ABSTRACT
The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28. This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.
IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella. However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri. Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.
INTRODUCTION
The bacterial flagellum is a rotary nanomachine embedded in the cell envelope. The motor is powered by the electrochemical ion gradient formed across the cytoplasmic membrane. The rotating part has three well-defined structures: the basal body, which expands from the cytoplasm to the outer membrane; the hook that is the first extracellular structure; and the helical filament that thrusts the cell forward through rotation of the flagellar motor (1, 2). The rotor is surrounded by a stator formed by multiple transmembrane subunits of MotA/MotB, or PomA/PomB in bacteria with H+- or Na+-driven motors, respectively (3–7). These proteins form a channel that conveys ions across the membrane and couples ion flow to rotation (8–10). In Escherichia coli and Salmonella enterica serovar Typhimurium (from here on Salmonella), the basal body is formed by four rings and a rod that expands from the cytoplasm to the outer membrane (OM), while the membrane/supramembrane (MS) ring is formed by 26 subunits of FliF that is located at the inner membrane (IM) (11, 12). At the cytoplasmic side the MS ring connects with the C-ring that is formed by FliG, FliM, and FliN; these proteins have been implicated in torque generation and morphogenesis (13–15). The periplasmic side of the MS ring couples with the flagellar rod through the adaptor protein FliE (16, 17). The rod formed by FlgB, FlgC, FlgF, and FlgG spans the cell wall and the OM crossing the P and L rings, which act as bushings for the rotating rod (18–21). FlgI and FlgH form the P and L rings, respectively (22). It has been recently proposed that L-ring formation contributes to dislodging the scaffolding protein FlgJ from the tip of the growing rod; this enables the transition from rod to the polymerization of the hook (23). The hook is the first extracellular structure, and it is formed by approximately 120 subunits of FlgE. The flagellar filament is formed by thousands of flagellin subunits and connects to the hook by means of the hook-associated proteins FlgK and FlgL (12, 24, 25).
Recently, electron cryotomography (ECT) of cells from different bacterial species allowed in situ visualization of the intact flagellum. These studies have revealed that the basal body has a central core structure that is conserved; however, around it a great diversity of additional components were observed (5, 7, 26, 27). The protein composition of these additional elements is still mostly unknown. In this regard, it has also been recognized that the presence of certain flagellar proteins is restricted to specific bacterial groups, and their characterization is incipient. This is the case for FlgT and FlgP, which are both absent in E. coli and Salmonella. FlgT is present in several species of Vibrio and Aeromonas, whereas FlgP has been reported to be present in species of Vibrio and in Campylobacter jejuni (28–32).
FlgT forms the H ring that covers the L and P rings and which is instrumental in supporting the high swimming velocities reported for several species of Vibrio (28). FlgP is a lipoprotein that was first identified and characterized in Vibrio cholerae and C. jejuni (29, 30, 32). In V. cholerae the absence of FlgP provokes a reduction in the number of flagellated cells, and morphologically the filaments were shorter than those observed in wild-type cells (29, 32). In contrast, a flgP mutant strain of C. jejuni was able to assemble a normal flagellum that showed a paralyzed phenotype (Mot–) (30). Recently, flagella of a flgP mutant of V. fischeri and C. jejuni were observed by ECT, and the reconstructed images were compared to those of the wild-type cells. It was suggested that FlgP is a component of a structure named basal disk, which seems to be in contact with the OM (7). The basal disk is required to form other flagellar structures such as the medial and proximal rings in C. jejuni or to recruit the stator complexes in V. fischeri (7). These authors also observed that in the flgP mutant of C. jejuni the flagellum is formed, which agrees with previous reports; however, in contrast to what had been reported for V. cholerae, the flgP mutant of V. fischeri seldom forms a flagellum (7).
Rhodobacter sphaeroides is an alphaproteobacterium with two different flagellar systems (33). Transcription of the fla1 set produces a single subpolar flagellum that is expressed constitutively under the growth conditions commonly used in the laboratory (33, 34). The products encoded by the fla2 set produce several polar flagella (33, 35). However, the expression of the fla2 genes is achieved only under very particular conditions. Fla2 flagella were detected in a mutant strain lacking the master activator of the fla1 genes that acquired a gain of function mutation in the histidine kinase CckA (36). Phylogenetic analysis of these flagellar gene systems suggested that the fla1 set was acquired by R. sphaeroides from an ancestral gammaproteobacterium, whereas the fla2 set is vertically inherited (33).
The fla1 system of R. sphaeroides includes the flgT and flgP genes. FlgT forms the periplasmic H-ring that covers the P and L rings, similar to the observed situation in Vibrio species; however, the phenotype of the flgT mutant strain differs from that of these bacteria, given that in V. cholerae and V. alginolyticus the absence of FlgT results in a reduction of the number of flagellated cells, but in R. sphaeroides the absence of FlgT yields a Mot– phenotype, where the flagellum is paralyzed (37–40).
In this study, we characterized FlgP from R. sphaeroides. This protein is absolutely required for the assembly of the Fla1 flagellum, and its absence results in the lack of hook and filament. FlgP was detected in isolated wild-type filament basal body structures, and evidence is presented suggesting that FlgP interacts with FlgH and FlgT.
(This study was conducted by C. Pérez-González in partial fulfillment of the requirements for a Doctorado en Ciencias Biomédicas degree from the Universidad Nacional Autónoma de Mexico, Mexico City, Mexico.)
RESULTS
FlgP is required for swimming and filament assembly.In R. sphaeroides the gene flgP encodes a 177-amino-acid polypeptide. The N terminus shows a short sequence similar to the consensus sequence recognized by the signal peptidase II (SPaseII) that has a predicted cleavage site between Ala20 and Cys21, with Ala at position +2. The identity of the residue at the +2 position after the cleavage site indicates if the polypeptide will be retained at the IM or directed to the OM (41, 42). Therefore, the presence of Ala at this position, suggests that FlgP is localized in the OM (Fig. 1A). This protein shows a 25/27% identity with homologs in V. cholerae and C. jejuni. In R. sphaeroides flgP is the first gene of a putative operon formed by flgP, flgT, flgA, flgM, and RSWS8N_05380 orf (Fig. 1A). The first three genes show overlapping of the translation stop and start codons. flgT encodes the protein that forms the H ring, which is present is some bacterial species (28), FlgA is a chaperone that assists in the assembly of the L ring and, FlgM is the anti-σ28 factor required to transcribe the late flagellar genes. RSWS8N_05380 encodes a 123-amino-acid protein that has not been reported to be involved in either flagellar biogenesis or swimming.
The role of FlgP on swimming in R. sphaeroides. (A) Genetic context of flgP and domain composition. SPII, signal protease II; LPP20, pfam PF02169. Amino acid sequence alignment of FlgP with homologs from C. jejuni and V. cholerae. The invariant cysteine in the lipobox is shown in pink. (B) Swimming of the ΔflgP::aadA mutant and complementation test. (C) Immunodetection of FlgT and FlgP in total cell extracts of the ΔflgP::aadA mutant and strains expressing FlgP, FlgT, and FlgPT from the promoter of pRK415. WT, wild-type strain WS8N.
To characterize the role of FlgP in the flagellar biogenesis of R. sphaeroides, a mutant strain in flgP (ΔflgP::aadA) was constructed. This mutant was unable to swim and introduction of the plasmid that expresses flgP (pRK_flgP) did not restore swimming; however, when the plasmid pRK_flgPT (that drives the expression of flgP and flgT) was introduced, swimming was recovered (Fig. 1B). These results suggest that the allele ΔflgP::aadA generates a polar effect on the expression of flgT. Nevertheless, the role of FlgP on swimming was clearly appreciated in the ΔflgP/pRK_flgT strain, since the expression of flgT did not restore swimming, suggesting the absence of flgP by itself is responsible of this defect. The presence in these strains of FlgP and FlgT was verified by Western blotting (Fig. 1C) and, as expected, FlgT is absent in the strain carrying the ΔflgP::aadA allele but is present in the nonmotile ΔflgP/pRK_flgT strain. Furthermore, FlgP was detected in the wild-type strain and also in the ΔflgP strain harboring pRK_flgP and pRK_flgPT (Fig. 1C). Given that in this bacterium the flagellar hierarchy is controlled by the master activator FleQ (43), the absence of FlgP in the ΔfleQ mutant strain indicates that flgP is expressed as a part of the flagellar regulon (Fig. 1C).
Overlapping of the translation stop and start codons of flgP, flgT, and flgA suggests that these genes form an operon. We noticed that the ΔflgP::aadA allele affects the expression of flgT but not the expression of flgA, since the ΔflgP::aadA/pRK_flgPT strain was fully motile (and the lack of FlgA would have produced a Fla– phenotype). From these observations, it could be hypothesized that the absence of FlgT in the ΔflgP::aadA mutant could be caused by translational coupling. This possibility would also explain why the insertion of the nonpolar gene aadA (44) could affect the expression of the downstream gene; alternatively, in the absence of FlgP, FlgT could be unstable. Unfortunately, several attempts to isolate a flgP mutant without a resistance marker were unsuccessful; therefore, we carried out experiments to characterize the ΔflgP (ΔflgP::aadA) mutant always in the presence of the plasmid pRK_flgT. In this strain the amount of FlgT detected by Western blotting is similar to the amount detected in WS8N (Fig. 1C), suggesting that FlgT should not be limiting for flagellum formation and functioning.
Filaments were easily detected in the microscope in the wild-type strain, but this structure was not observed in ΔflgP and ΔflgP/pRK_flgT strains, indicating that these two mutant strains are Fla– (Fig. 2). In contrast, the filament was visible in ΔflgP/pRK_flgP cells. Given that a strain lacking of flgT shows a Mot– phenotype (37), the presence of the flagellar filament in the ΔflgT mutant was expected (Fig. 2). From these results, we conclude that the absence of FlgP causes the loss of the flagellar filament. This phenotype is different from that reported for C. jejuni and V. cholerae (29, 30) but is similar to what was observed in V. fischeri (7).
Flagellar filament detection. Cells were stained with DAPI and observed by fluorescence microscopy as indicated in Materials and Methods. WT, wild-type strain WS8N. Scale bars, 1 μm.
Role of FlgP in the formation of other flagellar structures.Given that the flagellar filament is absent in the ΔflgP/pRK_flgT mutant cells, we proceeded to investigate whether the flagellar hook would be assembled in the absence of FlgP. For this, ΔflgP/pRK_flgT cells were labeled with an anti-FlgE antibody previously conjugated with Alexa Fluor 488 and then observed by fluorescence microscopy. The hook was readily observed as small fluorescent foci that are placed laterally on wild-type strain cells (Fig. 3A). In contrast, the ΔflgP/pRK_flgT mutant strain did not show fluorescent foci, indicating that the hook is not formed in the absence of FlgP (Fig. 3A).
Immunofluorescent detection of the flagellar hook in complete cells. (A) Cells were incubated in the presence of anti-FlgE prestained with Alexa Fluor 488 and observed by fluorescence microscopy. WT, wild-type strain WS8N. Scale bars, 1 μm. (B) Western blots of total cell extracts of the indicated strains, using anti-FlgE, anti-FliC, and anti-CheY4 antibodies. WT, wild-type strain WS8N.
A simple explanation for the lack of hook and filament in the ΔflgP/pRK_flgT strain could be that FlgE and FliC were not being synthesized. This possibility was tested by probing total cell extracts by Western blotting using anti-FliC and anti-FlgE antibodies (Fig. 3B). The intensity of the signal for FlgE was similar in the wild-type cells and ΔflgP/pRK_flgT mutant, indicating that the absence of the hook is not due to a lack of protein. However, the amount of FliC was severely reduced in the mutant strain (ΔflgP/pRK_flgT). This is the logical outcome of a lack of hook, since the anti-sigma factor FlgM cannot be exported from the cells, and transcription by σ28 of the late flagellar genes does not take place (43, 45–47). The expression of CheY4 was used to corroborate this notion, given that it is also σ28 dependent (48). As expected, a severe reduction in the amount of CheY4 was observed in the ΔflgP/pRK_flgT strain (Fig. 3B).
In ΔflgP/pRK_flgT cells, the hook was not detected but FlgE was observed in total cell extracts, raising the possibility that FlgE could be exported from the cell but not assembled. To test this idea, the supernatants of WS8N and ΔflgP/pRK_flgT cultures were probed by Western blotting for the presence of FlgE. No protein was detected in these samples, indicating that FlgE is not exported (data not shown). As a control, the cells of these cultures were vigorously vortexed to promote the mechanical shearing of the flagella. After centrifugation, the supernatants were tested by Western blotting. In these samples, FlgE was detected in the culture supernatant of WS8N but not in the ΔflgP/pRK_flgT strain (see Fig. S1 in the supplemental material), confirming the notion that, in the absence of FlgP, FlgE is not exported or assembled.
To determine whether the MS ring is formed in the absence of FlgP, we evaluated the localization of the GFP-FliF fusion protein as an indirect evidence of the presence of this structure. As shown in Fig. 4A, GFP-FliF was detected as a single focus in wild-type and ΔflgP cells, suggesting that the formation of the MS ring is not affected by the absence of FlgP.
GFP-FliF detection by fluorescence microscopy. (A) Wild-type (WT), WS8N, and ΔflgP::aadA/pRK_flgT cells. Scale bars, 1 μm. (B) Immunodetection of FlgE by Western blotting in the indicated genetic background.
It has been shown that in Salmonella FlgE is degraded in strains where the rod genes have been deleted. This was caused by the high sensitivity of FlgE to periplasmic proteases (49). We tested by Western blot analysis whether FlgE could be detected in rod mutants, type III export apparatus mutants, and the ΔflgP/pRK_flgT mutant. Total cell extracts of these strains revealed the presence of FlgE in the wild-type strain, in the export apparatus mutants (fliE, fliR, and fliO), and also in the ΔflgP mutant (ΔflgP/pRK_flgT) and its absence in the rod mutants (ΔflgB and ΔflgC) (Fig. 4B). A reduction of FlgE in the ΔflgI mutant was noted; this is in accordance with the results previously reported for Salmonella (49). The mutants lacking the flagellar master regulator, fleQ, and the hook gene flgE were included as negative controls. The presence of FlgE in ΔflgP/pRK_flgT cells (Fig. 4B) suggests that in the absence of FlgP the rod is completed since FlgE is not degraded by the periplasmic proteases, as has been previously reported (49).
FlgT is another flagellar protein localized in the periplasm, it is required to form the H ring that covers the P and L rings. In R. sphaeroides the absence of FlgT did not affect flagellar biogenesis (37). To determine whether FlgT is present in the flagellum in the absence of FlgP, we took advantage of the fact that the flagellar protein MotF (present exclusively in R. sphaeroides) requires the presence of FlgT to be localized (50). Therefore, we determined if green fluorescent protein (GFP)-MotF is localized in the absence of FlgP. We used strain SF3 (ΔflgT) carrying the plasmids pINDd_flgT and pRK_GFP-MotF as a positive control for this experiment. We observed the presence of fluorescent foci in these cells, suggesting that GFP-MotF is localized in the flagellar structure (Fig. 5A). However, no fluorescent foci were detected in ΔflgP cells carrying pINDd_flgT and pRK_GFP-MotF, suggesting that in the absence of FlgP the H ring could be destabilized or severely affected. The presence of FlgT and GFP-MotF in total cell extracts of these strains was detected by immunoblotting (Fig. 5B).
GFP-MotF detection by fluorescence microscopy. (A) Cells expressing FlgT from pINDd and GFP-MotF from pRK415. Scale bars, 1 μm. (B) Immunoblotting detection of FlgT and GFP-MotF in total cell extracts of the indicated strains. WT, wild-type strain WS8.
FlgP is an integral protein of the basal body and is stable in different genetic backgrounds.We isolated filament basal body structures of WS8N cells and used this preparation for electron microscopy (EM) and for immunodetection of FlgP. An example of the EM images obtained from intact flagellar structures, shows the H ring covering the L and P rings, as previously reported (37) (Fig. 6A). In this same sample, we were able to detect FlgP by immunoblotting, indicating that this protein is a part of this structure (Fig. 6B).
Presence of FlgP in isolated filament basal body preparations and stability of FlgP in the absence of other flagellar components. (A) Sample of an isolated flagellum from the wild-type strain. The arrow denotes the H ring. (B) Immunoblotting of total cell extracts using anti-FlgP antibodies (left lanes) and isolated flagella (right lanes). (C) Stability of FlgP in different genetic backgrounds. Western blots of total cell extracts were probed with anti-FlgP antibodies. WT, wild-type strain WS8N.
In C. jejuni FlgP was generally absent or present at low levels in whole-cell lysates of proximal rod (flgB and flgC) and type III export apparatus (flip and flhA) mutants (7), indicating that FlgP stability was dependent on other flagellar components. In contrast, we detected FlgP in rod (flgB and flgC) and type III export apparatus (fliE and fliR) mutants (Fig. 6C), demonstrating that in R. sphaeroides this protein is not particularly unstable.
FlgP interacts with FlgT and FlgH.Given that FlgP is part of the flagellar basal body structure, we evaluated possible interactions of FlgP with other periplasmic flagellar proteins, such as FlgH, FlgT, and MotF. The interactions of the periplasmic region of FlgP with the periplasmic regions of FlgH, FlgT, and MotF were tested using a yeast two hybrid assay. In this assay, we observed that FlgP interacts with FlgH and FlgT (Fig. 7). The possible interaction between FlgP with MotF could not be evaluated given the autoactivation of these proteins (Fig. 7). It should be noted that Gal4BD-FlgP by itself allows growth in the absence of histidine; therefore, only strong interactions that alleviate adenine auxotrophy were considered positives (Fig. 7). In this assay, the interaction of FlgP with FlgP does not occur, since a yeast strain expressing AD-FlgP and BD-FlgP recovered the prototrophy only for histidine (data not shown). Interestingly, FlgH showed a positive interaction with FlgT (Fig. 7).
Double hybrid assay. Yeast were transformed with the plasmids indicated at the left. The pairs Gal4AD T-antigen with Gal4 p53, and Gal4AD T-antigen with Gal4 BD Lam served as positive and negative controls, respectively (labeled “AC” [assay controls]). Transformant cells were seeded on the medium indicated at the top. To rule out spurious activation of the reporter genes, yeast cells transformed with a control plasmid, either Gal4 AD T-antigen or Gal4 BD p53, and a flagellar gene in the BD or AD plasmid. The growth of these cells was tested and corresponds to the six combinations in the lower part of the figure (Controls).
The interaction of FlgP with FlgP was evaluated by blue native polyacrylamide gel electrophoresis (PAGE). Two different species of FlgP were detected: one with an apparent molecular mass around 36 kDa that may represent the monomeric form and another that was around 56 kDa (Fig. 8A), suggesting that under these conditions FlgP could form a dimer. In these experiments, we also detected the monomeric and dimeric forms of FlgT (54 and 113 kDa, respectively). When FlgP and FlgT were mixed together, a weak but conspicuous band of around 90 kDa appeared, which was interpreted to represent the oligomeric complex formed by the association of FlgP with FlgT. Since high-molecular-weight markers were used in this native electrophoresis, the apparent molecular masses of the complexes may be overestimated. Denaturing two-dimensional SDS-PAGE (lanes 2 to 4) show that the bands observed in the native gels belong exclusively to FlgP, to FlgT, or to the FlgP/FlgT mixture (Fig. 8B).
Blue native gel electrophoresis. (A) Portions (100 μg) of His6×-FlgPp (lane 2), FlgTp-His6× (lane 3), or a mixture of both proteins (lane 4) were dialyzed overnight in PBS (pH 7.4), mixed with sample buffer, and loaded into a native 4 to 12% acrylamide gradient gel. Lane 1 was loaded with molecular weight protein markers. Gel casting and electrophoresis were performed as described previously (44). (B) Parallel lanes of each sample of interest from panel A were cut and subjected to denaturing two-dimensional SDS-PAGE (14%). The presence of FlgT in the complex formed with FlgP is indicated by an arrow.
The interaction of FlgP with FlgT was also detected by pulldown using glutathione S-transferase (GST)–FlgP as a bait (Fig. 9A). In contrast, the interaction between FlgP and FlgH could not be detected using this assay, given that FlgH showed nonspecific interactions with GST (data not shown). Nonetheless, we were able to corroborate the interaction between FlgP and FlgH by far-Western analysis (Fig. 9B). For this experiment, GST-FlgP was incubated with the blotted proteins His6×-FlgH and His6×-GFP. The presence of complexes was tested using anti-FlgP antibodies (Fig. 9B). The blotted proteins were also incubated with GST, and the absence of unspecific complexes was confirmed using anti-GST (Sigma) antibodies (Fig. S2).
FlgP interaction with FlgT and FlgH. (A) Pulldown assay using glutathione-agarose beads containing either GST-FlgP or GST, followed by incubation with FlgT. After a washing step, the presence of FlgT retained by the tested proteins was revealed by Western blotting. (B) Interaction between FlgP and FlgH tested by far-Western analysis. Purified His6×-FlgP, His6×-FlgH, and His6×-GFP were subject to 12% SDS-PAGE and blotted on nitrocellulose. The membrane was incubated with GST-FlgP for 2 h. After a washing step, the presence of FlgP was detected using anti-FlgP antibodies.
DISCUSSION
Several new ancillary proteins different from the well-characterized flagellar components of E. coli and Salmonella have been identified in various bacterial species. These new elements in some cases improve flagellar rotation by recruiting a larger number of stator complexes (7); in others, they contribute to remodel other bacterial structures such as the outer membrane (OM) (51). FlgP is absent in E. coli and Salmonella but is present in several species of Vibrio and many Epsilonproteobacteria (29, 30, 52). ECT images suggest that FlgP could form a basal disk located underneath the OM. It has also been suggested that the basal disk (FlgP) attaches to the motor via the P ring (FlgI) in C. jejuni and in V. fischeri through the H ring (FlgT) (7).
In R. sphaeroides, the absence of FlgP causes a Fla– phenotype that contrasts with the phenotypes observed in other species so far studied. In C. jejuni, a ΔflgP mutant forms flagella (30), whereas a reduction in the number of flagellated cells was observed for the ΔflgP mutant in V. cholerae (29). This suggests that in R. sphaeroides the role of this protein is not redundant; perhaps the absence of flagellar rings, such as the T ring (present in Vibrio) or the proximal and medial disks (found in C. jejuni), makes flagellar assembly impossible in the absence of FlgP.
A closer inspection of the ΔflgP strain of R. sphaeroides revealed that the flagellar hook is not assembled even though the presence of the hook protein FlgE was detected by Western blotting. In addition, the fact that flagellin (FliC) and the chemotactic protein CheY4 were not detected in total cell extracts of the ΔflgP mutant indicates that the anti-sigma factor FlgM is not exported. This is consistent with the well-established idea that a structural defect in the hook basal body (HBB) impairs secretion of FlgM (45–47). Nevertheless, in the absence of FlgP, the flagellar rod is assembled, and FlgE was not detected in the culture supernatant. Therefore, the absence of FlgP could be altering the rod to filament transition. This idea is in accordance with the images observed by ECT for the flgP mutant of V. fischeri, where the rod and the P, L, H, and T rings are formed, but the presence of the hook is not evident (7).
As a result of the absence of FlgT (H ring) in R. sphaeroides, MotF is not localized in the flagellar motor (37). Furthermore, we have observed in the present study that in the absence of FlgP, MotF is not recruited to the flagellum, suggesting that the H ring is not formed or that the architecture of the H ring is modified. In agreement with the possible remodeling of these rings as they are being assembled, it was observed in V. fischeri that in the absence of FlgP, all the rings were formed (i.e., P, L, T, and H rings), but the stator complexes were not recruited, suggesting that FlgP could modify the architecture of the H and T rings (7).
It is conceivable that the flagellar structure of R. sphaeroides has a basal ring similar to that observed in C. jejuni and V. fischeri. In this regard, our results indicate that FlgP can interact with itself, suggesting that it could be a part of an oligomeric structure, such as the basal disk. Besides this interaction, FlgP also interacts with FlgT, indicating that the OM is in close contact with the H ring through FlgP. In spite of the strong interaction between FlgP and FlgT, recruitment of FlgP to the growing structure should not be dependent only on FlgT, given that the flagellum is formed in its absence (37) but not in the absence of FlgP. This indicates that even in the absence of FlgT, FlgP must be recruited to the growing flagellum. This recruitment could be achieved by a FlgP-FlgH interaction. This is in contrast to the observed situation in V. fischeri, where it was proposed that the H ring provides a platform for assembly of FlgP (7).
We observed that FlgP is stable in the different flagellar mutants tested. In contrast, in C. jejuni, FlgP was not detected in total cell extracts of proximal rod and type III export apparatus mutants (7). This difference could be explained by a different intrinsic stability of these proteins or by the presence of ancillary flagellar proteins, such as FlgQ and FlgO (52), that are absent in R. sphaeroides. FlgQ is required for FlgP stability in C. jejuni (30), whereas FlgO of Vibrio seems to be a part of the H ring and is located at the OM similar to FlgP (29).
To explain the Fla– phenotype of the ΔflgP strain, we propose that the L ring could be remodeled by the basal disk. This event could be part of the check point that involves the removal of the rod scaffolding protein FlgJ to enable hook assembly. It is possible that in other species in which the flagellar hook can be assembled in the absence of FlgP, other proteins collaborate to form an L ring suitable for the removal of FlgJ or that FlgH could accomplish this task by itself, as occurs in E. coli and Salmonella. A model summarizing the findings reported in this is presented in Fig. 10.
Model showing the possible role of FlgP during flagellar biogenesis in R. sphaeroides. The right side depicts the wild-type flagellum where FlgP (orange) interacts with FlgH (pink) and FlgT (violet). The hook (yellow) is formed. The left side shows that, in the absence of FlgP, the hook is not assembled. A possible perturbation in the formation of the L ring (formed by FlgH) prevents the progression of the flagellar biogenesis beyond the outer membrane. In the absence of FlgP, the possible presence of FlgJ (blue) at the tip of the growing flagellum is shown. In addition, FlgT is shown in light violet, indicating that it may also be absent.
MATERIALS AND METHODS
Strains, plasmids, growth conditions, and oligonucleotides.All strains and plasmids used in this study are listed in Table 1 . R. sphaeroides was grown in Sistrom’s minimal medium at 30°C (53). Cultures were grown heterotrophically in Erlenmeyer flasks with orbital shaking (200 rpm). E. coli was grown in Luria broth at 37°C (54). When needed, the following antibiotics were used at the indicated concentrations: 25 μg/ml kanamycin, 50 μg/ml spectinomycin, and 1 μg/ml tetracycline. For E. coli, the antibiotics used were 100 μg/ml ampicillin, 50 μg/ml kanamycin, 50 μg/ml spectinomycin, 30 μg/ml gentamicin, 25 μg/ml chloramphenicol, and 10 μg/ml tetracycline. The oligonucleotides used are also listed in Table 1.
Strains and plasmids used in this studya
Molecular biology techniques.Standard methods were used to isolate and analyze chromosomal or plasmid DNA (54). Restriction and other DNA-modifying enzymes were purchased either from New England BioLabs (NEB, Ipswich, MA), Roche (Basel, Switzerland), or Invitrogen (Carlsbad, CA). Plasmids used for sequencing were purified using an Illustra plasmidPrep minispin kit (GE Healthcare Life Sciences, Buckinghamshire, UK). DNA was amplified with the appropriate oligonucleotides using PrimeSTAR HS DNA polymerase (TaKaRa Bio, Inc., Mountain View, CA) according to the manufacturer’s recommendations.
Isolation of mutant strains.The CP1 strain (ΔflgP::aadA) was obtained by cloning together two PCR products that amplified the upstream and downstream regions of flgP in pTZ19R. The 756-bp product from the upstream region of flgP was obtained using the oligonucleotides orf_11 and RvRSP0035B, whereas the downstream product of 1,293 bp was obtained with the oligonucleotides FwRSP0035B and orf_12. These PCR products were joined through a BglII site designed in the oligonucleotides and cloned in pTZ19R. A 1.4-kb PCR fragment carrying the aadA gene, which encodes the streptomycin/spectinomycin adenylyltransferase (Spcr) (44), was cloned into pTZ_ΔflgPup-down previously digested with BglII. The resultant plasmid, pTZ_ΔflgP::aadA, was digested with XbaI, and the fragment carrying the allele ΔflgP::aadA was subcloned in plasmid pQJ200mp18. To obtain the CP2 strain (ΔflgH::aadA), a similar strategy to that used to obtain CP1 was followed; in this case, the 700-bp product from the upstream region of flgH was obtained using the oligonucleotides Fw_flgH and Rvi_flgH, whereas a downstream product of 1,008 bp was obtained with Fwi_flgH and Rv_flgH. These products were joined through a BamHI site designed in the oligonucleotides and cloned in pTZ19R_BamHI−. The fragment carrying the aadA gene was cloned in pTZ_flgHup-down previously digested with BamHI. The fragment carrying ΔflgH::aadA was subcloned in pJQ200mp18.
Plasmid constructions.pRK415_flgP plasmid carries a 1,086-bp fragment that includes the coding region of flgP (534 bp), 501 bp upstream and 51 bp downstream. pRK_flgPT carries a 2,300-bp fragment that includes the coding regions of flgP and flgT (1,083 bp), 501 bp upstream of flgP and 186 bp downstream of the stop codon of flgT. pINDd_flgT was obtained by cloning the 1,083-bp PCR product encoding the complete polypeptide of FlgT in pINDd.
Protein purification.The coding region of flgP lacking the segment corresponding to the signal peptide was amplified by PCR using the oligonucleotides 0035fwBAD and 0035RvBAD and cloned in pBAD-HisB. The resultant plasmid was introduced to the strain LMG194 strain. A culture of this strain was grown until midexponential phase and induced with 0.2% arabinose for 6 h at 28°C. The culture was harvested and resuspended in buffer containing 50 mM Tris, 5% glycerol, 50 mM NaCl, and 1 mg/ml lysozyme (pH 8). The cell suspension was sonicated on ice with three bursts of 10 s. Cell debris were removed by centrifugation. The supernatant was mixed with nickel-nitrilotriacetic acid (Ni-NTA)–agarose beads (1/250 of the original culture volume) and incubated by 1 h on ice, with occasional mixing. The beads were washed with 3 volumes of phosphate-buffered saline (PBS; pH 7.4). The protein was eluted in PBS containing 20% glycerol and 200 mM imidazole. The purity of the His6×-FlgPp (periplasmic region of FlgP) was evaluated by SDS-PAGE and Coomassie blue staining. The sample was dialyzed overnight using PBS (pH 7.4). To purify FlgH, the fragment encoding from Ala58 to the stop codon of flgH (FlgHp) was amplified by PCR using the oligonucleotides flgH-pGABfw and flgH-pBADB and cloned in pET28a. The purification protocol was similar to the procedure used to purify His6×-FlgPp, except that induction of the cell culture was carried out with 0.25 mM IPTG for 4 h at 28°C. FlgTp-His6X and His6×-MotFp were purified using the procedures previously described (37, 50). The plasmid expressing glutathione S-transferase fused to the mature polypeptide of FlgP (GST-FlgPp) was constructed by cloning in pGEX-4T-2, the PCR product obtained with the oligonucleotides 0035FwpGEX and 0035RvpGEX that encompasses the coding region of flgP, excluding the segment that encodes the signal peptide sequence. The resultant plasmid was used to transform the E. coli strain Rosetta. The resultant cells were grown in Luria broth medium (30 ml) supplemented with chloramphenicol and ampicillin at 37°C to an optical density at 600 nm (OD600) of 0.6. The culture was induced by the addition of IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 0.25 mM, cultured for 4 h at 30°C, and collected by centrifugation. The cell pellet was resuspended in 1 ml of PBS containing 20% glycerol (pH 7.4) and 1 mg/ml lysozyme. The cell suspension was let stand on ice for 1 h before sonication with three bursts of 9 s. Cell debris was removed by centrifugation. The supernatant was mixed with 100 μl of glutathione-agarose beads and incubated for 1 h on ice, mixing by inversion every 10 min. The beads were washed with 3 volumes of PBS. GST-FlgPp protein was eluted in elution buffer (50 mM Tris HCl, 10 mM reduced glutathione [pH 8]). GST was purified according to a similar protocol.
Immunoblotting and antibody production.Samples were obtained from heterotrophically grown cultures at an OD600 of 0.6. At this point, the cells were harvested and lysed by boiling in a solution containing 2% SDS, 1% β-mercaptoethanol, and 50 mM Tris (pH 7.5). Western blotting was performed as previously described (55). Briefly, these samples were separated using 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked with 2% fat-free milk in TBST (20 mM Tris, 150 mM NaCl, containing 0.1% Tween 20 [pH 7.4]) for 12 h. After a wash in TBST, the membranes were incubated with the primary antibody in the same buffer, as indicated. Removal of excess primary antibody was carried out by washing the membrane three times in TBST. The secondary antibody (phosphatase alkaline-conjugated, anti-mouse IgG secondary antibody) diluted 1:30,000 was incubated with the membrane in TBST for 1 h. After being washed, the membranes were incubated with CDP-Star (Thermo Fisher Scientific, Waltham, MA) reagent for detection on X-ray films. Polyclonal antibodies were raised in female BALB/c mice against His6×-FlgPp and His6×-FlgHp according to previously reported protocols (55).
Microscopy.Slides were covered with an agarose pad containing Sistrom’s culture medium. Images were taken with a Hamamatsu Orca-ER camera and a Nikon E600 microscope. The flagellum was stained DAPI (4′,6′-diamidino-2-phenylindole) according to a previous report (50). Immunofluorescent detection of FlgE was carried out by fixing cells from a heterotrophically grown culture, where cells were swimming (OD600, ∼0.6) with paraformaldehyde (3%); after 20 min at room temperature, the paraformaldehyde was removed by centrifugation. The cell pellet was resuspended in 1/10 of the original volume in PBS–1% bovine serum albumin (BSA). Anti-FlgE γ-globulins were stained with Alexa Fluor 488 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Yeast two-hybrid assays.The assays were performed according to the directions of the manufacturer (Clontech). In summary, yeast cells of the strain AH109 were cotransformed with pGBKT7 and pGADT7 plasmids expressing the mature polypeptide of FlgP, or FlgH, fused to the activation (AD) or DNA binding (BD) domain of Gal4. The DNA fragment encoding the periplasmic regions of FlgP and FlgH was obtained by PCR using the oligonucleotides 0035fw pGA-pGB and 0035rvBAD or the oligonucleotides flgH-pGABfw and flgH-pBADB, respectively. The plasmid expressing pGBD-flgT was previously reported (37). pGBKT7 and pGADT7 plasmids restore prototrophy for leucine (LEU) and tryptophan (TRP), respectively. For interaction assays, the AH109 strain carrying the appropriated plasmids was grown overnight in synthetic defined (SD) minimal medium supplemented with histidine (HIS) and adenine (ADE), washed in SD medium without HIS and ADE, diluted at OD600 of 0.5, and serially diluted in the same medium. From these dilutions, 10-μl aliquots were seeded on plates containing SD supplemented with HIS and ADE (–LEU –TRP), ADE (–LEU –TRP –HIS), or HIS (–LEU –TRP –ADE).
Blue native PAGE.A total of 100 μg of His6×-FlgPp, FlgTp-His6×, or a mixture of both proteins was dialyzed overnight in PBS (pH 7.4), mixed with sample buffer, and loaded in a native 4 to 12% acrylamide gradient gel. Gel casting and electrophoresis were performed as described before (56). The lines of each sample were cut and subjected to denaturing two-dimensional SDS-PAGE (14%).
Pulldown interaction assay.Next, 5 μg of GST-FlgPp or GST bound to glutathione-agarose beads in PBS (pH 7.4) was mixed with FlgT-His6× in a 1:1 molar ratio. The volume was adjusted to 250 μl with PBS containing 10% glycerol and 3% BSA. The mixture was incubated for 2 h at 4°C, with mixing by inversion every 10 min. The beads were collected by centrifugation for 1 min at 3,000 rpm. The supernatant was discarded, and the beads were washed three times with 1 ml of PBS. To elute the protein, the beads were resuspended in 100 μl of buffer containing 100 mM Tris (pH 8) and 10 mM reduced l-glutathione. After 10 min, the sample was centrifuged for 1 min at 5,000 rpm. An aliquot (10 μl) of the supernatant was analyzed by Western blotting with Penta-His antibodies (Qiagen).
Far-Western assay.Far-Western blotting was performed according to a method reported previously (57). Briefly, 5 μg of His6×-FlgHp and His6×-GFP protein was mixed with an equal volume of 2× Laemmli sample buffer without β-mercaptoethanol, loaded onto 12% acrylamide (wt/vol), and electrophoresed at 100 V. Proteins were transferred electrophoretically to nitrocellulose membranes as described previously (55). Membranes were incubated in blocking buffer (5% nonfat dry milk in PBS [pH 7.4] and 0.05% Tween 20) for 1 h with shaking. Blocked membranes were incubated with 17 μg of purified GST-FlgPp or GST proteins in 10 ml of blocking buffer for 1 h with shaking. Membranes were washed three times for 5 min in PBS (pH 7.4) and 0.05% Tween 20, followed by incubation with anti-FlgP or anti-GFP antibodies diluted 1:5,000, for 1 h, and then washed again three times for 10 min in PBS–0.05% Tween 20, followed by incubation with anti-mouse AP antibody (Sigma, 1:30,000) for 45 min. Antibody binding was detected by incubation with CDP-Star reagent according to the manufacturer´s instructions and visualized by autoradiography.
ACKNOWLEDGMENTS
We are indebted to Aurora Osorio, Javier de la Mora, Teresa Ballado, Jorge Omar García Rebollar, and Miriam Vázquez for technical assistance. We thank the Molecular Biology Unit at IFC-UNAM for sequencing facilities.
C.P.-G. was supported by a fellowship from CONACyT (2013 to 2018). This study was partially supported by DGAPA-UNAM (PAPIIT-IN204317) and CONACyT (CB2014-235996).
FOOTNOTES
- Received 6 December 2018.
- Accepted 12 December 2018.
- Accepted manuscript posted online 17 December 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/JB.00752-18.
- Copyright © 2019 American Society for Microbiology.
