore, accumulation of proline residues in the central part of the protein is pointless.
The complex flagellar filaments of S. meliloti and R. lupini are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their stability, which prevents a switch of handedness (6, 50). It is conceivable that filaments composed of heterodimers formed by four and three different flagellin subunits, respectively, place different demands on the flagellar export channel than single-subunit filaments place. This possibility is supported by the fact that S. meliloti FliK, when expressed from plasmid pRU2930 (a derivative of pQE60) (Table 1) and introduced into E. coli HCB7 and Salmonella strain SJW880, is not able to complement the fliK polyhook phenotype. Furthermore, S. meliloti FliK is not secreted by either of the two enterobacterial strains (data not shown).
There are two hypothetical models for the function of FliK after completion of the hook structure, the quantized measuring cup model proposed by Aizawa and colleagues (28) and the molecular ruler model (17, 33). In the first model, the hook proteins accumulate to fill the C ring and are then secreted to form a hook having a precise length. In the ruler model, the N- and C-terminal ends of FliK are anchored to the tip of the hook (strictly speaking, to the hook cap FlgD) and to the basal body, respectively. When FliK is fully stretched because the hook has grown to its full length, it transmits information to FlhB, switching the secretion specificity. Preliminary binding studies using affinity chromatography and Far Western blot analysis revealed an interaction of S. meliloti FliK with the C-ring component FliM, which would be more in agreement with the measuring cup model but would not contradict the ruler model (data not shown). Interaction of FliK with the C-ring proteins might shed light on the mechanism of hook length control and will be the topic of future studies.
ACKNOWLEDGMENTS
We thank Helmut Krause and Titus Franzmann for excellent practical assistance and Howard C. Berg and Karen Fahrner for providing strains HCB7 and SJW880.
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Scha914/1-3) and Fonds der chemischen Industrie to M.H.
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