Department of Molecular Biophysics and
Biochemistry, Yale University, New Haven, Connecticut
06520-8114,1 and Izumi Campus, Meiji
University, Suginami, Tokyo 168-0064, Japan2
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INTRODUCTION |
The MS ring of the flagellar basal
body of Salmonella is usually thought of in terms of its
role in motor function: as a mounting plate for the rotor element of
the motor/switch, also known as the cytoplasmic C ring. However, with
increasing attention being paid to the process of flagellar protein
export, which occurs by a type III pathway (7) and entails
delivery of the export substrates into the lumen of the nascent
structure, a second role for the MS ring is emerging, namely, as the
structure that houses the membrane components of the export apparatus.
The MS ring has a complex appearance, with two rings (M and S) and a
collar projecting beyond the cytoplasmic membrane into the periplasmic
space, yet it is constructed from subunits of a single 61-kDa protein,
FliF. It is estimated that there are about 26 subunits arranged as an
annulus that has a central pore about 10 nm in diameter
(10). The export apparatus has six integral membrane
components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) in addition to some
soluble components (FliH, the ATPase FliI, and putative chaperones
FliJ, FlgN, FliS, and FliT) (1, 2, 4, 16, 18, 19, 22). We
have argued (2, 15) that the logical location for the
membrane components of the export apparatus is in a patch of
specialized membrane within the core of the MS ring, so the apparatus
can deliver its substrates into the lumen. Thus far, two of these
components (FliP and FliR) have been shown to be associated with the
basal body (2); in the case of FliR, immunoelectron
microscopy positioned it in the vicinity of the cytoplasmic face of the
MS ring.
If the model of the MS ring enclosing the export apparatus is correct,
the question arises whether there are specific interactions between the
two structures, or whether the export apparatus is better thought of as
a floating island. This report describes a number of examples where a
specific FliF mutation can be suppressed by mutations in an integral
membrane component of the export apparatus, FlhA, suggesting that the
MS ring and the export apparatus do in fact interact.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
The fliF
parental mutant strains are derived from SJW1103 (34). The
pseudorevertants derived from these fliF mutants were mapped
by P22-mediated transduction to flhA in flagellar region II.
The characteristics of the parental strains and their pseudorevertants are listed in Table 1. Chromosomal DNA
from the mutant and pseudorevertant strains was prepared by the method
of Woo et al. (32). Plasmids containing the
fliF mutant insert or the flhA isolated second site mutant inserts were cloned into pTrc99A1de4 (2),
using NdeI and BamHI sites created by PCR with
mutant chromosomal DNA as target.
Luria broth and soft tryptone motility plates are described in
reference 29. Nutrient gelatin agar is described in
reference 33.
DNA techniques.
PCR and cloning were performed as described
previously (18) except that the Taq polymerase
was from Qiagen (Valencia, Calif.). DNA sequencing was carried out with
the modified T7 DNA polymerase Sequenase version 2.0 (USB Corp.,
Cleveland, Ohio) and various primers synthesized using an ABI model 392 DNA-RNA synthesizer (Perkin-Elmer, Foster City, Calif.).
Preparation of soluble and membrane protein fractions.
The
cells were inoculated into 100 ml of Luria broth and grown overnight at
37°C with shaking. After collection by centrifugation, cells were
resuspended in 5 ml of 10 mM Tris-HCl (pH 8.0)-200 mM dithiothreitol
and sonicated (Branson model 250 Sonifier, Danbury, Conn.). The cell
lysates were centrifuged (13,000 × g, 10 min, 4°C)
to pellet undisrupted cells; then the cell lysates were centrifuged at
130,000 × g for 1 h. The supernatant and pellet
fractions, which contained the soluble proteins and membrane proteins,
respectively, were collected separately.
Preparation of periplasmic and culture supernatant
fractions.
The periplasmic and culture supernatant fractions from
SJW1103 (wild type), SJW2706 (fliF), MM0608 (fliF
flhA), and SJW156 (flgD) were prepared as described
elsewhere (18).
Preparation of hook-basal bodies.
Hook-basal bodies were
prepared from SJW1364 (flhA) carrying pMM106, a pET19b-based
plasmid encoding N-His-FLAG-FlhA, as described by Fan et al.
(2).
Antibodies.
Monoclonal anti-FliF antibody and polyclonal
anti-FlgD antibody are described in references 5 and 23,
respectively. Polyclonal antibodies against FliG and FliN were a gift
from K. Oosawa and S.-I Aizawa, Teikyo University, Japan. Anti-FLAG M2
monoclonal antibody was obtained from Sigma (St. Louis, Mo.).
Antibodies were detected using an ECL (enhanced chemiluminescence)
immunoblotting detection kit from Amersham (Little Chalfont, United Kingdom).
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RESULTS |
Isolation of pseudorevertants of fliF mutants.
Pseudorevertants were isolated from a total of 33 fliF
mutants by streaking single colonies out on 8% gelatin agar,
incubating them for up to 4 days, and looking for small swarms emerging
from the streak. Cells from these swarms were picked and purified as single colonies. The locations of the suppressing mutations were broadly determined in terms of the major flagellar regions: I, II and
III, which are at about 26, 42, and 43 centisomes, respectively (26). (Flagellar region III in fact consists of two
subregions, IIIa and IIIb, with a region unrelated to flagellar
function in between [11].)
None of the fliF mutants yielded suppressor mutations lying
in region I. For 30 of them, all of the suppressor mutations lay in
region III. Since fliF itself lies in region IIIb, many of these suppressors may have been intragenic, and none were analyzed further in this study. The remaining three fliF
mutants (SJW2706, SJW2713, and SJW2715) between them gave rise to
a total of 24 examples of intergenic suppressors in region II, in
addition to a total of 229 examples of suppressors in region III. Finer
deletion mapping of two of the region II suppressors indicated that
they lay in or close to flhA, a gene which encodes an
integral membrane component of the flagellar export apparatus
(18); DNA sequence analysis later confirmed that all of
the suppressors we identified lay in flhA (see below).
Motility of SJW2706 (fliF) and its fliF
flhA pseudorevertants.
DNA sequence analysis of the three
parental fliF strains revealed that they were identical (see
below). Strain SJW2706 (fliF) was chosen for further study.
SJW2706 swarmed very poorly at 30°C, whereas the pseudorevertants,
illustrated by MM0608, swarmed considerably better (Fig.
1), although still only at about 20% of
the wild-type rate (estimated after 15 h at 30°C [data not
shown]). When examined in liquid culture by dark-field light
microscopy, SJW2706 was essentially nonmotile, with only an occasional
cell swimming feebly. Few if any flagella could be seen. With MM0608
and the other pseudorevertants that were examined, ca. 20 to
50% of the cells were swimming. Swimming speed was less than that of
wild-type cells, and there were only ca. two to three flagella per
swimming cell. Wild-type SJW1103, in contrast, was vigorously motile,
with well-formed bundles containing ca. five to seven flagella. Thus,
the swarming behavior can be simply explained by essentially total
failure to assemble flagella in the case of the parental
fliF mutant and partial success in assembling flagella in
the case of the pseudorevertants.
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FIG. 1.
Swarming abilities of SJW1103 (wild type [wt]),
SJW2706 (parental fliF mutant, fliF*), MM0608
(pseudorevertant, fliF* flhA*), and MM1608
(flhA* single-site mutant derived from MM0608). The plates
were incubated for the times indicated; note the much longer times used
for the fliF* mutant and the pseudorevertant than for the
wild type and the flhA* strain.
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The parental FliF mutation and the suppressing FlhA mutations.
FliF is a protein containing 560 amino acids, or 559 after cleavage of
the N-terminal methionine (9). (In this report, numbering
starts with Met-1; the sequence in databases such as SwissProt starts
with Ser-2.) The fliF mutation in all three parental strains
was found to be the same, an in-frame deletion of six nucleotides (bp
521 to 526) about a third of the way into the gene, resulting in loss
of two amino acids, A174 and S175.
Flagellar region II contains a mixture of flagellar, motor, chemotaxis,
and receptor genes. Classical genetic deletion mapping had indicated
that for at least two of the region II pseudorevertants, the
suppressing mutations lay in or near flhA. DNA sequence
analysis was carried out on the suppressor alleles of all 24 pseudorevertants and resulted in identification of 19 of the mutations
(Table 2). They were all missense
mutations, lying within flhA, as the deletion mapping had
suggested. Two of the mutations were encountered several times, so that
only 10 were distinct. Three of those were different nucleotide changes
within the same codon, two of them giving the mutation F138L and the
other giving F138C. Thus, amino acid changes were seen at only eight
FlhA positions.
FlhA is a large integral membrane protein containing 692 residues
(17). It has an N-terminal transmembrane domain extending to about residue 328 and a C-terminal soluble domain comprising the
remainder of the protein. All of the mutations lie within the membrane
domain and in fact are confined to a subregion of it extending from
residues 57 to 272.
Comparison of swarming abilities of the pseudorevertants.
With
the knowledge of the second-site flhA mutations responsible
for suppression, we examined whether these could be ranked or compared
in any useful way in terms of their abilities to suppress. After
15 h at 30°C, among those giving the poorest swarming was MM0663, whose FlhA mutation was S272N. It swarmed at about 75% of the
rate of MM0608 (V268L) (whose swarming was shown in comparison with the
wild-type strain and the parental fliF mutant in Fig. 1).
The best swarming was observed with MM0624 (I148S), which swarmed at
about 140% the rate of MM0608. Thus, although there were differences,
they were small compared with the roughly fivefold difference between
the wild-type strain and the pseudorevertants as a group.
Construction and properties of second-site flhA
mutants.
The second-site mutations from a subset of the
pseudorevertants (MM0608, MM0609, MM0610, MM0621, MM0635, MM0652, and
MM0654) were transferred by phage P22-mediated transduction into KK1012 (lacking region II [14]) to construct strains containing
only the second-site flhA alleles: MM1608, MM1609, MM1610,
MM1621, MM1635, MM1652, and MM1654, respectively. (The flhA
mutations of MM1635, MM1652, and MM1654 subsequently proved to be
identical.) These strains, illustrated by MM1608 (Fig. 1), swarmed at
essentially wild-type levels, indicating that these second-site
flhA mutations by themselves have no phenotype.
Complementation, dominance, and multicopy properties of the
suppressor flhA mutations.
To examine the properties
of the suppressor flhA alleles expressed in
trans, the wild-type flhA gene and several of the
suppressor flhA alleles were cloned into pTrc99A. Even
without induction, pTrc99A-based plasmids result in expression levels
that are considerably higher than those from the chromosome
(21). The insert consisted of the entire flhA
gene (bases 1 to 2079, with no flanking material from either
flhB or flhE [Table 1]). These plasmids were
introduced into SJW2706 (fliF), MM0608 (fliF
flhA), and a flhA null mutant, SJW1364. The resulting
transformants were inoculated onto soft tryptone agar plates and
incubated at 30°C (Fig. 2).
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FIG. 2.
Swarm test of the complementation and dominance
properties of wild-type (wt) FlhA (middle row) and suppressor mutant
FlhA (FlhA*) from pseudorevertant MM0608. pTrc99A-based plasmids
producing these proteins were used to transform the strains indicated
at the top. SJW2706 is the parental mutant (fliF*), MM0608
is a pseudorevertant (fliF* flhA*), and SJW1364
is a flhA null mutant. v, pTrc99A vector. Transformed cells
were incubated on tryptone semisolid agar plates for the times
indicated.
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Plasmids carrying the second-site suppressor flhA alleles
(illustrated in Fig. 2 by pMMhA1608) had the ability to suppress to a
considerable degree the FliF defect of SJW2706, giving essentially the
pseudorevertant phenotype; thus, overproduced mutant FlhA was dominant
over wild type for interaction with mutant FliF. Conversely, pMM130
(wild-type FlhA) only slightly inhibited swarming of MM0608; in other
words, even at chromosomal levels of expression, mutant FlhA
outcompetes overproduced wild-type FlhA for interaction with mutant
FliF. All second-site suppressor FlhA proteins complemented the
flhA null mutant, confirming the results obtained with the isolated second-site mutants, namely, that the suppressor
flhA mutations by themselves have no phenotype.
Complementation, multicopy, and dominance properties of the
parental FliF mutant protein.
We wished to know whether the
parental FliF mutant protein would, in high copy number, improve the
function of the parental mutant itself, and also whether it would exert
negative dominance over wild-type cells (generating poorly motile
first-site phenotype) or second-site flhA mutants
(generating pseudorevertant phenotype).
The parental mutant fliF allele from SJW2706 was cloned into
pTrc99A to give plasmid pMMiF2706, which was then used to transform various hosts. The swarming of the parental mutant SJW2706 or two
pseudorevertants that were tested (MM0608 and MM0635) was not improved
by overproduction of mutant FliF (data not shown). In the wild-type and
second-site mutant backgrounds, the level of swarming was reduced
markedly, to about 40% of that of the same cells transformed with
vector (Fig. 3). Thus, although there was
clearly dominance, it was not complete, since that would have resulted
in the much slower swarming of the parental mutant (in the case of the
wild-type host) or the pseudorevertant (in the case of the second-site
host).
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FIG. 3.
Swarm test of the dominance properties of mutant FliF
(FliF*) from SJW2706. A pTrc99A-based plasmid, pMMiF2706, producing
this protein was used to transform the strains indicated at the top.
SJW1103 is wild type (wt); MM1608 and MM1635 are second-site mutants
(flhA*). v, pTrc99A vector. Transformed cells were incubated
on tryptone semisolid agar plates for 5 h at 30°C.
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The parental FliF mutation does not affect C-ring assembly.
fliF null mutants fail to assemble the C ring, which is a
peripheral membrane structure consisting of FliG, FliM, and FliN; thus,
the C ring associates with the membrane only via the MS ring
(13). Formation of the C ring is essential not only for motor function but also (for poorly understood reasons) for export and
further flagellar assembly. To examine whether the parental FliF
mutation affects C-ring formation, cells were lysed and separated into
a high-speed pellet fraction (containing membrane and
membrane-associated material) and a supernatant fraction (containing
soluble cytoplasmic proteins). The fractions were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting with anti-FliF, anti-FliG, and anti-FliN antibodies
(Fig. 4). SJW1103 (wild type) and null
mutants SJW1684 (fliF) and SJW1364 (flhA) were
used as controls.
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FIG. 4.
Distribution of the MS-ring protein FliF and the C-ring
motor/switch proteins FliG and FliN between the high-speed pellet, or
membrane, fraction (m) and the supernatant fraction (s) of cells of
wild-type SJW1103 (wild type [wt]), a fliF null mutant
SJW1684 (fliF), the first-site fliF mutant
SJW2706 (fliF*), and a flhA null mutant SJW1364
(flhA). The positions of molecular mass markers (in
kilodaltons) are shown on the left.
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FliF was detected in the membrane fractions of wild-type cells (lane
1), the parental fliF mutant (lane 5), and the
flhA null mutant (lane 7) but not in that of the
fliF null mutant (lane 3). Both FliG and FliN were detected
predominantly in the membrane fractions of wild-type cells (lane 1),
the parental fliF mutant (lane 5), and the flhA
null mutant (lane 7). Only in the case of the fliF null
mutant were FliG and FliN found mostly in the soluble fraction (lane
4). Thus, the parental FliF mutation does not appear to significantly
affect either assembly of the MS ring itself or assembly of the C ring
onto the MS ring. This result and the fact that the suppressor
mutations lie in the flhA gene suggest that the parental
FliF mutation directly impairs the assembly or function of the export
apparatus within the central pore of the MS ring.
The parental fliF mutant is defective in export and
second-site flhA mutations partially suppress the
defect.
We next examined whether the failure of the parental
fliF mutant to swarm is a result of failure to export
substrates and whether the second-site suppressor flhA
mutations restore the ability to export. FlgD, the hook-capping
protein, was chosen as a test substrate. Periplasmic and culture
supernatant fractions of SJW2706 and MM0608 were prepared as described
before (18), and immunoblotting was carried out with
polyclonal anti-FlgD antibody. SJW1103 (wild type) and SJW156
(flgD) were used as positive and negative controls, respectively.
FlgD was not detected in either the periplasmic or the culture
supernatant fractions of the parental fliF mutant (Fig.
5, lanes 3 and 4). We conclude that its
poor flagellation and motility result directly from poor flagellar
protein export. FlgD was exported into both fractions by the intergenic
fliF suppressor mutant (lanes 5 and 6), indicating that the
second-site flhA mutation can suppress the defect in
flagellar protein export of the parental fliF mutant. However, suppression was not complete: both the overall level of FlgD
export and the amount found in the supernatant (lane 6) versus the
periplasm (lane 5) were much lower than with wild-type cells (lanes 2 and 1). The latter aspect can be explained by the fact that impaired
export of periplasmic components (the rod proteins) results in doubly
impaired export of external components like FlgD.
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FIG. 5.
Export of the hook-capping protein FlgD to the periplasm
(p) and culture supernatant (s) of cells of wild-type (wt) SJW1103, the
first-site fliF mutant SJW2706 (fliF*), the
pseudorevertant (fliF* flhA*), and the
flgD null mutant SJW156 (flgD). Samples were
subjected to SDS-PAGE and immunoblotted with polyclonal anti-FlgD
antibody. Size in kilodaltons is indicated at the left.
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Attempt to obtain biochemical evidence for FlhA association with
the basal body.
Two membrane components of the export apparatus
(FliP and FliR) have been shown previously to be associated with the
basal body (2). The genetic evidence described above
provides strong reason to believe that FlhA interacts with the
basal-body MS-ring protein FliF. We sought biochemical evidence for
such an interaction, by preparing hook-basal bodies from a
flhA mutant transformed with a plasmid producing FlhA tagged
with the FLAG epitope (6) and, as a control, wild-type
cells transformed with vector. The samples were then subjected to
SDS-PAGE and immunoblotting. With monoclonal anti-FliF antibody, a
strong band at the position expected for FliF was seen with both
hook-basal body samples. With monoclonal anti-FLAG antibody, there was
a weak signal at the position expected for FLAG-tagged FlhA in the case
of the sample from the mutant transformed with the plasmid producing
FLAG-tagged FlhA and no signal with the control (data not shown). Thus,
we have obtained weak biochemical evidence for FlhA being in physical
association with the basal body.
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DISCUSSION |
Both the first-site FliF mutation and its suppressors are
rare.
In our initial survey, we found that the majority of
fliF mutants giving rise to suppressors produced ones that
were in flagellar region III and may well have been intragenic. Only 3 out of 33 fliF mutants gave suppressors that were in a
different flagellar region and therefore necessarily were intergenic,
and even here the intergenic suppressors were much less common than
ones in the same region. Since these three mutants proved to be
identical, the true numbers we obtained are 1 out of 31. Of the 19 suppressor mutations identified, two were encountered several times
(Table 2). Also, three different mutations were found within a
particular codon (number 138), but these gave rise to only two distinct
amino acid changes. The end result is that we found nine distinct amino acid changes, involving eight positions. Thus, both the parental and
the suppressing mutations appear to be fairly rare events, indicating
that the interaction between the MS ring and the export apparatus may
be quite a restricted one.
Position of the parental FliF mutation.
As is evident from the
complex shape of the MS ring (30), the FliF protein
subunits from which it is built must themselves have a complex
architecture. This makes it difficult to predict where the
two-amino-acid deletion (A174 and S175) in the mutant FliF protein is
located within its three-dimensional structure. However, on functional
grounds (suppression by a mutation in FlhA), it seems reasonable to
propose that the deletion may be on the inner surface of the annular
pore of the MS ring (Fig. 6). Apparently, it does not greatly affect the intersubunit interface within the MS
ring, or the interface between the MS ring and the C ring, since both
of these structures assemble in the FliF mutant (Fig. 4).
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FIG. 6.
Cartoon interpreting the suppression of a mutational
defect in the MS-ring protein FliF by a mutation in one of the membrane
components of the flagellar protein export apparatus, FlhA. Other
membrane components of the export apparatus are indicated by exp and a
dashed oval. The export apparatus, which translocates export substrates
from the cytoplasm into the lumen of the nascent flagellar structure
(rod, etc.), is believed to be located in a patch of membrane within
the pore that exists within the MS ring. Suppression is postulated to
be a result of physical interaction (double-headed arrow) between the
inner annular surface of the MS ring and the transmembrane region of
FlhA or, in one case, near the interface between the transmembrane
region and the soluble domain. The C ring is a part of the motor that
is mounted onto the MS ring. CM, cytoplasmic membrane; cyto, cytoplasm;
peri, periplasm.
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Since a mutant producing an essentially full-length fusion between FliF
and the motor/switch protein FliG (a known cytoplasmic protein) has
close to wild-type function (3), it is clear that the C
terminus of FliF lies in the cytoplasm. The protein must therefore
cross the membrane an even number of times. From topology prediction
programs (e.g., TopPred2 [31]), FliF is predicted to
have either two or three transmembrane segments, centered at about
positions 37, 199, and 467 of the 560-residue protein (9); the first of these is much more strongly predicted to exist than the
other two. Ueno et al. (30), in a study with
trypsin-digested FliF and C-terminally truncated FliF, assumed that the
only two spans were those at 37 and 467 but gave no clear justification for ignoring the possibility of a span at position 199. FliF of Caulobacter crescentus (24) is 26% identical
to FliF of Salmonella and can be aligned with reasonable
confidence throughout most of its sequence. Jenal and Shapiro
(8) have analyzed the topology of Caulobacter
FliF by 
-lactamase and -galactosidase fusions and concluded that
Q129 (equivalent to Salmonella E137) and E332 (equivalent to
Salmonella E367) lie in the periplasm. The result with E332
would seem to preclude a second span as early in the Salmonella sequence as position 199. However, given the
complex shape of the FliF subunit which gives rise to the quite bulky M-ring feature of the MS ring, it may be that FliF has an unusual transmembrane structure and crosses the membrane more often than the
two times indicated by computer algorithms. With this reservation, we
tentatively conclude that the parental mutation (deletion of amino
acids 174 and 175) lies in the periplasmic domain of FliF.
Conservation of FliF sequence among species is generally quite weak,
based on CLUSTAL W alignments (28) of the 11 FliF
sequences available in the SwissProt database (version 39). However,
the parental FliF deletion (A174 and S175) belongs in one of the
regions that is most strongly conserved, and within this fairly
conserved region it contributes to a short conserved motif
A-S(A)-V(I)-X-V(L/I), where A is conserved in all 11 sequences, S and the first V are both conserved in 10 of the 11 sequences, and the second V is conserved in 3 of the 11 sequences.
Thus, it appears to represent an important part of the FliF sequence,
such that deletion could cause a significant conformational change and
so affect the interface with FlhA.
Positions of the suppressor FlhA mutations.
FlhA is a
692-amino-acid integral membrane protein with a large soluble domain
encompassing the C-terminal half of the protein (residues 328 to 692)
(Fig. 7). Within the N-terminal half, the membrane protein topology algorithm TopPred2, using a span length of 19 amino acids, predicts eight 
-helical transmembrane spans (TM1 to
TM8) at the following approximate residue positions: TM1, 21 to 39;
TM2, 46 to 64; TM3, 68 to 86; TM4, 124 to 142; TM5, 211 to 229; TM6,
251 to 269; TM7, 288 to 306; and TM8, 309 to 327. TM7 is less strongly
predicted than the others and is predicted weakly or not at all by some
other algorithms (e.g., TMHMM [27]). Two experimental
arguments, however, support its existence, which would place the
C-terminal domain in the cytoplasm rather than in the periplasm.
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FIG. 7.
Schematic illustration of the predicted transmembrane
organization of FlhA, the integral membrane component of the export
apparatus that gave rise to suppression of mutations in the MS-ring
protein FliF. Positions of residues at the beginning and end of
predicted terminal and loop regions are indicated. Mutations identified
in this study are indicated by boldface letters and asterisks. CM,
cytoplasmic membrane; cyto, cytoplasm; peri, periplasm.
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First, PhoA fusion analysis of the FlhA homolog for export of virulence
factors, Salmonella InvA (which is 34% identical overall to
FlhA and 47% identical in the transmembrane region), supports the
existence of eight transmembrane spans (Jorge Galán, personal communication); specifically, three fusions at positions corresponding to predicted periplasmic loops 1, 2, and 3 gave a phosphatase-positive result (indicating they are located in the periplasm), while two fusions following predicted TM8 (and therefore lying beyond the transmembrane region) gave a phosphatase-negative result (indicating they are located in the cytoplasm).
Second, in a previous analysis of interactions among components of the
export apparatus, the cloned soluble C-terminal domain of FlhA was
found to interact with known cytoplasmic components, namely, FliH,
FliI, and FliJ (20).
As might have been expected from the fact that they are suppressing a
defect in a largely transmembrane structure, the MS ring, all of the
FlhA mutations identified in this study lie within its N-terminal
membrane-spanning domain (Table 2 and Fig. 7), extending from residue
57 (in predicted TM2) through residue 272 (in the cytoplasmic loop
following predicted TM6). Thus, of the eight predicted transmembrane
spans of FlhA, only TM2 to TM6 and the loops associated with them have
thus far provided examples of suppression. A reasonable hypothesis
would be that these spans form a surface that is exposed to the inner
surface of the MS-ring annulus.
The FlhA mutations themselves (Table 2) are conservative or moderately
so. Several of them involve replacement of one hydrophobic residue by
another. Some involve replacement of a hydrophobic residue by a polar
one. None involve replacement or introduction of a charged residue.
Also, they typically reflect the degree of conservation that exists at
the corresponding positions (using CLUSTAL W alignment) in the 10 FlhA
sequences in the SwissProt database (version 39). This situation seems
consistent with the need to suppress a defect in the FliF-FlhA
interaction without severely disrupting the role of FlhA in export.
This latter point is reinforced by the fact that the second-site FlhA
mutations themselves (in a wild-type FliF background) have wild-type
motility (Fig. 1).
Since the export apparatus is believed to be located in a membrane
patch within the annular structure of the MS ring, it seems likely that
the pseudorevertants with a second mutation in FlhA are compensating
for the FliF mutation by altering a physical interface between the two
proteins (Fig. 6). However, the situation is unusual in that the MS
ring consists of ca. 26 subunits of FliF, presumably all with
equivalent quaternary interactions. On steric grounds, it is impossible
for all 26 FliF subunits to be simultaneously in pairwise interaction
with 26 FlhA molecules, since it has been estimated that the pore of
the MS ring annulus can accommodate only about 70 membrane-spanning
segments (2) and each FlhA subunit is predicted to contain
8 membrane-spanning segments (and there are five other membrane
components of the export apparatus to be accounted for). It seems
likely that there will be a single copy of the export complex,
containing some low number of FlhA subunits
perhaps just one. Probably
because of a quite tight geometry, a FlhA subunit will interact with
whichever FliF subunit it happens to be close to.
The single parental FliF mutation has given rise to suppressors in
predicted periplasmic loops, transmembrane spans, and cytoplasmic loops
of FlhA, and so it is clear that they cannot all correspond to
immediately proximal interactions. Rather, we imagine that the FliF
mutation causes a displacement that can be compensated for in various
ways by the FlhA mutations in its N-terminal transmembrane domain. The
absence of any examples in the C-terminal cytoplasmic sequence suggests
it may constitute a distinct domain that is insensitive to the detailed
state of the MS ring.
A cartoon illustrating our overall interpretation of the FliF-FlhA
suppression data is given in Fig. 6.
Interpretation of multicopy and dominance effects.
The
multicopy and dominance effects observed, and our interpretation of
them, are illustrated in cartoon form in Fig.
8. For convenience, mutant FliF and FlhA
will be referred to as FliF* and FlhA*, respectively. We observed
no examples of simple multicopy effects. In other words, overproduction
of FliF* in the absence of FliF had no effect on swarming [cf. cases
(v) and (ii) or cases (vi) and (iii)]. The same was true of
overproduction of FlhA* in the absence of FlhA [cf. cases (vii) and
(iii)].
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FIG. 8.
Schematic illustration of effects of FliF and FlhA
overproduction (as a result of expression from pTrc99A-based plasmids)
on swarming in various host backgrounds. (i) Wild-type, untransformed
or transformed with vector; (ii) parental FliF* mutant, untransformed
or transformed with vector; (iii) FliF* FlhA* pseudorevertant,
untransformed or transformed with vector; (iv) second-site FlhA*
mutant, untransformed or transformed with vector; (v) parental FliF*
mutant with FliF* overexpressed; (vi) FliF* FlhA* pseudorevertant
with FliF* overexpressed; (vii) FliF* FlhA* pseudorevertant with
FlhA* overexpressed; (viii) wild-type with FliF* overexpressed;
(ix) second-site FlhA* mutant with FliF* overexpressed; (x) FliF*
FlhA* pseudorevertant with FlhA overexpressed. F, wild-type FliF;
F*, mutant FliF; A, wild-type FlhA; A*, mutant FlhA. Chromosomal
expression is indicated by the smaller font, while plasmid expression
is indicated by the larger font. Swarming ability is indicated
qualitatively by the diameter of the black circles, categorized by four
levels: wild-type, intermediate, pseudorevertant, and parental. The
data are interpreted in terms of a physical interaction between FliF
and FlhA subunits, where the strength of the interaction is indicated
by the thickness of the connecting bar. Wild-type proteins are
indicated by light shading, and mutant proteins are indicated by dark
shading. In cases (viii) and (ix), the possibility of a mixed
(FliF|FliF*) multimer is indicated (see text).
|
|
There were, however, two interesting examples with regard to dominance.
The first was where FliF* was overproduced in either a wild-type
background [case (viii)] or a FliF FlhA* (second-site mutant)
background [case (ix)]. In both cases, the wild-type level of
swarming of the host was considerably reduced, but not to the level
expected if FliF* - FlhA [case (ii)] or FliF*-FlhA* [case (iii)] interactions were dominant. Thus, the result was intermediate between dominant negative and recessive, with copy number effects offsetting interaction effects.
The second was where FlhA was overproduced in a FliF* FlhA*
(pseudorevertant) background [case (x)]. If FlhA had been dominant negative, this should have produced a FliF* FlhA, or parental mutant,
phenotype [case (i)]. In fact, the pseudorevertant phenotype was
retained; thus, the FliF*-FlhA* interaction proved resistant to the
elevated FlhA levels.
The result of case (x) suggests that FliF* and FlhA interact with
each other to a negligible extent (a result also supported by the
parental mutant phenotype with respect to export and motility). The
result from case (viii), where FliF* has a marked negative effect on
the strong positive interaction between FliF and FlhA, might appear to
contradict this. We think the resolution of this apparent contradiction
may lie in the fact that FliF is a homomultimer. Both FliF and FliF*
can form this multimer; thus, if both proteins are being produced, a
mixed multimer could form, with an intermediate level of interaction
with FlhA [case (viii)] or FlhA* (case (ix)].
An alternative explanation would be that FliF* sequesters other
components with which it interacts, such as the C ring, the export
apparatus, or the rod proteins. However, since we know that FliF*
permits C-ring assembly (Fig. 4), and also that overproduction of
FliF* does not inhibit the motility of pseudorevertants, we think
this explanation is less likely.
Thus, although we have not demonstrated conclusively that FliF and FlhA
physically interact, all of the above results are consistent with such
an interaction, whose strength can be ranked as follows (Fig. 8):
FliF-FlhA = FliF-FlhA* > (FliF |FliF*)-FlhA = (FliF|FliF*)-FlhA* > FliF*-FlhA* > FliF*-FlhA.
Is FlhA unique in suppressing mutation in FliF?
There are six
integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR)
that are associated with flagellar protein export. Why has only FlhA
emerged from this suppression study? To avoid analyzing potential
examples of intragenic suppression, we set aside the many suppressor
mutations that were in the same flagellar region (region III) as the
parental fliF one. However, this also meant that we
eliminated the possibility of encountering suppressors in the four
export genes that lie in region III: fliO, fliP, fliQ, and
fliR. Thus, of the export genes, only suppression by
flhA and flhB could have been detected by our
strategy. It is still noteworthy, however, that of the region II
suppressors, no examples were found in flhB.
In independent studies using a different parental FliF mutant, H. Komatsu and K. Oosawa (personal communication) have found examples of
intergenic suppression involving the Mot and switch proteins. Taken
together, the data suggest that the inner pore of the MS ring interacts
with the export apparatus, while its outer circumference and its
cytoplasmic face interact with the motor.
We thank Hitomi Komatsu and Kenji Oosawa for sharing data prior
to publication on suppression of FliF mutations by mutations in the Mot
and switch proteins, and we thank Jorge Galán for providing
unpublished data on the topology of Salmonella InvA.
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Abstract
Introduction
Present address: Protonic Nanomachine Project, ERATO, JST, Seika,
Kyoto, 619-0237, Japan.
