The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor

Bacterial strains, plasmids and growth condition.Bacterial strains and plasmids used in the present study are listed in Table 1. V. alginolyticus was cultured in VC medium (0.5% [wt/vol] Bacto tryptone, 0.5% [wt/vol] yeast extract, 0.4% [wt/vol] K₂HPO₄, 3% [wt/vol] NaCl, 0.2% [wt/vol] glucose) or in VPG500 medium (1% [wt/vol] Bacto tryptone, 0.4% [wt/vol] K₂HPO₄, 500 mM NaCl, 0.5% [wt/vol] glycerol) at 30°C. E. coli was cultured in LB broth (1% [wt/vol] Bacto tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl). In the second selection to isolate mutants, V. alginolyticus was cultured at 30°C in plates containing sucrose (1% [wt/vol] polypeptone, 30 mM NaCl, 55 mM KCl, 10% [wt/vol] sucrose, 1.25% [wt/vol] agar). Chloramphenicol was added to final concentrations of 2.5 μg/ml for V. alginolyticus and 25 μg/ml for E. coli. Kanamycin was added to final concentrations of 100 μg/ml for V. alginolyticus and 50 μg/ml for E. coli. Ampicillin was added to a final concentration of 50 μg/ml for E. coli.

Swarming assay.VPG500 semisolid agar (1% [wt/vol] Bacto tryptone, 0.4% [wt/vol] K₂HPO₄, 500 mM NaCl, 0.5% [wt/vol] glycerol, 0.25% [wt/vol] Bacto agar) was used for motility assays of V. alginolyticus. A 1-μl aliquot of an overnight culture was spotted onto VPG500 semisolid agar, followed by incubation at 30°C for the desired time.

Immunoblotting.The samples were suspended with sodium dodecyl sulfate (SDS) loading buffer and boiled at 95°C for 5 min, and SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed as described previously (49). Antisera against PomA (PomA1312), PomB (PomB93), MotX (MotXB0080), MotY (MotYB0079), and FliF (FliFB0424) were prepared previously (47, 49; R. Ogawa et al., unpublished data). Antisera against FlgT (VA15390B0472) were prepared (see below), and a His probe (Santa Cruz) was purchased. Horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz) was used as the secondary antibody.

Introduction of plasmids into V. alginolyticus.Transformations were carried out by electroporation as described previously (19).

Isolation of flagellar basal bodies.The isolation of the flagellar basal bodies was carried out as described previously with several modifications (42). EDTA for forming spheroplasts was modified to a final concentration of 5 mM and, in the following step, MgSO₄ was added to a final concentration of 10 mM.

Electron microscopy.The isolated flagellar structures were negatively stained with 2% uranyl acetate and observed with a JEM-2010 electron microscope (JEOL, Japan).

High-intensity dark-field microscopy.Flagella were observed by using a dark-field microscope (Olympus model BHT) equipped with a 100-W mercury lamp (Ushio USH-102). Images was recorded by using a charge-coupled device camera (Sony model SSC-M370) and a DVD video recorder (Panasonic model DMR-E100H).

flgT gene cloning, plasmid construction, and disruption.The flgT gene and flanking regions (−541 to +1660) were amplified from purified chromosomal DNA from strain VIO5 by PCR and then subcloned in pGEM-T (Promega), and the resultant plasmid was named pTH103. The flgT gene and upstream sequence (−30 to −1) with a BamHI site at the 5′ end and SacI site at the 3′ end was cloned in pSU41 or pKJ502, and the resultant plasmids were named pTH104 and pTH106, respectively. The DNA sequence of a hexahistidine tag was attached by site-directed mutagenesis (Stratagene), and the resultant plasmids were named pTH105 and pTH107, respectively. For purification of FlgT proteins, the flgT-His₆ gene with an NdeI site at the 5′ end and a BamHI site at the 3′ end was cloned in pET3a (Novagen), and the resultant plasmid was named pTH108. The flgT deletion mutant was generated by homologous recombination using a suicide vector as described previously (42). The sequence upstream of the flgT coding region (−541 to −1) with a SacI site at the 5′ end and the downstream sequence (+1135 to +1144) at the 3′ end, and the downstream sequence of the flgT coding region (+1135 to +1660) with upstream sequence (−10 to −1) at the 5′ end and a SacI site at the 3′ end were amplified by PCR. Their PCR products were used as primer and template in the following PCR. A deletion fragment was amplified by PCR and subcloned into pGEM-T, and the resultant plasmid was named pTH109. The flgT deletion fragment was inserted into the SacI site of pKY704, followed by insertion of the sacB gene, which was obtained from pHFS401, in the XbaI site, and the resultant plasmid was named pTH110. pTH110 was used to transform E. coli SM10λpir. The deletion allele was introduced into VIO5 or KK148 by a conjugation-based method. The resultant strains were named TH6 and TH7, respectively.

Determination of N-terminal amino acid sequence.The proteins were separated by SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore) and stained with Coomassie blue R250. The MotX, MotY, and FlgI bands were excised. The N-terminal amino acid sequences were determined by Aproscience (Tokushima, Japan), using the Edman degradation method.

Antibody raised against FlgT.BL21(DE3)/pLysS cells harboring pTH108 were cultured in LB broth at 30°C, and FlgT proteins were induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at early exponential phase. Cells were collected by centrifugation, resuspended in buffer A (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 25% [wt/vol] sucrose), incubated on ice for 10 min, diluted with 2 volumes of buffer B (100 mM NaCl, 1.5 mM sodium-EDTA [pH 8]), and incubated on ice for 20 min. The supernatant was recovered by centrifugation (10,000 × g for 20 min), MgCl₂ and imidazole were added to final concentrations of 2 and 20 mM, respectively, and then the periplasmic fraction was recovered by ultracentrifugation (100,000 × g for 60 min). The periplasmic fraction was applied to HisTrap FF (GE Healthcare), and the bound proteins were eluted with a 20 to 500 mM linear gradient of imidazole (using the buffers 20 mM Tris-HCl [pH 7.5] and 100 mM NaCl containing 20 or 500 mM imidazole). FlgT-His₆ was purified with a HiTrap Q column (GE Healthcare) and Superdex 200HR 10/30 (GE Healthcare) as necessary. Purified FlgT was separated by SDS-PAGE, stained with Coomassie blue R250, and excised. Rabbit anti-FlgT antibody was produced by Biogate (Tokushima, Japan).

Coelution assay to detect the interaction between FlgT and MotY.The coelution assay was carried out by the purification protocol described above with slight modifications. YM14 cells, which are sigma54⁻ mutant (rpoN mutant) cells that do not express the flagellar genes, harboring pTH106 or pTH107 were cultured in VPG500 broth at 30°C. The pH of Tris buffer was changed from 7.5 to 8.0.

Identification of a novel component in the Vibrio basal body.We purified the wild-type hook-basal bodies from the flhG mutant (KK148), which produces multiple polar flagella (25). The fractions of purified hook-basal bodies were analyzed by SDS-PAGE and stained (Fig. 2). We obtained a similar band profile, and the most intense band of 50 kDa was the hook protein, FlgE, as previously reported (42). To confirm the basal body components, we determined the N-terminal amino acid sequences of some of the protein bands, which appeared to be MotX, MotY and FlgI (Fig. 2). The sequences of the 25- and 30-kDa bands were NVADV and VMGKR, which correspond to the sequences of MotX and MotY without the putative N-terminal signal sequences, respectively. The 38-kDa band contained two sequences, ARIKD and SWYEV, which correspond to the sequences of FlgI without the signal sequence and a hypothetical gene product (VA15390 of V. alginolyticus strain 12G01). This result suggests that the hypothetical gene product, VA15390, is a component of the Vibrio basal body. A homolog of VA15390 has been identified as a gene required for motility in V. cholerae and was named FlgT (9). FlgT homologs from various species were aligned, and the secondary structure of FlgT from V. alginolyticus strain VIO5 was predicted by using PSIPRED (8) (Fig. 3). FlgT homologs are likely to exist in only Vibrio, Shewanella and related species, which are known to possess MotX and MotY. The deduced flgT products have a signal sequence for secretion and the amino acid sequence of FlgT detected from the basal body fraction had lost the signal sequence. This suggests that FlgT is cleaved between Ala²³ and Ser²⁴ and is translocated into the periplasmic space. FlgT was predicted to have an α/β mixed structure in the N-terminal half and be rich in β-strand structures in the C-terminal half. There are two conserved cysteine residues that might form a disulfide bond for protein stabilization.

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FIG. 2.

Identification of the proteins containing the purified hook-basal body fraction. The proteins of the hook-basal body from KK148 were separated by SDS-PAGE. The N-terminal amino acid sequences of the putative MotX, MotY or FlgI bands were determined by Edman degradation.

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FIG. 3.

Alignment of the amino acid sequences of V. alginolyticus strain VIO5 FlgT and homologous proteins from various species. The sequence alignment was generated with Genetyx software (Genetyx Corp.). Abbreviations: VaFlgT, FlgT of Vibrio alginolyticus strain VIO5; VP0767, hypothetical protein of Vibrio parahaemolyticus RIMD 2210633; VC2208, FlgT of Vibrio cholerae El Tor N16961; Il1154, hypothetical protein of Idiomarina loihiensis L2TR; SO_3258, hypothetical protein of Shewanella oneidensis MR-1. White letters in black boxes indicate residues that are identical in all sequences. Letters in gray boxes show residues that matched in at least three of the five sequences. The arrowhead shows the site of signal sequence cleavage. Asterisks show the conserved cysteine residues. Green boxes indicate β-strands predicted by PSIPRED, and orange wavy lines indicate predicted α-helixes.

Motility of ΔflgT cells.To examine the role of FlgT in the Vibrio flagellar system, we deleted the flgT gene and examined the motility of the ΔflgT cells based on swimming ability in semisolid agar and in liquid medium (Fig. 4 and data not shown). The ΔflgT cells scarcely showed any ability to swim in 4 h (Fig. 4A) and slightly expanded in semisolid agar by 9 h (Fig. 4B) compared to strain NMB191 (Mot⁻, ΔpomAB). Although most of the ΔflgT cells did not have a flagellum (Fla⁻), only a small fraction had a flagellum (Fla⁺) (Fig. 4C). We did not observe released flagella with attached basal bodies as reported previously for V. cholerae (29). This might suggest that the strength of the membrane between the two species is not the same. When we observed flagella using high-intensity dark-field microscopy, cells of wild type (VIO5) were more than 90% flagellate, and all of the flagellate cells were motile; on the other hand, the flgT mutant cells were ca. 30% flagellate, and only ca. 10% of the flagellate cells were motile but very slow. Therefore, FlgT seems to partially (not critically) contribute to both flagellar formation and rotation.

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FIG. 4.

Motility of ΔflgT cells in semisolid agar. (A) Cells were inoculated into VPG500 semisolid agar and incubated at 30°C for 4 h. WT, VIO5; ΔflgT, TH6; vector, pSU41; FlgT, pTH104; FlgT-His6, pTH105. (B) Cells were inoculated into VPG500 semisolid agar and incubated at 30°C for 9 h. ΔflgT, TH6; ΔpomAB, NMB191. (C) Cells of VIO5 (WT) and TH6 (ΔflgT) grown in panel A were negatively stained with 2% potassium phosphotungstate and observed with a JEM-2010 electron microscope (JEOL, Japan). Bar, 2 μm.

Basal body structure purified from the ΔflgT cells.Because FlgT seemed to be a component of the basal body, we speculated that FlgT forms a substructure in the basal body. To investigate this possibility, we purified the basal bodies from the ΔflgT cells of a multiple-flagellated strain (KK148) and observed them by electron microscopy. The basal bodies of the ΔflgT cells had smaller LP rings than those of wild type, and the T ring beneath the LP ring was lost, suggesting that FlgT is a component protein of the ring structure outside of the LP ring (Fig. 5). Therefore, the ring that has been referred to as the Vibrio LP ring could be separated into a ring that is lost in the flgT mutant and the conventional LP ring formed by FlgH and FlgI. The new ring was named the H ring (Fig. 1). Next, to examine whether depletion of FlgT affects the other components of the basal body (MotX, MotY, and FliF), we analyzed whole-cell lysates or basal body fractions by immunoblotting using anti-MotX, MotY, FliF, or FlgT antibodies. In whole-cell lysates, MotY and FliF were expressed at similar levels regardless of the presence or absence of FlgT, but MotX was decreased in the absence of FlgT (Fig. 6). In the basal body fraction, FliF in the ΔflgT cells was detected at lower levels than in the wild-type cells. We measured the band densities of FliF from the wild-type and the ΔflgT cells. The intensity of the band from the ΔflgT cells was reduced to 18 or 51% compared to the intensity of wild type in two independent experiments. FliF in the basal body fraction was detected as smeared bands, suggesting that the N- or C-terminal region of FliF was degraded during the purification step. On the other hand, MotX and MotY were barely detectable in ΔflgT cells. These results suggest that the H ring is involved in basal body formation and is necessary to assemble the T ring composed of MotX and MotY (Fig. 6).

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FIG. 5.

Electron microscopy images of the hook-basal bodies purified from V. alginolyticus. The hook-basal bodies were negatively stained with 2% (wt/vol) uranyl acetate and observed with a JEM-2010 electron microscope (JEOL, Japan). Bar, 20 nm. WT, KK148; ΔmotX ΔmotY, TH3; ΔflgT, TH7. The diagrams of the flagellar basal bodies isolated from the various strains are shown below the pictures.

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FIG. 6.

Detection of FlgT, FliF, MotX, and MotY in the purified hook-basal body fraction. Whole-cell lysate (WC) and the purified hook-basal bodies (HBB) of V. alginolyticus were subjected to SDS-PAGE, followed by immunoblotting with anti-FlgT, FliF, MotX, and MotY antibodies. Asterisks show degraded products of FliF proteins in the basal body. WT, KK148; ΔflgT, TH7; ΔmotX ΔmotY, TH3; FlgT/ΔflgT, pTH104/TH7.

Interaction between FlgT and MotY.We speculated that FlgT interacts directly with MotX or MotY, which has been suggested to directly interact with the basal body (23). To determine whether this interaction occurs, a hexahistidine tag-fused FlgT and MotY were produced in sigma54⁻ mutant (mutant rpoN) cells that do not produce the other flagellar components (Fig. 7). As for MotX, we cannot purify it as a soluble protein (34, 35), and thus we could not test for interaction between MotX and FlgT. After the periplasmic fraction was applied to a HisTrap column and washed, the bound proteins were eluted with imidazole. Although most of the MotY protein was detected in the flowthrough fraction, MotY was also coeluted with FlgT-His₆ but not with plain FlgT. The elution profile of MotY corresponded to that of FlgT-His₆ eluted by the liner gradient of imidazole. The present results support a direct interaction of MotY with FlgT. Under these experimental conditions, only FlgT and MotY were produced, so the interaction between them may be weak, and other flagellar proteins might be necessary for more stable interaction to form the ring structures.

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FIG. 7.

Coelution assay of MotY with FlgT-His₆. FlgT or FlgT-His₆ proteins with MotY proteins were expressed from pTH106 or pTH107 in strain YM14 (rpoN mutant). The samples were subjected to SDS-PAGE, followed by immunoblotting with anti-FlgT and MotY antibodies. P, periplasmic fraction; F, flowthrough fraction; E, eluted fraction.