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Journal of Bacteriology, August 2000, p. 4207-4215, Vol. 182, No. 15
Department of Molecular Biophysics and
Biochemistry, Yale University, New Haven, Connecticut
06520-8114,1 and Izumi Campus,
Meiji University, Suginami, Tokyo 168-0064, Japan2
Received 29 February 2000/Accepted 15 May 2000
We isolated and characterized spontaneous mutants with defects in
the 147-amino-acid Salmonella protein FliJ, which is a
cytoplasmic component of the type III flagellar export apparatus. These
mutants, including ones with null mutations, have the ability to form
swarms on motility agar plates after prolonged incubation at 30°C;
i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the
N-terminal 73 amino acids of FliJ, one-half of the protein. At 30°C,
overproduction of this mutant protein improved, to wild-type levels,
both motility and the ability to export both rod/hook-type (FlgD; hook
capping protein) and filament-type (FliC; flagellin) substrates. At
42°C, however, export was inhibited, indicating that the mutant FliJ
protein was temperature sensitive. Taking advantage of this, we
performed temperature upshift experiments, which demonstrated that FliJ
is directly required for the export of FliC. Co-overproduction of FliJ
and either of two export substrates, FliE or FlgG, hindered their
aggregation in the cytoplasm. We conclude that FliJ is a general
component of the flagellar export apparatus and has a chaperone-like
activity for both rod/hook-type and filament-type substrates.
The Salmonella flagellum
is a complex structure; most of its components lie beyond the
cytoplasmic membrane (8) and, therefore, have to be
exported. With the exception of the basal-body P- and L-ring proteins,
these components utilize a specialized export system called the type
III flagellar export apparatus. Many other type III systems exist for
the export of virulence factors (4).
The substrate specificity of the flagellar export apparatus changes on
completion of the hook structure (9-11), and on the basis
of this difference, its export substrates are divided into two classes.
One is the rod/hook-type export class, comprising FliE (basal-body
protein); FlgB, FlgC, and FlgF (proximal rod proteins); FlgG (distal
rod protein); FlgE (hook protein); FlgD (hook capping protein); and
FliK (hook length control protein). The other is the filament-type
export class, comprising FlgK (first hook-filament junction protein),
FlgL (second hook-filament junction protein), FlgM (anti-sigma factor),
FliC (flagellin), and FliD (filament capping protein).
Six membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) were
found to be required for all flagellar protein substrates tested to be
translocated across the plane of the cytoplasmic membrane
(11). These membrane components are believed to be located
in a patch of membrane within the annular pore of the basal-body MS
ring (2). Two soluble proteins (FliH and FliI) were also
found to be required. Recently, we provided evidence for a number of
interactions among export components and between export components and
their substrates (12).
FliJ is another cytoplasmic protein implicated in export: mutants with
defects in FliJ fail to export rod/hook-type substrates into the
periplasm. However, for technical reasons, we could not test whether
FliJ also might be required for the export of filament-type substrates.
This possibility was raised by our subsequent finding that FliJ binds
to both filament-type substrates and rod/hook-type substrates
(12). We also found that FliJ interacts with the soluble
components FliH and FliI and the soluble cytoplasmic domains FlhAC and FlhBC of the export apparatus,
strengthening the argument that FliJ itself is involved in the export process.
FliJ is a small protein consisting of 147 amino acids (19).
It shares several structural features with the Salmonella
flagellar proteins FliS, FlgN, and FliT, which are putative chaperones
for the filament-type substrates FliC, FlgK and FlgL, and FliD,
respectively (3, 26), and with other members of the type III
cytoplasmic chaperone family, including the Yersinia Syc
(for specific Yop chaperones) proteins (4, 23). These
chaperones are small proteins (ca. 15 to 20 kDa) with a high proportion
of charged residues; they are predicted to have a large amount of
In addition to possessing these characteristics, FliJ has a sequence
with a high probability of
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of FliJ in Flagellar Protein Export in
Salmonella
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-helical structure, including potential amphipathic helices toward
their C termini.
-helical coiled-coil structure near its N
terminus (24) (Fig. 1A), which
suggests that FliJ is likely to engage in such an interaction, either
with itself, to form a homodimer, or with its substrates.
View larger version (14K):
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FIG. 1.
(A) Estimation of the total (dimer plus trimer)
probability that a given residue of the 147-amino-acid wild-type
sequence of FliJ will participate in an -helical coiled-coil
structure. The probability for residues 14 through 42 is 
80%. Data
were calculated by the MultiCoil program of Wolf et al.
(24). In the schematic representation of the wild-type FliJ
sequence (bottom), the region with a high probability of having
coiled-coil structure is shaded. (B) Mutant FliJ proteins analyzed in
this study. Sequence present is shown in solid bars, with the beginning
and ending residues indicated. SJW104 has an in-frame deletion. SJW135
and SJW277 are nonsense mutants. The SJW104 and SJW277 FliJ products
are referred to throughout this paper as FliJ(
13-24) and FliJ-N73,
respectively.
Stephens et al. (17) hypothesized that FliJ of Caulobacter crescentus might be a chaperone. We tentatively proposed (11) that FliJ of Salmonella might be a chaperone specific for rod/hook-type export substrates, based on its role in their export. In the present study, we showed that FliJ is also required for the export of filament-type substrates and that it prevents aggregation of export substrates in the cytoplasm.
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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
1. The fliJ mutants were
spontaneous, essentially nonflagellated derivatives of wild-type
Salmonella strain SJW1103 (25) and were mapped by
transduction using bacteriophage P22. L broth (LB), M9, and soft
tryptone agar plates were used as described previously (10,
11). Ampicillin and Casamino Acids (CA) were added to the media
at final concentrations of 100 µg/ml and 0.3%, respectively.
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DNA techniques. PCR and cloning were performed as described previously (11). DNA sequencing was carried out with the modified T7 DNA polymerase Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) and various synthetic primers.
Preparation of soluble cellular fractions and culture
supernatants.
Overnight 60-µl cultures of SJW135gK carrying
pTrc99A, pMM404, pRCJ104, or pRCJ277 were inoculated into 3-ml volumes
of M9 plus CA medium containing ampicillin and incubated at either 30 or 42°C with shaking. With pRCJ104 and pRCJ277,
isopropyl-
-D-thiogalactopyranoside (IPTG) was also added
at the concentrations indicated (see Fig. 4). When the cells reached an
optical density at 600 nm (OD600) of 1.0 to 1.2, 1.5 ml of
each culture was centrifuged and the culture supernatants were
collected. The cells of each culture were resuspended in 300 µl of
B-PER reagent (Pierce, Rockford, Ill.) by up-and-down pipetting. After
1 min of vortexing, centrifugation was carried out at 20,800 × g for 5 min and the pellet was discarded, leaving the soluble
intracellular fraction (cytoplasm plus periplasm) in the supernatant.
The proteins in the intracellular fraction and the culture supernatant
were each precipitated with 10% trichloroacetic acid, suspended in
Tris-saturated sodium dodecyl sulfate (SDS) loading buffer, and boiled
for 3 min as described previously (11).
Immunoblotting. Immunoblotting with polyclonal anti-FlgD and anti-FliC antibodies and monoclonal anti-FLAG (Sigma, St. Louis, Mo.) and anti-His (Pierce) antibodies was performed as described previously (11).
Temperature upshift experiment. A 60-µl volume of an overnight culture of SJW135gK carrying pTrc99A, pMM404, or pRCJ277 was inoculated into 3 ml of LB medium containing ampicillin (and, in the case of pRCJ277, 1 mM IPTG). The culture was grown at 30°C with shaking until the cells reached an OD600 of 1.2 to 1.4 and then centrifuged, and the pelleted cells were washed twice with M9 plus CA medium containing ampicillin, prewarmed to 42°C. The cells were then suspended in 3 ml of the same medium and incubated at 42°C for 60 min in the absence of IPTG. Intracellular and supernatant fractions were prepared as described above and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting with anti-FliC antibody.
Pulse-labeling with [35S]methionine.
Overnight
cultures (60 µl) of SJW1368 [(cheW-flhD)] carrying
pMM1004, pMM1004iJ, pMM203, or pMM203iJ were inoculated into 3-ml
volumes of LB containing ampicillin and incubated at 37°C with
shaking. When the cells reached an OD600 of 0.8, 1.5 ml of each culture was centrifuged. The cells were washed twice with M9
medium containing ampicillin. Constant numbers of cells were resuspended in M9 medium containing ampicillin, and 150 µl of culture
was incubated at 37°C for 20 min. Then 1 µl of 1.5-µCi/µl [35S]methionine (Amersham Pharmacia, Piscataway, N.J.)
was added, and incubation was continued for another 5 min. After
centrifugation, the pelleted cells were suspended in 30 µl of 50 mM
Tris-HCl (pH 8.0) containing 1 mM EDTA and 1% SDS and heated at
100°C for 3 min. A 470-µl volume of TNE (50 mM Tris-HCl [pH 8],
150 mM NaCl, 0.1 mM EDTA) containing 0.1% Thesit (Roche Molecular
Biochemicals, Indianapolis, Ind.), 2.5 µl of a 1-mg/ml solution of a
trypsin inhibitor (Sigma), and 1 µl of a solution of anti-FLAG
antibody were added. After overnight incubation of the cells at 4°C
on a rotator, 5 mg of protein A-Sepharose CL-4B (Pharmacia, Piscataway, N.J.) was added and incubation was continued for another 1 h at 4°C on a rotator. After centrifugation, the pellets were washed twice
with 500 µl of TNE containing 0.1% Thesit and once with 500 µl of
50 mM Tris-HCl (pH 8.0). The pellets were resuspended in 50-µl
volumes of SDS loading buffer and boiled for 3 min. The immunoprecipitated samples were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, Mass.). After the membranes were dried, they were exposed to
X-ray film.
Cell fractionation into aggregated and soluble materials.
Volumes (60 µl) of overnight cultures of SJW1368
[(cheW-flhD)] carrying pMM1004, pMM1004iJ, pMM203, or
pMM203iJ were inoculated into 3-ml volumes of LB containing ampicillin,
and the cells were grown at 37°C with shaking. Then IPTG was added to
a final concentration of 1 mM, and incubation was continued for another
3 h. A 1.5-ml volume of each culture was centrifuged. The pelleted
cells were resuspended in 300 µl of B-PER reagent by up-and-down
pipetting. After 1 min of vortexing, centrifugation was carried out at
20,800 × g for 5 min. The supernatants (soluble
fraction) and pellets (insoluble fraction) were collected separately.
The pellets were each resuspended in 300 µl of 50 mM Tris-HCl (pH
8.0)-0.5% NaCl.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of spontaneous fliJ mutants.
In an
attempt to understand the role of FliJ in the export process, we
isolated a set of 13 spontaneous fliJ mutants. Those used
explicitly in this study are listed in Table 1. All of the fliJ mutations resulted in severe inhibition of swarming on
motility agar plates (Fig. 2A). However,
after prolonged incubation, the mutants all formed distinct swarms
(Fig. 2B), indicating that they had a leaky motile phenotype; this was
most pronounced in the case of SJW277. In contrast, a mutant with a
defect in FlhA, which is a general component of the flagellar export
apparatus (11), did not swarm at all.
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Sequence analysis of the fliJ mutants. The fliJ mutant alleles were amplified by PCR, and the amplified fragments were sequenced by using various internal primers.
All of the fliJ mutations were fairly severe. They consisted of in-frame deletion, nonsense, and frameshift mutations; there were no missense mutations. The mutations for those strains used in the present study are represented graphically in Fig. 1B. The most severe mutation was that in SJW135, which generated an ochre (TAA) stop codon at position 15, and so presumably SJW135 is a null mutant. Since this strain, like the others, displayed a leaky motile phenotype (Fig. 2B), we conclude that FliJ, though important, is not absolutely required for the flagellum-specific export process. We also conclude that the mutation in SJW135 does not have a polar effect on the only downstream gene in the operon, fliK, since this would result in a polyhook phenotype (long hooks without filaments) and total failure to swarm (16, 18). The fliJ mutation in SJW277, the strain which swarmed significantly better than the others (Fig. 2B), created an amber (TAG) stop codon at position 74, resulting in an N-terminal fragment of 73 amino acids (FliJ-N73) that lacks the C-terminal 73 amino acids; the fragment thus consists of exactly half of the wild-type sequence.Complementation effects of mutant FliJ proteins on
motility.
The motility phenotype of SJW277 suggested that
the N-terminal region of FliJ is important for its function
but that its C-terminal region might be dispensable to some degree.
To test this hypothesis, we cloned into pTrc99A the
fliJ alleles from SJW1103 (wild type), SJW104 [which
contains a 12-codon in-frame deletion near the 5' end of the gene and
encodes FliJ(13-24) (Fig. 1B)], and SJW277 (encoding FliJ-N73) to
give pMM404, pRCJ104, and pRCJ277, respectively. These plasmids were
introduced into SJW135 (fliJ), and the resulting transformants were inoculated on plates of soft tryptone agar with or
without 0.1 mM IPTG. In the absence of IPTG, pRCJ277 (FliJ-N73) complemented the motility of SJW135 to a considerable degree (Fig. 3, left panel), causing significantly
better motility than that of SJW277 (Fig. 2A); even when
uninduced, pTrc99A-based plasmids generally result in
higher-than-chromosomal expression levels (13). Addition of
IPTG improved the complementation to almost the wild-type level.
pRCJ104, in contrast, did not complement at all, even in the presence
of IPTG, indicating that damage to the N-terminal sequence of FliJ has
severe consequences. pMM404 (wild-type FliJ) complemented fully in the
absence of IPTG but, consistent with previous observations
(12), complemented poorly in the presence of IPTG; i.e., it
exerted a negative or inhibitory multicopy effect.
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) 0.1 mM IPTG.
Complementation effects of mutant FliJ proteins on the export of
flagellar components.
We next examined whether the stimulation of
motility caused by overproduction of FliJ-N73 was a consequence of
stimulation of the export of flagellar components. We used SJW135gK
(fliJ flgK::Tn10) as the host to
prevent filament assembly and thus facilitate detection of the export
of substrates. We chose the hook capping protein FlgD as an example of
a rod/hook-type substrate and FliC (flagellin) as an example of a
filament-type substrate (11). SJW135gK was transformed with
pTrc99A, pMM404 (wild-type FliJ), pRCJ277 (FliJ-N73), and pRCJ104
[FliJ(13-24)], and intracellular fractions and culture
supernatants were prepared. After SDS-PAGE, immunoblotting was carried
out with anti-FlgD and anti-FliC antibodies.
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13-24)] (Fig. 4D), even though intracellular levels of the
protein were higher than those of the wild type (Fig. 4C), in which
FlgD is presumably depleted by the export process. Note that failure to
export any of the substrates that assemble prior to penetration of the
outer membrane (i.e., the rod proteins) will by itself result in
failure of FlgD to be exported into the external medium; however, in a
previous study (11), we have shown that the presence of an
FliJ(13-24) allele results in the failure of FlgD to be exported
even into the periplasm.
With SJW135gK carrying pRCJ277, the intracellular levels of flagellin
(FliC) increased with the IPTG concentration (Fig. 4E), reaching a
constant level by 50 µM. This increase is presumably a result of
export of the anti-sigma factor, FlgM, when substrate specificity
switches upon completion of the hook (5, 6). The
extracellular levels of flagellin also increased with the IPTG
concentration (Fig. 4F) but never fully reached the wild-type level,
even at 1 mM IPTG. Comparing the IPTG dependencies of intracellular and
exported FliC levels in Fig. 4E and F, we conclude that a large part,
but probably not all, of the stimulation of FliC export is a
consequence of enhanced FliC synthesis. Thus, although overproduction of FliJ-N73 results in stimulation of the export of a filament-type protein (FliC) as well as that of a rod/hook-type protein (FlgD), it is
difficult to judge from these experiments the extent to which the
effect of FliJ on FliC export is direct.
With pRCJ104 [FliJ(13-24)], no FliC was detected in either the
intracellular fraction or the culture supernatant, regardless of the
IPTG concentration (data not shown). The absence of this protein in the
intracellular fraction presumably reflects a failure to export the
flagellum-specific anti-sigma factor FlgM and to activate
fliC gene expression (5, 6).
High temperature inhibits the export function of FliJ-N73.
Preliminary work had indicated that the swarm behavior of
fliJ mutants, including SJW277, displayed some temperature
sensitivity (data not shown). To examine the effect of high temperature
on the function of FliJ-N73 with respect to protein export, we took SJW135gK/pRCJ277 cell cultures that had been grown at 42°C in the
presence of various IPTG concentrations, prepared the intracellular fractions and culture supernatants, and performed immunoblotting with
anti-FlgD. With FliJ-N73, intracellular levels of FlgD (Fig. 5A, lanes 3 to 7) were independent of the
IPTG concentration and were higher than those with uninduced wild-type
FliJ (lane 2). With FliJ-N73, no FlgD was detected in the culture
supernatants, even at 1 mM IPTG (Fig. 5B, lanes 3 to 7), in contrast to
the situation with wild-type FliJ (lane 2). We conclude that although overproduced FliJ-N73 can support a wild-type level of export function
at 30°C, it cannot function at 42°C.
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Temperature shift experiment with cells carrying pRCJ277. Because flagellin synthesis (5, 6) and export (10) are dependent on the completion of hook assembly, we were concerned that the stimulation of flagellin export by FliJ-N73 (Fig. 4F) might be an indirect effect caused by stimulation of hook protein export and assembly and FlgM export. We were able to take advantage of the temperature sensitivity of FliJ-N73 to examine whether FliJ directly stimulates flagellin export.
SJW135gK cells transformed with either pTrc99A, pMM404, or pRCJ277 were grown at 30°C in LB (plus 1 mM IPTG for pRCJ277 only). The cells were then washed twice in M9 plus CA containing ampicillin (prewarmed to 42°C), resuspended in the same medium, and incubated at 42°C for 60 min in the absence of IPTG. The intracellular fraction and culture supernatant were then subjected to SDS-PAGE and immunoblotting with anti-FliC antibody. Even though the intracellular level of FliC for cells transformed with pRCJ277 (Fig. 6A, lane 3) was the same as that of cells transformed with pMM404 (lane 2), the amount in the culture supernatant was much lower in the former (Fig. 6B, lane 3) than the latter (lane 2), establishing that FliJ is directly involved in the export of filament-type substrates.
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Steady-state levels of FliJ-N73 are lower than those of wild-type
FliJ.
The need for IPTG induction with FliJ-N73 in order to reach
wild-type levels of substrate export and swarming may mean that the
truncated protein is less functional or less stable. To examine this
issue, we first attempted to use anti-FliJ antibody, but we found that
the intracellular levels of FliJ-N73 were too low to be detected.
We therefore constructed pTrc99A-based plasmids pMM406, pMM411,
and pMM410, which encode N-terminally His-tagged versions of wild-type
FliJ, FliJ(13-24), and FliJ-N73, respectively. We transformed
SJW135 with these plasmids and examined the intracellular levels of the
His-tagged forms of FliJ by immunoblotting with anti-His antibody (Fig.
7). Even after induction with 1 mM IPTG, the amount of FliJ-N73 (lane 7) was considerably lower than that of
wild-type FliJ produced under noninducing conditions (lane 1). We
conclude that the primary reason for the reduced efficacy of FliJ-N73
is its low intracellular level. Pulse-chase data (not shown) also
indicate that it is less stable than wild-type FliJ.
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13-24) under noninducing conditions
(lane 2) was only slightly less than that of wild-type FliJ and
increased with induction (lanes 3 and 4); this establishes that
FliJ(13-24) is intrinsically incapable of sustaining substrate export (Fig. 4) or swarming (Fig. 2 and 3).
Effect of the level of full-length FliJ on motility and protein export. Studies of FliJ-N73 yielded valuable information about the roles of FliJ, especially its role in the export of filament-type proteins. However, we also wanted to know the effect of subnormal levels of wild-type FliJ on export and motility. Manipulation of FliJ levels by using IPTG was pointless since uninduced cells transformed with pMM404 (carrying the wild-type gene) swarmed as well as wild-type SJW1103 cells (cf. Fig. 3, left panel, and Fig. 2A).
We therefore decided to lower the uninduced level of FliJ by introducing an opal codon into the gene and relying on the ca. 2% natural misreading of this as a Trp codon (13, 14). In previous studies, there had been suitable Trp codons in the gene of interest that could be replaced by an opal codon; this was not the case with fliJ. At position 4, which in the wild-type gene is a His codon, we therefore created an opal codon (i.e., we made an H4opal substitution). This should then result in a null product (H4opal) 98% of the time and a conservative His
Trp substitution (H4W) 2% of the time.
The resulting pTrc99A-based plasmid, pMM450, restored wild-type
swarming (Fig. 8A) and wild-type FlgD and
flagellin export levels (Fig. 8B and C, lanes 3) to SJW135
(fliJ) cells, even under noninducing conditions. We
attribute this to the fact that the level of uninduced expression from
a pTrc99A-based plasmid is about 20-fold higher than the level of
chromosomal expression (13), almost offsetting the 50-fold
decrease resulting from the opal mutation.
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FliJ affects inclusion body formation by export substrates. FliJ shares several structural features with type III cytoplasmic chaperones (see the introduction). It might, therefore, have an activity which prevents export substrates from premature aggregation in the cytoplasm. To investigate this possibility, we used FliE and FlgG as export substrates, because overproduction of these proteins results in formation of inclusion bodies (reference 12 and data not shown). We constructed pMM1004, pMM1004iJ, pMM203, and pMM203iJ, which are pTrc99A-based plasmids encoding N-His-FLAG-FliE, N-His-FLAG-FliE plus N-His-FliJ, N-His-FLAG-FlgG, and N-His-FLAG-FlgG plus N-His-FliJ, respectively (Table 1). Where fliJ was present, it was the downstream gene.
We first tested whether FliJ affects the synthesis of these export substrates by carrying out radiolabeling experiments with [35S]methionine, using SJW1368 [(cheW-flhD)] as the host to avoid contributions from
chromosomal gene expression. After being radiolabeled, the cells were
spun down, dissolved in SDS buffer, and diluted, and the FLAG-tagged
proteins were immunoprecipitated with anti-FLAG antibody.
N-His-FLAG-FliE and FlgG synthesis was not inhibited by N-His-FliJ
(Fig. 9A, lanes 2 and 4); in fact, levels
were slightly higher in the presence of this protein than in its
absence (lanes 1 and 3). N-His-FliJ was also detected (lanes 2 and 4),
indicating that it coimmunoprecipitated with the FLAG-tagged proteins;
it was not detected in their absence (data not shown). This result supports our previous observation, by affinity blotting, that FliJ
interacts with export substrates (12).
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(cheW-flhD)] cells carrying pMM1004, pMM1004iJ,
pMM203, or pMM203iJ were solubilized with B-PER reagent and
fractionated by centrifugation. The soluble and pellet fractions were
then subjected to SDS-PAGE and immunoblotted with anti-FLAG antibody.
For cells with pMM1004 or pMM203, most of the FliE or FlgG was found in
the pellet fraction (Fig. 9B, lanes 1 and 5), confirming previous
observations that these proteins form inclusion bodies when
overproduced. However, in the presence of FliJ (plasmids pMM1004iJ and
pMM203iJ), the amount of aggregated material was severely decreased, to
below the detection threshold in the case of FliE (lane 3) and to close
to the detection threshold in the case of FlgG (lane 7). The level of
FlgG in the soluble fraction was also reduced by FliJ (cf. lanes 6 and
8), but not by so much; in fact, the amount in the soluble fraction
(lane 8) now exceeded that in the pellet fraction (lane 7). The level of FliE in the soluble fraction (lane 4), as with that in the pellet
fraction (lane 3), was too low to be detected.
Thus, our data strongly suggest that FliJ prevents export substrates
from premature aggregation. The decrease in the soluble amounts
presumably indicates that they are susceptible to protease digestion,
perhaps because, of the export components, only FliJ is being overproduced.
In parallel experiments with FlgD and FlgE, which are stable in the
cytoplasm and do not form aggregates even when overproduced, co-overproduction of FliJ had no effect on their intracellular levels
(data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Most flagellar components are translocated across the cytoplasmic membrane via the type III flagellar export apparatus. We have previously established that at least six membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) and two cytoplasmic proteins (FliH and FliI) constitute this apparatus (11).
In this study, we examined the role of a third cytoplasmic protein, FliJ, in the export process. We provided evidence that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity in terms of preventing substrate aggregation.
All fliJ mutants display a leaky motile phenotype. All of the spontaneous fliJ mutants that we isolated displayed a leaky motile phenotype (Fig. 2). This was true even of SJW135, which encodes only the N-terminal 14 amino acids and so presumably can be regarded as a null mutant. In other words, although FliJ is required for efficient export of substrates, export can occur, with a low probability, in its absence.
The N-terminal 73 amino acids of FliJ are necessary and sufficient for full function. One mutant, SJW277, formed significantly larger swarms than the rest (Fig. 2). Its fliJ allele encodes just the N-terminal 73 amino acids (FliJ-N73) (Fig. 1B). Overproduction of this polypeptide resulted in stimulation of both motility and export of flagellar components to wild-type levels (Fig. 3 and 4). Thus, the N-terminal 73 amino acids of FliJ can supply full function. This idea is strongly supported by the fact that an N-terminally His-tagged variant of FliJ-N73, encoded by plasmid pMM410, complemented swarming to wild-type levels even without induction (T. Minamino and R. M. Macnab, unpublished results). We do not understand the role of the C-terminal region of FliJ in its function but suggest that it may contribute to maintaining the proper conformation and stability of the N-terminal region.
The N-terminal region is essential for FliJ function. Overproduction of a version of FliJ lacking residues 13 to 24 did not improve either its motility (Fig. 3) or its export (Fig. 4).FliJ acts as a general component in the export pathway. We originally suggested that FliJ might be a chaperone specific for rod/hook-type proteins (11) but then found that FliJ bound to both rod/hook-type and filament-type proteins (12). This raised the question of whether FliJ might also be required for export of filament-type proteins.
We found that FliJ-N73 overproduction stimulated the export of flagellin (FliC) as well as that of the hook-capping protein (FlgD) (Fig. 4). However, this result should be treated with caution, since the stimulation of FliC export could be attributed at least in part to stimulation of hook completion (which is needed for export of filament-type substrates to commence) and to stimulation of FliC synthesis (Fig. 4E). (The latter effect is presumably a consequence of export of the anti-sigma factor FlgM, which can only occur after completion of hook assembly [5, 6].) We therefore took advantage of the fact that FliJ-N73 behaves as a wild type when overproduced at 30°C (Fig. 3 and 4) but as a mutant when produced at 42°C (Fig. 5). Recall that export of filament-type proteins is dependent on a switch in the substrate specificity that occurs only on completion of the hook structure. In a temperature shift experiment, we found that even though hook assembly had been completed at the permissive temperature and that intracellular levels of FliC were normal, a shift to the restrictive temperature severely inhibited FliC export (Fig. 6). Therefore, we feel confident in concluding that FliJ is a general component of the flagellar export apparatus, which is required for efficient export of both rod/hook-type and filament-type export substrates.FliJ inhibits substrate aggregation in the cytoplasm. Several export substrates, including FliE and FlgG, form aggregates when overproduced. Co-overproduction of FliJ greatly reduced aggregation (Fig. 9B). Even though the co-overproduction of FliJ did not reduce FliE or FlgG synthesis, it did cause a decrease in their steady-state intracellular levels. Because monomeric flagellar proteins are not completely folded and their termini are disordered (20-22), they may be more sensitive to proteolysis than if they are aggregated. Thus, at least under overproduction conditions, FliJ is unable to protect the substrates, perhaps because other essential components are also not being overproduced.
The molecular characteristics of FliJ versus those of other type
III chaperones.
In the flagellar export pathway, FlgN and FliT
specifically bind to FlgK and FlgL and to FliD, respectively
(3), and are important for their export; FliS is believed to
play a similar role for FliC (26). They are therefore
believed to be specific chaperones for these proteins. Similarly, in
the type III Yersinia Yop export pathway, some of the Yop
proteins associate with specific chaperones called Syc proteins in
order to be maintained in an export-competent conformation (1,
23). These chaperones have several common features: small size,
high charge, acidic pI (ca. 4.5 to 5.2), and the potential to form an
amphipathic -helix that has been proposed to mediate interactions
with their targets.
-helical residues; and a predicted amphipathic -helix
in the region of residues 85 to 105. However, it has an additional,
distinctive characteristic. We noted above that the N-terminal region
of FliJ is essential for its function. Within this region is a strongly
predicted, sharply bounded, coiled-coil structure (>80% probability
for residues Ala-14 through Leu-42) (Fig. 1A). The flagellar and Syc
chaperones differ considerably in terms of their propensity to form
coiled-coil structures, especially if analyzed by the more stringent
pairwise correlation algorithm of Wolf et al. (24) rather
than by the simpler, individual probability algorithm of Lupas et al.
(7). The Syc chaperones show essentially no predicted
coiled-coil structure. Both FlgN and FliT do have predicted coiled-coil
structure near the central portion of their sequences, FlgN at around
the 40% probability level and FliT at around the 20% level; FliS
shows zero probability throughout its structure. Thus, the data for
FliJ are striking, in terms of both the strength of the prediction
(>80%) and the N-terminal location of the predicted region. We do not
understand the significance of these differences. It should, however,
be remembered that a lack of coiled-coil structure is not necessarily
equivalent to a lack of amphipathic helical interactions. One obvious
question regarding FliJ is whether the predicted coiled coil is within a homodimer. Experiments addressing these and related questions are
under way.
What are the relative roles of FliJ and of FlgN and FliT? Unlike FlgN and FliT, FliJ does not display substrate specificity, because it binds to both rod/hook-type and filament-type export substrates (12) and is necessary for their transport (this study). Therefore, we conclude that FliJ is a general flagellar chaperone. FliJ binds to the other cytoplasmic export components, FliH and FliI, including the cytoplasmic domains of FlhA (FlhAC) and FlhB (FlhBC) (12). Perhaps its role is more intimately related to the export process itself and proteins like FlgN, FliS, and FliT play an earlier role in protecting their substrates in the time between their synthesis and their export.
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ACKNOWLEDGMENTS |
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We thank May Kihara and Gabrielle Miller for technical assistance and Gillian Fraser for helpful discussions.
This work was supported by USPHS grant AI12202.
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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}yale.edu.
Present address: International Institute for Advanced Research,
Matsushita Electric Industrial Company, Ltd., 1-7 Hikaridai, Seika,
Kyoto, 619-0237, Japan.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, August 2000, p. 4207-4215, Vol. 182, No. 15
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