JOURNAL OF BACTERIOLOGY, May 1996, p. 2954–2959
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology

Vol. 178, No. 10

Characterization of the Flagellar Hook Length Control Protein
FliK of Salmonella typhimurium and Escherichia coli
IKURO KAWAGISHI,† MICHIO HOMMA,† ANDREW W. WILLIAMS,‡ AND ROBERT M. MACNAB*
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114
Received 27 December 1995/Accepted 14 March 1996

During flagellar morphogenesis in Salmonella typhimurium and Escherichia coli, the fliK gene product is
responsible for hook length control. A previous study (M. Homma, T. Iino, and R. M. Macnab, J. Bacteriol.
170:2221–2228, 1988) had suggested that the fliK gene may generate two products; we have confirmed that bothproteins are products of the fliK gene and have eliminated several possible explanations for the two forms. We
have determined the DNA sequence of the fliK gene in both bacterial species. The deduced amino acidsequences of the wild-type FliK proteins of S. typhimurium and E. coli correspond to molecular masses of 41,748
and 39,246 Da, respectively, and are fairly hydrophilic. Alignment of the sequences gives an identity level of
50%, which is low for homologous flagellar proteins from S. typhimurium and E. coli; the C-terminal sequenceis the most highly conserved part (71% identity in the last 154 amino acids). The central and C-terminal
regions are rich in proline and glutamine residues, respectively. Linker insertion mutagenesis of the conserved
C-terminal region completely abolished motility, whereas disruption of the less conserved N-terminal andcentral regions had little or no effect. We suggest that the N-terminal (or N-terminal and central) andC-terminal regions may constitute domains. For several reasons, we consider it unlikely that FliK is functioning as
a molecular ruler for determining hook length and conclude that it is probably employing a novel mechanism.
length rather than multiple units of a defined length (7). Polyhook mutants not only fail to control hook length but also failto add filament to the tips of the polyhooks. Pseudorevertantsof polyhook mutants which are still largely defective in hooklength control but have regained the ability to assemble filament (polyhook-filament mutants) have been isolated (33).
The gene responsible for the polyhook mutant phenotype is
not the hook structural gene, flgE, but another flagellar gene,
fliK (28, 33). The FliK protein has not been detected within theflagellar structure, and the means by which it controls hooklength is not known. The organization of the subunits in polyhooks appears to be identical to that in normal hooks, andindeed polyhooks have been used for biochemical characterization of the hook protein (13, 14) and for analysis of the hookstructure (27, 36). Also, simple overexpression of flgE in vivo
can result in abnormal hook length (12). Thus, it seems unlikely that biochemical modification of the hook protein by
FliK is the means of length control.
Several biological structures are believed to have theirlength determined by a molecular-ruler mechanism, in which a
protein in an elongated state determines the number of subunits of a globular protein that assemble via quaternary interactions into a filamentous structure; these structures include
the tail of phage lambda (with the GpH protein as the ruler)(16, 17) and the thin (F-actin) filaments of muscle (with nebulin as the ruler) (21). We have determined the fliK gene sequence in both S. typhimurium (fliKS) and E. coli (fliKE) andconclude from the deduced amino acid sequence that FliK isunlikely to be a molecular ruler. The sequence instead suggeststhat FliK may be a two- or three-domain protein, and insertion
mutagenesis experiments indicate that the C-terminal region(which is the most highly conserved region) is especially important for function. The possibility that the N-terminal (orN-terminal and central) and C-terminal domains might correspond functionally to two separable processes is explored in an
accompanying paper (37), in which mutations responsible forpolyhook and polyhook-filament phenotypes are analyzed.

Bacterial flagella of Salmonella typhimurium and Escherichia
coli consist of three distinct parts, the filament, the hook, andthe basal body (for a review, see reference 23). The basal bodycontains an axial rod, as well as four ring structures, two located in the outer membrane and the periplasm (the L and P
rings, respectively), one embedded in the inner membrane (theMS ring), and one in the cytoplasm mounted onto the MS ring(the C ring). The flagellar filament extends up to 10 mm fromthe cell and is the propulsive component of the system. The
hook connects the filament to the basal body, transmitting therotation of the rod by the flagellar motor and acting as aflexible coupling or universal joint.
The hook of the bacterial flagellum has a fairly well-defined
length. A recent quantitative analysis (7) has established amean length of 55 nm for the wild-type hook, with a standard
deviation of about 10%. This is in contrast to the filament,
which has no active length control (although its rate of elongation decreases with length and it is susceptible to accidental
shortening by breakage). The hook probably needs to be of aparticular length, because if it was too short it would not
generate a sufficient bend angle and if it was too long it would
not be able to transmit torque efficiently to the filament. The
control of hook length does not appear to be intrinsic to its
structure, since purified hook monomer can polymerize indefinitely in vitro (15).
Mutants that possess hooks of indeterminate length havebeen isolated. These hooks have been called polyhooks, although the term is somewhat misleading since each polyhookconsists of a single abnormally long structure of arbitrary* Corresponding author. Mailing address: Department of Molecular
Biophysics and Biochemistry, Yale University, New Haven, CT 065208114. Phone: (203) 432-5590. Fax: (203) 432-9782. Electronic mailaddress: robert_macnab@qm.yale.edu.
† Present address: Department of Molecular Biology, Nagoya University, Nagoya 464, Japan.
‡ Present address: Department of Microbiology and Immunology,
University of Kentucky, Lexington, KY 40536-0084.
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FIG. 1. Nucleotide sequences of the 59 and 39 portions of the hook length control gene fliK in S. typhimurium (a) and E. coli (b), together with the flanking sequences,namely, the 39 end of fliJ (35) and the 59 end of fliL (18, 19). The central portions of the fliK sequences (. . . . . . . . . .) are not shown. The deduced amino acidsequence of FliK is shown below the corresponding DNA sequence. Potential ribosome binding sites (Shine-Dalgarno sequences [S-D]) are indicated by underlining.
Potential stem-loop sequences that could function in transcription termination of the fliFGHIJK operon are indicated by horizontal arrows. The putative flagellumspecific promoter for fliL (pfliL) is also indicated by underlining. The amino acid sequences that were deleted in plasmids pAW11 and pAW12 (see the text) are shownin brackets in panel a; only the rightmost boundary of the pAW12 deletion is shown.

MATERIALS AND METHODS
Chemicals and enzymes. BglII linkers (dCAGATCTG, dGAAGATCTTC, anddGGAAGATCTTCC) (BglII sites are in boldface) were obtained from New
England Biolabs, Inc. (Beverly, Mass.). Universal primers for DNA sequencing
were obtained from standard commercial sources. All other chemicals and enzymes were of at least reagent grade and were obtained from standard commercial sources.
Recombinant DNA techniques. Restriction enzymes, T4 DNA ligase, andDNA polymerase Klenow fragment were used according to the manufacturers’
recommendations. Isolation of plasmid DNA was carried out according to standard procedures (29). Subclones of fliKS and fliKE were constructed by standard
techniques (29). Deletion derivatives of these clones for DNA sequencing wereobtained either by digesting replicative forms of phage DNA with suitable restriction enzymes, treating them with Klenow enzyme, and then ligating themwith T4 DNA ligase or by the single-strand sequential method (3) using theCYCLONE I Biosystem (International Biotechnologies, Inc., New Haven,Conn.). The dideoxynucleotide method (30), using the modified T7 DNA polymerase (34) Sequenase (U.S. Biochemical, Cleveland, Ohio) and occasionallyusing dITP in the place of dGTP, was employed for all DNA sequencing.
Sequence manipulation and analysis. Routine sequence manipulations werecarried out by using the personal computer package DNANALYZE (Gregory
Wernke, University of Cincinnati Medical School). Sequence comparisons wererun at the National Center for Biotechnology Information, Washington, D.C., byusing the program TBLASTN and the combined nonredundant database (31August 1995 version).
Minicell analysis. Minicells from strain UH869 (11) were isolated, and plasmid-encoded proteins were labeled with [35S]methionine as described previously(24). For pulse-chase labeling, a 150-ml aliquot of minicells (;2 3 109 cells ml21)
was incubated with 15 mCi of [35S]methionine and chased with 5 ml of an 8-mg
ml21 solution of unlabeled methionine and the chase was terminated by additionof 16 ml of 50% trichloroacetic acid. Samples were then subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography as described previously (38).
Nucleotide sequence accession numbers. The nucleotide sequence data re-ported in this paper have been deposited with GenBank under accession numbers L43492 (S. typhimurium) and L43491 (E. coli).

RESULTS
Construction of plasmids carrying fliKS and fliKE. Publisheddeletion mapping data had indicated where fliK should belocated in cloned DNA fragments from the S. typhimurium andE. coli chromosomes (2, 9). fliKS was subcloned from pMH11
into pUC19 as a 1.7-kb SalI-SacI fragment, yielding plasmid
pIK600. fliKE was subcloned from pDB7 into pUC19 as a
1.4-kb PstI-BamHI fragment, yielding plasmid pIK800. Bothplasmids were able to complement an E. coli fliK null mutant,
MS694 (32), in a swarm plate assay.
Nucleotide sequences of the DNA fragments containing fliKSand fliKE. The nucleotide sequences of the fliK-complementing
DNA fragments in plasmids pIK600 and pIK800 were determined on both strands. The 59 and 39 regions of the S. typhimurium and E. coli fliK sequences, together with the corresponding deduced amino acid sequences, are presented in Fig.
1.
In reporting the sequence of fliLE and fliME (19), Kuo andKoshland included a small amount of 39 sequence of fliKE andthe intergenic region between fliKE and fliLE. Our sequenceand the published one agree, with two exceptions: the published sequence has G instead of A at position 1086 (not shownin Fig. 1b) (corresponding to arginine rather than glutamine)
and it has an additional base (T) after position 1198 (Fig. 1b).

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J. BACTERIOL.

FIG. 2. Alignment of the amino acid sequences of FliK from S. typhimurium (above) and E. coli (below). Identities are indicated by vertical dashes, conservativechanges (among I-L-V-M-F, F-Y, A-G, D-E, H-K-R, N-Q, or S-T) are indicated by dots, and gaps are indicated by hyphens. The central region of FliK contains a large
number of prolines (p). Note also the high glutamine content of the C-terminal region. Vertical arrows 1 to 3 show the sites of insertion of oligopeptides into the E.
coli sequence, as described in the text, as follows: 1, pIK812; 2, pIK808; and 3, pIK815.

At the position of the first discrepancy, we find that both S.
typhimurium and E. coli have glutamine at the center of theconserved sequence QQQ; at the position of the second discrepancy, we find that the S. typhimurium and E. coli sequences
are highly similar (GNGAVDIFA[stop] and GNSGVDIFA[stop], respectively), whereas the frameshift in the published
E. coli sequence gives a quite different sequence (WQQRR[stop]). We therefore consider that our sequence is correct.
Identification of the fliKS and fliKE genes. The identificationof the open reading frames shown in Fig. 1 as fliKS and fliKE is
supported by several lines of evidence. (i) The orientation of
the open reading frames agrees with the known orientation of
the fliFGHIJK operon (20). (ii) The open reading frames startwith an ATG codon which overlaps (ATGA) with the termination codon of the upstream gene, fliJ (35); such overlap is acommon feature for flagellar genes within the same operon(22). (iii) The 59 portion of the following open reading frames
is identical to that of the published DNA sequences of fliLS(18) and fliLE (19), in agreement with the known organizationof flagellar genes in this region. (iv) The open reading frames
are preceded by a good ribosome binding site consensus(AGGA; 7 bp before the ATG codon for both species). (v) The
sizes of the open reading frames are approximately those expected from the apparent molecular masses of the gene products (see below). (vi) The deduced amino acid sequences offliKE and fliKS show considerable similarity (see below). (vii)
For both species, the open reading frames were assessed ascoding regions (at confidence levels of 96% for S. typhimurium
and 88% for E. coli) by the program TESTCODE (5); codon
adaptation indices (31) calculated for fliKS and fliKE are 0.26
and 0.30, respectively, indicating a low to moderate level ofsynthesis, as is typical for other flagellar proteins.

The intergenic region between the fliK and fliL coding regions is 104 bp long in both S. typhimurium and E. coli andcontains a stem-loop sequence (Fig. 1) that may represent atermination signal for the fliFGHIJK operon. It also contains
the putative promoter for the fliLMNOPQR operon and apotential ribosome binding site for the fliL gene (18, 19).
The deduced amino acid sequences of FliKE and FliKS. The
deduced molecular masses of FliKS and FliKE are 41,748 Da(405 residues) and 39,246 Da (375 residues), respectively. The
deduced molecular mass of FliKS (41,748 Da) is similar to theapparent value of 44 to 46 kDa estimated by SDS-PAGE (9;also this study). However, the deduced molecular mass of

FIG. 3. Phenotype of fliKE mutant alleles constructed in vitro. Cells of an E.
coli fliK amber mutant strain, MS694, were transformed with plasmids carrying
wild-type (pIK800) and mutant (pIK812, pIK808, and pIK815) alleles of fliKE(see the text). The mutant alleles contain short in-frame insertions at the residues
indicated in this figure and in Fig. 2. Cells were inoculated in triplicate onto
semisolid tryptone agar plates and incubated for 8 h at 308C, and their motility
was scored.

VOL. 178, 1996

FIG. 4. Deletion analysis of the two forms of FliKS. An autoradiograph ofradiolabeled minicells containing plasmids with either the wild-type fliK allele(pIK600) or mutant alleles (pAW11 and pAW12) with in-frame deletions of thecodons shown; pUC19 was used as a control. The two forms of FliK are indicatedby arrows and by the labels FliK for pIK600 and pAW11 and FliKD for pAW12.
With pAW11, there is a small but significant shift in the positions of both formsof FliK compared with wild-type FliK; with pAW12 there is a large shift of about
the expected magnitude. Molecular masses (in kilodaltons) of standards areshown to the left.

FliKE (39,246 Da) is considerably lower than the value of 54
kDa reported as its apparent molecular mass (2). We haveconfirmed in minicell analysis experiments with plasmid
pIK800 that FliKE does have an anomalously low mobility in
SDS-PAGE gels (data not shown); the reason for this is unknown.
Comparison of the sequences with the combined nonredundant database at the National Center for Biotechnology Information yielded highly significant similarity between the S. typhimurium and E. coli sequences (see below). The only othersignificant similarity found was a weak one to the deduced
product of Bacillus subtilis orf7 (a presumed gene within a large
flagellar operon), as was noted previously by Albertini et al. (1)in a comparison with our unpublished S. typhimurium and E.
coli sequences.
Hydropathy analysis (4) indicates that FliK is unlikely to bea membrane protein (data not shown), consistent with its cellular location in minicell expression experiments (9). Most of
the flagellar structural proteins that are external to the cell (theso-called axial family, including the hook and filament proteins) have certain sequence similarities, including N- and Cterminal hydrophobic repeats and limited consensus sequences(8, 10). FliK does not have any of these features, and so it is not
likely to be a member of the axial family.
The central one-third of the protein is proline rich. For
example, the 96-residue stretch between residues 139 and 234
of FliKE contains 17 prolines, or 18 mol%, whereas the remainder of the sequence has a proline content of only 5 mol%.
The C-terminal part of the protein is glutamine rich, with one
stretch of about 100 residues having a content of over 20
mol%.
Alignment of the deduced amino acid sequences from thetwo species (Fig. 2) indicates an identity level of around 50%.
This level, though highly significant statistically, is much lower
than those for most other flagellar proteins, where values ofgreater than 80% are common. Also, the alignment entailed
introduction of substantial gaps, especially in the central regionof the sequence. The N-terminal and central portions of FliK
are the least conserved parts. Thus, the first 251 residues are
only 36% identical and contain only a few appreciable
stretches of identity (the longest being 12 consecutive residues

FliK CHARACTERIZATION

2957

centered around residue 140). The N-terminal sequence issomewhat better conserved than the central portion of themolecule. In contrast, the last 154 residues are 71% identicaland contain only a single one-residue gap. This is also thebest-conserved part of the sequence in comparisons with theputative FliK protein (Orf7) of B. subtilis (1).
From the features described above, FliK can be considered
to consist of three regions. The N-terminal region, representing roughly one-third of the sequence, is not strongly conserved and contains some gaps. The central one-third of thesequence is even less conserved, contains extensive gaps, and isproline rich. The C-terminal one-third of the sequence is wellconserved, has essentially no gaps, and is glutamine rich. These
data suggested to us that FliK might consist of two or moredomains.
The unusually high glutamine content of the C-terminal
region suggests that it may have a specialized function. Glutamine-rich activation domains exist in a number of eukaryotic
transcription factors (26), but we have no evidence that FliK isfunctioning in transcriptional control.
Construction and characterization of oligonucleotide linker-insertion mutant alleles of fliKE. In an initial attempt toaddress the importance of the various regions of FliK, weconstructed three in-frame linker-insertion mutant alleles ofthe fliKE gene in plasmid pIK800. pIK812 has a 12-mer BglII
linker inserted at the HpaI site, pIK808 has a 10-mer BglII
linker at the Klenow fragment-treated BstEII site, and pIK815
has an 8-mer BglII linker at the Klenow fragment-treated
HindIII site. These linkers result in the insertion of shortamino acid sequences at residues 98, 208, and 324 of the
375-residue sequence, as shown by arrows 1 to 3 in Fig. 2.
Cells of the fliKE null mutant MS694 were transformed withthese plasmids, and the transformants were tested for motility(Fig. 3). The insertions had increasingly severe effects as they
were placed progressively closer to the C terminus: pIK812
gave essentially wild-type swarming, pIK808 gave about 50% of
wild-type swarming, and pIK815 gave no swarming at all.
FliKS occurs in two forms. Previous experiments (9) had
suggested that expression of the S. typhimurium fliK gene (andpossibly also that of the E. coli gene [see Fig. 8 of reference 9])
resulted in two products with similar apparent molecularmasses and isoelectric points, the protein with the higher apparent molecular mass being the predominant form. To test
whether both proteins do in fact derive from expression offliKS, we constructed two in-frame deletion derivatives of plasmid pIK600, making use of existing restriction sites within thegene. pAW11 utilized two BclI sites and resulted in removal ofcodons 2 to 7 (Fig. 1a). pAW12 utilized two MluI sites, which
after filling in with Klenow fragment and blunt-end ligation
resulted in the deletion of codons 285 to 375.
Minicell expression experiments with pIK600 confirmed theoriginal finding of two products with apparent molecularmasses of 44 and 43 kDa (Fig. 4, second lane from the left).
When the deletion plasmids were used, there were subsequent
shifts in the mobilities of both bands. The shifts for pAW11(third lane) and pAW12 (fourth lane) corresponded to apparent losses of about 1 and 9.5 kDa, respectively, close to the
expected values. These results demonstrate that the 44- and
43-kDa proteins are both products of the fliK gene. Pulse-chaselabeling indicated that the 44-kDa form was the precursor of
the 43-kDa form (data not shown).
These results also establish that the two forms do not reflect(i) N-terminal peptide cleavage (since the position of the second band in pAW11 would not be expected to shift in thiscase), (ii) modification in the immediate vicinity of the N
terminus, or (iii) modification within a substantial part of the C2958

KAWAGISHI ET AL.

terminus. Removal of the N-terminal methionine is unlikely
because while such removal is common with bacterial proteins,
it is not observed when—as is the case with both wild-type FliKand the mutant FliK of pAW11—the second residue is isoleucine (25).
DISCUSSION
The two forms of FliK. Previous studies had suggested that
fliK might be giving rise to two products closely similar in sizeand pI (9). We have now demonstrated that both proteins areproducts of the fliK gene. We have eliminated several possible
mechanisms for generation of the second form, such as modification or cleavage of the N terminus or modification of a
91-residue region near the C terminus. If the process involved
can be identified, it may be possible to block it and determine
whether it is important for hook length control or filament
assembly.
FliK is unlikely to be a molecular ruler. Several filamentous
structures, such as phage tails (16, 17) and thin filaments inmuscle (21), have their lengths controlled by a molecular-ruler
mechanism. It appears that the rulers in these cases are predominantly stretches of polypeptide in an a-helical state.
While the molecular size of FliK (ca. 400 residues) is in theright range to be an a-helical molecular ruler for the hook (400
3 0.15 5 60 nm), its amino acid sequence makes it highly
unlikely that it functions in this way. Predictions of secondary
structure show no pronounced tendency towards a-helix, andthe central region, with its high proline content, is almost
certainly not a-helical. Furthermore, the highly asymmetriccomposition and degree of conservation of the molecule are
not characteristics that one would expect to be associated witha molecular ruler.
There are at least two other reasons to doubt that FliK
functions as a molecular ruler. (i) It would need to be responsible for terminating elongation rather than enabling it, since
its absence results in abnormally long hooks rather than zerolength hooks (33). (ii) It would have to function externally tothe cell, and yet it has never been found in that location. We
suspect, therefore, that the length of the bacterial flagellar
hook is controlled by a novel mechanism, one distinct fromthose that have been described for other structures, such as
phage tails, thin filaments, and (with DNA or RNA rather thanprotein as the ruler) filamentous phages such as M13 (6).
Possible domain structure of FliK. The amino acid sequenceof the FliK protein suggests that it consists of three regions.
The fact that the central region is poorly conserved and of
substantially different sizes in the two species seems to argue
against any highly specific function, and its high proline content suggests that it may not have large amounts of welldefined secondary structure. This initially led us to hypothesizethat FliK is a two-domain protein with the central portion of
the sequence acting as a hinge or connector between the two;
however, the linker insertion mutagenesis results indicate thatthe functional distinction between the N-terminal and central
regions may not be an absolute one, since disruption of at least
part of the N-terminal region can be tolerated without loss offunction. Disruption of the proline-rich region caused someloss of function. However, the insertion was towards the end of
the region, at a point where sequence conservation between S.
typhimurium and E. coli begins to increase rapidly (Fig. 2),
suggesting that the insertion might have an effect on the stability of the C-terminal region. Disruption of the conserved
C-terminal region itself caused total loss of function.
Thus, there are indications that the FliK protein may have adomain structure. Is there any experimental evidence that FliK

J. BACTERIOL.

possesses multiple functions? FliK mutants are characterized
by a double failure: the inability to terminate the hook at its
proper length and the inability to initiate filament assembly.
The existence of intragenic suppressor mutants with double
mutations in fliK which still fail in hook length control but
succeed in filament assembly at the tips of the polyhooks seems
to argue in favor of the hypothesis that FliK is a bifunctional
protein. In the companion paper (37), we explore this hypothesis by examining the nature of mutations responsible for thepolyhook phenotype and by characterizing the suppressor mutations that permit filament addition.
ACKNOWLEDGMENTS
We thank Stavros Stavropoulos for his contribution to the sequencing of the E. coli fliK gene, Phil Matsumura (University of Illinois,
Chicago) for the gift of plasmid pDB7, and May Kihara for technicalassistance.
This work was supported by USPHS grant AI12202.
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