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Journal of Bacteriology, March 1999, p. 1388-1394, Vol. 181, No. 5
Department of Molecular Biophysics and
Biochemistry, Yale University, New Haven, Connecticut 06520-8114
Received 29 September 1998/Accepted 11 December 1998
Until now, identification of components of the flagellar protein
export apparatus has been indirect. We have now identified these
components directly by establishing whether mutants defective in
putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space.
Hook-type proteins could be exported to the periplasm of
rod mutants, indicating that rod protein export does not
have to precede hook-type protein export and therefore that both
types of proteins belong to a single export class, the rod/hook-type
class, which is distinct from the filament-type class. Hook-capping
protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI,
FliO, FliP, FliQ, and FliR for their export to the periplasm.
In the case of flagellin as an export substrate, because of the
phenomenon of hook-to-filament switching of export specificity, it was
necessary to use temperature-sensitive mutants and establish whether
flagellin could be exported to the cell exterior following a
shift from the permissive to the restrictive temperature. Again, FlhA,
FlhB, FliH, FliI, and FliO were required for its export. No suitable
temperature-sensitive fliQ or fliR mutants were
available. FliP appeared not to be required for flagellin export, but
we suspect that the temperature-sensitive FliP
protein continued to function at the restrictive temperature if
incorporated at the permissive temperature. Thus, we conclude that
these eight proteins are general components of the flagellar
export pathway. FliJ was necessary for export of hook-type
proteins (FlgD and FlgE); we were unable to test whether FliJ is needed
for export of filament-type proteins. We suspect that FliJ may be a
cytoplasmic chaperone for the hook-type proteins and
possibly also for FliE and the rod proteins. FlgJ was not required for
the export of the hook-type proteins; again, because of lack
of a suitable temperature-sensitive mutant, we were unable to test
whether it was required for export of filament-type proteins. Finally,
it was established that there is an interaction
between the processes of outer ring assembly and of penetration of the
outer membrane by the rod and nascent hook, the latter process being of
course necessary for passage of export substrates into the external
medium. During the brief transition stage from completion of rod
assembly and initiation of hook assembly, the L ring and perhaps the
capping protein FlgD can be regarded as bona fide export components,
with the L ring being in a formal sense the equivalent of the outer
membrane secretin structure of type III virulence factor export systems.
Many species of bacteria,
including Salmonella, swim by means of flagella. The
flagellum consists of the motor structure and basal body, which are
embedded in the cell surface, and the hook and the helical filament,
both of which are external to the cell. If we define the term
"external structure" in an even more restrictive sense to mean
lying beyond the cytoplasmic membrane, there are at least nine such
structures: the basal body rod, the periplasmic P ring, the outer
membrane L ring, the hook, the hook cap, two hook-filament junction
zones, the filament, and the filament cap. The proteins corresponding
to these substructures are listed in Table
1. All of these proteins have to cross
the cytoplasmic membrane, and several of them must cross the outer
membrane as well. Also included in Table 1 is FliE, a basal body
protein (27) whose precise location and function are not
known but which is postulated to interface with, or even be part of,
the rod (16).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Components of the Salmonella Flagellar
Export Apparatus and Classification of Export Substrates
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
External flagellar substructures and the proteins
from which they are composed
Over the years, several observations have made it clear that export of these flagellar proteins occurs by a rather unusual process. The first was the demonstration that the addition of new monomers of flagellin to a growing flagellar filament occurs at the distal end, i.e., the end furthest from the cell surface (5, 12). The second was that in the process of flagellar morphogenesis, substructures are successively added from most cell proximal to most cell distal (13, 16, 31, 32). Taken together, these observations indicate a successive addition of protein subunits from most cell proximal to most cell distal. A third observation was that the flagellar filament is not a solid cylinder but has a central channel (22, 26, 34); the same has subsequently been found to be true of the hook (25) and the rod (33). A fourth observation was that all exported proteins except those of the P and L rings (8, 10) do not undergo signal peptide cleavage during the export process (7, 9) and therefore cannot be proceeding by the general secretory pathway.
These observations led to a general acceptance of the idea that subunits reach their assembly point by traveling down the channel. This, however, did not explain how the subunits enter the channel in the first place, i.e., how they cross the plane of the cytoplasmic membrane. A specialized export apparatus seemed to be called for. In this study, we have not investigated the location or structure of the apparatus. Suffice to say that a reasonable hypothesis, which is beginning to gain experimental support (6), is that the apparatus exists within a central pore in the MS ring of the basal body; this ring is located in the cytoplasmic membrane and therefore is in a position to deliver exported substrates to the lumen of the growing flagellar structure.
The aim of this study was to positively identify the components of the export apparatus. There is currently suggestive evidence for several such components but little if any data in the way of direct proof. One line of evidence is a default one. The flagellar gene system has been studied exhaustively, and the functions of many of the more than 40 genes are known (see, e.g., reference 21). These functions include structural ones (hook protein, flagellin, etc.) and regulatory ones (sigma factor, anti-sigma factor, etc.). After assignment of these functions there was a residue of around 10 genes of unknown function, and as the probable existence of a specialized export apparatus became increasingly apparent, it was natural to consider these unassigned genes as candidates for encoding the apparatus.
An experiment involving the ability of various temperature-sensitive mutants to regrow sheared filaments at the restrictive temperature (35) suggested that three of these genes, flhA, fliH, and fliI, might indeed be involved in the export process. Studies of flagellar morphogenesis (13, 16, 17) are also consistent with several of these genes being involved in export, but they do not clearly distinguish between the issues of export and assembly, or of export across the cytoplasmic membrane and export across the outer membrane.
Finally, it has become evident in recent years that export of a variety of virulence factors by pathogenic bacteria has characteristics in common with export of flagellar proteins, and they now carry the common designation of type III export pathways (reviewed in reference 11). Most significantly, the proteins thought to be involved in flagellar protein export and those thought to be involved in virulence factor export show a high degree of similarity. Thus, the tentative assignment for one system lends credence to the tentative assignment for the other. This, however, does not constitute proof.
We therefore felt that direct evidence was needed and decided to make our criterion for export as simple as possible: is the presence of a given protein required for a given flagellar protein substrate to cross the cytoplasmic membrane? As far as flagellar structure was concerned, the only other condition besides integrity of the export apparatus itself was the integrity of the MS ring and the C ring, since they are presumed to provide a physical support structure for the export apparatus.
In the case of flagellin (and presumably the other filament-type proteins; see below), the simple approach of measuring periplasmic content was not possible because the export apparatus will not export these proteins until it has received a signal to switch its specificity, and this signal is generated only upon completion of an external structure, the hook. We were therefore forced to adopt a modified temperature shift protocol and measure the content of the cell exterior.
A question related to the issue of identification of export components is that of classes of export substrates. It is well established that hook-type proteins, i.e., the hook protein itself (FlgE) and the hook-capping protein (FlgD), belong to a different export class than the filament-type proteins, i.e., flagellin (FliC), the hook-filament junction proteins (FlgK and FlgL), the filament-capping protein (FliD), and the anti-sigma factor (FlgM). By defining these classes, we mean that there is an ordered export of hook-type proteins followed by export of filament-type proteins and that the switch in export specificity, mediated by FlhB, occurs at a well-defined point in the export/assembly process (20, 36). However, it has not been clear whether the rod proteins (FlgB, FlgC, FlgF, FlgG, and possibly FliE) belong to a distinct class from the hook-type proteins. This is a question of intrinsic interest, and it was also important in the present study in terms of finding a positive control for export of substrates to the periplasm.
Since export across the outer membrane, at least once it has been fully penetrated by the nascent structure, probably consists simply of diffusion of subunits through a passive channel, it should probably not be thought of as part of the primary export process, nor should structures such as the rod, hook, or filament be thought of as components of the export apparatus. However, we have found that the events immediately surrounding the stage of rod completion and hook initiation have some very interesting features that suggest a genuine export process and a genuine export structure, which in some regards resembles the secretin structure associated with export of virulence factors by the type III pathways.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains, plasmids and media.
The strains and
plasmids used in this study are listed in Table
2. Luria broth (LB) contained 10 g
of Bacto Tryptone (Difco, Detroit, Mich.), 5 g of yeast extract,
and 5 g of NaCl per liter. M9 medium contained (per liter) 100 ml
of 10× M9 salts (10 g of NH4Cl, 59 g of
Na2HPO4, 30 g of
KH2PO4, 5 g of NaCl), 1 ml of 0.1 M
CaCl2, 1 ml of 1 M MgSO4, 20 ml of 50%
glycerol, and 1 mg of vitamin B1 per ml. Ampicillin was
added to the medium at a final concentration of 100 µg/ml.
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Preparation of the periplasmic contents and culture
supernatants.
To examine FlgD and FlgE export, pUC18 was
introduced into various nonflagellate (Fla
) mutants (in
order to generate 
-lactamase as a control for the periplasmic
fraction). These transformants were incubated at 37°C in M9 medium
containing ampicillin. When the cells reached an optical density at 600 nm of 1.0 to 1.2, 1.5 ml of culture was centrifuged and the cells and
culture supernatants were collected separately. The cells were washed
twice with TN buffer (10 mM Tris-HCl [pH 8.0], 0.3 M NaCl) and then
suspended in 400 µl of sucrose buffer (20% sucrose, 100 mM Tris-HCl
[pH 8.0], 0.5 mM EDTA) and incubated at room temperature for 20 min.
After centrifugation, the cells were resuspended in 750 µl of 0.5 mM
MgCl2 and placed on ice for 10 min to release the
periplasmic fraction. The proteins in the supernatant and
periplasmic fractions were precipitated by 10% trichloroacetic
acid and suspended in Tris-saturated sodium dodecyl sulfate (SDS)
loading buffer.
Antibodies.
Polyclonal antibodies were used for detection of
FlgD, FlgE, FliC, and anti--lactamase (the latter from
5Prime
3Prime, Berkeley, Calif.).
Immunoblotting.
After the proteins in each fraction had been
separated by SDS-polyacrylamide gel (normally 12% acrylamide)
electrophoresis (PAGE), they were transferred onto a nitrocellulose
membrane (Schleicher & Schull, Keene, N.H.) with a transblotting
apparatus (Hoefer, San Francisco, Calif.). The membrane was then
blocked for 1 h with Tris-buffered saline plus 0.1% Tween 20 and
5% nonfat dry milk and probed with polyclonal anti--lactamase,
anti-FlgD, anti-FlgE, or anti-FliC. Immunodetection was performed
with an enhanced chemiluminescence immunoblotting detection kit
(Amersham International, Little Chalfont, United Kingdom).
Temperature shift-up experiments.
Temperature-sensitive
Fla mutants, which also contained a
flgK::Tn10 mutation, were transformed
with pDIC7 (a pTrc99B-based plasmid encoding fliC
[4]) and were grown overnight at 30°C in 1 ml of LB
containing ampicillin; 30 µl of the overnight culture was inoculated
into 1.5 ml of fresh LB containing ampicillin and grown at 30°C to an
optical density at 600 nm of 0.8. Then 1.5 ml of the culture was
transferred to the sampling tube, washed twice with 500 µl of M9
medium containing ampicillin and 20 µg of each of the common amino
acids except methionine per ml, and resuspended in 300 µl of the same
medium; 150 µl of this culture was incubated at 30°C for 20 min,
and the remainder was incubated at 42°C for the same length of time.
The cells were then labeled with [35S]methionine for 5 min each at 30 and 42°C and incubated in the presence of excess
unlabeled methionine for a further 20 min. The samples were
fractionated into whole cells and culture supernatant as before. After
immunoprecipitation of the cell lysates and culture supernatants with
polyclonal anti-FliC antibody, the labeled proteins in each sample were
separated by SDS-PAGE and analyzed by autoradiography.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Choice of export substrates. From the known exported flagellar proteins (Table 1), we chose as substrates for our analysis of the export apparatus the following proteins: FlgD (hook-capping protein) and FlgE (hook protein), both representative of the hook protein class, and flagellin (representative of the filament protein class).
Choice of putative export components. Initially, we confined ourselves to those proteins (FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR) which there was already reason to suspect were members of the export apparatus. Later, we extended the list to include proteins (FliE, FliJ, and FlgJ) whose function we felt was not well enough understood to warrant excluding them from a possible role in export. As controls, we examined the presumed need (for structural reasons) for the MS ring protein, FliF, and for the switch proteins of the cytoplasmic C ring, FliG, FliM, and FliN. Finally, we examined the role of the periplasmic P-ring protein and the outer membrane L-ring protein in the process of export across the outer membrane.
Protocol for determining whether a putative export component was required for export of a given export substrate. The basic idea for the hook-type proteins was to test whether they would cross from the cytoplasm to the periplasm in a strain with a defect in a suspected export component. After the cells were washed, the periplasmic fraction was released by sucrose osmotic shock. The protein content was concentrated by trichloroacetic acid precipitation, and the samples were subjected to SDS-PAGE, electrotransferred to nitrocellulose membrane, and probed with the relevant antibody. In some cases, where the signal was weak or simply to confirm the immunoblotting data, we also carried out radiolabeling experiments.
Hook-type proteins can be exported in the absence of rod protein export. It is well established that a major switch in the specificity of substrates being exported occurs during flagellar morphogenesis when the hook has reached its mature length. At that point, an integral membrane protein, FlhB, switches the specificity of substrates that it will accept from the hook-type proteins to the filament-type proteins (20, 36). One can readily rationalize this event, since flagellin is by far the most abundant exported substrate, and premature export would probably have severe logistical consequences. Less clear is whether a similar switch should occur at an earlier stage of morphogenesis, upon completion of the rod.
To test this, we examined whether hook-type proteins could be exported to the periplasm in a rod mutant. As illustrated by the immunoblot in Fig. 1, a representative hook-type protein, FlgD, was detected by anti-FlgD antibody in the periplasmic fraction of a rod mutant (flgB; lane 2) and a hook protein mutant (flgE; lane 8). Control experiments using anti-lactamase antibody yielded a strong
signal that was evenly balanced in the periplasmic fractions of all
samples (data not shown). In the case of the flgE mutant, significant amounts of FlgD were detected in the supernatant. Similar results were obtained with other rod mutants (flgF
and flgG) and with anti-FlgE instead of anti-FlgD (data not
shown).
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Components necessary for export of FlgD, the hook-capping
protein.
Using the same protocol, we tested for the presence of
FlgD in the periplasmic fraction of a variety of other mutants
(Table 3 and Fig.
2). It was detected in the
periplasmic fraction of the flgB rod mutant (lane 1; see
above) and of a strain with a mutation in flgJ (lane 11) but
not in the periplasmic fractions of strains with mutations in
flhA (lane 2), flhB (lane 3), fliH (lane 4), fliI (lane 5), fliO (lane 6),
fliP (lane 7), fliQ (lane 8), fliR
(lane 9), and fliJ (lane 10). The same results were obtained when the hook protein FlgE was used as the export substrate (data not
shown).
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Components necessary for export of FliC, flagellin. Flagellin presents a special case, since the switch in export specificity from hook-type proteins to filament-type proteins must occur before flagellin can be exported, and this switch is dependent on completion of the hook. Since we were now dealing with the external compartment rather than the periplasmic compartment, we used a flgK (first hook-filament junction protein; hook-associated protein 1) mutant as a control. This permitted assay of export of monomeric flagellin without the complication of filament assembly. The cells were grown at the permissive temperature of 30°C and shifted to the restrictive temperature of 42°C, and then export of flagellin to the external medium was monitored. This protocol assumes that preexisting copies of a temperature-sensitive component will become nonfunctional upon the temperature shift. This assumption may not always be justified, since a component tightly constrained by surrounding structure might continue to function normally, as we in fact found with FliP (see below).
We had in our collection temperature-sensitive strains with mutations in flhA, flhB, fliH, fliI, fliO, and fliP but not in fliQ, fliR, fliJ, and flgJ. We found flagellin in the culture supernatant of the control strain (wild type except for the flgK mutation) (Table 3 and Fig. 3, lane 1) but not in the supernatants of flhA, flhB, fliH, fliI, and fliO mutants (Table 3 and Fig. 3, lanes 2 to 6). Unexpectedly, we found flagellin in the supernatant of the fliP mutant (lane 7). However, in this strain (which is not the same one used for Fig. 2), the mutation (V42F) was at a position which would place it in the first membrane span of the mature protein, following signal peptide cleavage. (As a prokaryotic cytoplasmic membrane protein, FliP is unusual in undergoing such cleavage [28].) We suspect that this residue may be so highly constrained in the membrane that it retains function following the temperature upshift and so yields a false-positive result. We think it likely that FliP is needed for export of flagellin, as it is for other export substrates.
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The MS ring and the cytoplasmic C rings are required for export of all substrates. Morphological assembly studies (13, 16, 17) have indicated that the MS ring (FliF) and the cytoplasmic C ring (FliG, FliM, and FliN) are necessary for flagellar assembly to proceed to the external structures such as the rod. Consistent with this finding, we found that export to the periplasm of the substrates tested did not occur in fliF, fliG, fliM, and fliN mutants (data not shown). However, we interpret these results to mean that the MS and C rings provide the necessary structural environment for the export apparatus, not that they are themselves directly involved in export.
FliE is a prerequisite for hook-type protein export. FliE is a structural component of the basal body, but its location and function are not known. When the periplasmic fraction of a fliE mutant was examined with anti-FlgD antibody, no FlgD was detected (Fig. 4, lane 5).
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Export of hook-type proteins in outer ring (flgA, flgH, and flgI) mutants. Kubori et al. (16) found that flgD mutants were capable of assembling L rings onto the rod but far less so than wild-type cells, suggesting that the processes of L-ring assembly and addition of the hook-capping protein, FlgD, to a mature rod were somehow interconnected. This led us to question whether the interconnection might be reciprocal; in other words, might the existence of an L ring be important or necessary for export of hook-type proteins across the outer membrane? We therefore examined mutants defective in flgA (necessary for P-ring assembly), flgI (structural gene for the P ring, which is required for L-ring assembly), and flgH (structural gene for the L ring). The periplasmic and culture supernatant fractions from these mutants were subjected to immunoblotting with anti-FlgD antibody. FlgD was detected in the periplasmic fraction but not in the culture supernatant fraction (Fig. 5). The same result was obtained with FlgE (data not shown).
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Export of hook protein is impaired in flgD mutants. If the FlgD protein is important for L-ring formation, and if L-ring formation is important for export, the presence of a mutation in flgD might impair export of the other hook-type protein, FlgE. Consistent with this idea, we found that most flgD mutants resulted in FlgE accumulating exclusively in the periplasm. However, several strains with point mutations, such as SJW157, resulted in approximately equal amounts of FlgE in the periplasm and the external medium (data not shown), confirming results reported previously by Ohnishi et al. (29).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The rod and hook proteins belong to a single export class. It was known from previous work that the hook protein FlgE and its capping protein FlgD belonged to a different export class from the filament-type proteins (the hook-filament junction proteins FlgK and FlgL, flagellin [FliC], the filament-capping protein FliD, and the anti-sigma factor FlgM). In other words, export of the hook-type proteins proceeds at an earlier stage, during which export of the filament-type proteins is blocked because of the specificity of acceptance of substrates by the export component FlhB. Subsequently, the specificity is reversed such that hook-type proteins are rejected as export substrates and filament-type proteins are accepted. Since rod assembly precedes hook assembly, it seemed reasonable to ask whether the rod proteins might also represent a distinct export class. We found that hook-type proteins can be exported regardless of whether rod protein export is occurring. Thus, the rod proteins and the hook-type proteins appear to belong to the same export class, which we call the rod/hook-type class of export substrates.
This leaves another question, which is how rod proteins get assembled ahead of the hook-type proteins, if they can be exported simultaneously. This is a general question within a given export class and one to which we do not know the answer. Possibly some export substrates are kinetically accepted ahead of others. We know, for example, that interference can occur between exported substrates, since overproduction of FlgG has a dominant negative effect on flagellar assembly (data not shown). Alternatively, the sorting may occur at the assembly level so that exported hook protein is left unassembled while the rod structure is under construction.The flagellum-specific export apparatus contains at least eight
general components.
In this study, we have established that all
eight of the suspected major components of the flagellum-specific
protein export apparatus
FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and
FliRare indeed components of this apparatus.
FliJ is an export component, necessary for export of at least some
substrates.
FliJ, a cytoplasmic protein, was found to be necessary
for export of at least FlgD and FlgE. A plausible role would be as a
cytoplasmic chaperone, as has been suggested by Stephens et al.
(30) in the case of fliJ of Caulobacter
crescentus, since it shares some of the general properties of
other chaperones: small size, high charge density, and high predicted
-helical content. We were unable to test whether it is necessary for
export of flagellin, but we suspect not, since the role of a
flagellin-specific chaperone has been postulated for another protein,
FliS (39). Interestingly, fliJ has a leaky mutant
phenotype (38), so that export of its putative substrates
can occur with low probability in its absence; this is a feature that
has also been noted with fliS. We are currently trying to
obtain evidence for physical interactions between FliJ and the proteins
whose export it facilitates.
FliE, a basal-body component, is required for export of some substrates. FliE is a basal body protein whose location is not known with certainty but which is postulated to be a component of the rod (17). We found that it was required for the export of at least two other substrates, the hook protein FlgE and the hook-capping protein FlgD. The reason for this requirement is not known. One possibility could be that it interacts with the periplasmic domains of integral membrane components such as FliO or FliP and facilitates passage of the other substrates into the periplasmic space.
FlgJ is essential for rod assembly but not for export of hook-capping protein or hook protein. Until this study, essentially nothing was known about the FlgJ protein. Although not needed for export of the substrates tested, it is necessary for the flagellar assembly process, since flgJ mutants lack a rod structure (13, 16). One possibility would be that it functions as a scaffolding protein for rod assembly, in a similar fashion as FlgD does for hook assembly. Another would be that it interacts with the peptidoglycan layer. Consistent with the latter suggestion, Dijkstra and Keck (3) have noted that the FlgJ sequence contains a muramidase consensus motif.
The flagellar export apparatus. Although the primary purpose of this study was to identify export apparatus components and the range of export substrates that they each handle, it is perhaps worthwhile to make a few comments concerning the possible locations and functions of the various components (Fig. 6). The proposed location of the membrane-associated components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) is in a central pore within the MS ring of the flagellar basal body. There is physical evidence that such a pore exists (14), and there is evidence that the FliP and FliR proteins are associated with the basal body (6). We have recently established that FlhA is also associated with the basal body (23) and are currently seeking equivalent evidence for FlhB, FliO, and FliQ. FlhA and FlhB have large soluble domains that presumably protrude into the cytoplasm within the cavity that lies at the center of the cytoplasmic C ring of the motor. We suggest that export substrates, with the aid of possible cytosolic chaperones such as FliJ or FliS, are brought into contact with FliI (an ATPase) and FliH (function unknown), which then interface with the soluble domains of FlhA and FlhB and release their substrates. These substrates are then translocated across the plane of the membrane through a complex consisting of FliO, FliP, FliQ, FliR, and the transmembrane domains of FlhA and FlhB; the point at which ATP hydrolysis occurs is not known. FliK in some fashion enables FlhB to recognize when hook assembly is complete and to switch its export specificity to filament-class substrates. FliE may be located at the periplasmic side of the lumen and facilitate transfer of the export substrates into the lumen. The role of FlgJ is unclear; it does not seem to play a role in the export process but may participate in some aspect of assembly of subunits once they have been exported.
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The basal body L ring can be regarded as a component of the export apparatus at the stage of rod completion and hook initiation. We thought initially that our definition of the flagellar export apparatus should be confined to components that were responsible for translocating export substrates across the plane of the cytoplasmic membrane. Our rationale for using this restrictive definition was that subsequent events were merely diffusion down a tunnel. However, at one critical point in assembly, when the periplasmic rod has just been completed and the external hook is just being initiated, the structure has to penetrate the outer membrane; only after that has happened does the simple diffusion model once again apply.
The results from this study make it clear that the L ring is essential, or at least very important, for this penetration process and, further, that assembly of the L ring is to a considerable extent dependent on one of the export substrates themselves, namely, the hook-capping protein FlgD. We therefore suggest that for a brief period of time there is an additional element in the export apparatus, consisting of either the L ring alone or an L ring-FlgD complex. (The need for the P ring is probably a result of the fact that L-ring assembly appears to be dependent on preassembly of the P ring [16], not because the P ring participates directly in penetration of the outer membrane.) In type III secretion systems for virulence factors there exists in the outer membrane a large homomultimeric annular complex made of molecules called secretins. The function of this complex is believed to be to permit the passage of export substrates across the outer membrane. Also, a recent ultrastructural study of Salmonella has revealed a needle-like structure that contains known components of the type III virulence factor export system (15). Among these components was InvG, which is believed to be a secretin and whose homolog, HrcC of P. syringae, is known to be necessary for substrate to pass the outer membrane (2). It seems reasonable to postulate that InvG comprises the outer ring that is seen in the needle-like structure. We suggest that the L ring of the flagellar basal body may transiently correspond to a secretin-like annulus, in addition to its long-term function as part of a bushing to stabilize flagellar rotation.| |
ACKNOWLEDGMENTS |
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We thank Kazuhiro Kutsukake for the gift of unpublished plasmids, Shigeru Yamaguchi for the gift of unpublished strains and for unpublished information about the leakiness of fliJ mutants, and Gabriele Miller and May Kihara for technical assistance.
This work was supported by USPHS grant AI12202.
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ADDENDUM IN PROOF |
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Our understanding of the role of FlgJ in flagellar assembly has significantly advanced with the concurrent publication of a paper by Nambu et al. (T. Nambu, T. Minamino, R. M. Macnab, and K. Kutsukake, J. Bacteriol. 181:1555-1561, 1999).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114. Phone: (203) 432-5590. Fax: (203) 432-9782. E-mail: robert_macnab{at}qm.yale.edu.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 4. | Doi, H., and K. Kutsukake. Unpublished data. |
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Journal of Bacteriology, March 1999, p. 1388-1394, Vol. 181, No. 5
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