J. Biochem. 139, 113–121 (2006)
doi:10.1093/jb/mvj010

Regulation of Polar Flagellar Number by the flhF and flhG
Genes in Vibrio alginolyticus
Akiko Kusumoto, Kenji Kamisaka, Toshiharu Yakushi*, Hiroyuki Terashima,
Akari Shinohara and Michio Hommay
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602
Received August 26, 2005; accepted October 11, 2005

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selectedmutants on the basis of reduced swarming ability on soft agar plates. Among them, wefound two mutants with multiple polar flagella, and named them KK148 and NMB155. In
Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs,
respectively, are involved in regulation of flagellar placement and number, respectively.
We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense
mutation in flhG; cells expressing transgenic flhG recovered the swarming ability andhad a reduced number of polar flagella. NMB155 cells did not have a mutation in either
flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; incontrast, expression of flhG reduced both the number of polar flagella and the swarming
ability. These results suggest that FlhG negatively regulates the number of polar flagellain V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar
flagella and better swarming ability than KK148 cells expressing flhG alone, suggestingthat FlhG acts with FlhF.
Key words: bacterial flagella, FtsY, MinD, multiflagellate mutants.

Most motile bacteria have flagella that rotate by means ofa motor embedded in the cytoplasmic membrane and generate a driving force by rotating a helical filament, like ascrew, connected to the motor. There are two types of flagella: polar (mono and multi) and peritrichous. Escherichia
coli, Salmonella typhimurium (1), and Bacillus subtilis (2)have peritrichous flagella. Vibrio cholerae (3), Caulobacter
crescentus (4), and Pseudomonas aeruginosa (5) havea single polar flagellum. V. fischeri (6), Helicobacter
pylori (7), and P. putida (8) have multiple polar flagella.
V. alginolyticus and V. parahaemolyticus have both a single polar flagellum and peritrichous (or lateral) flagella (9).
The number and localization of flagella are regulateddifferently in different species. Specific polar localization
has been reported for several proteins and multicomponent
complexes (10, 11): Shigella flexneri IcsA and Listeria
monocytogenesis ActA, which form actin-organizing centers to mediate bacterial movement in host cells (12, 13);
E. coli chemoreceptor complexes, which are two-component
signal transduction proteins that respond to extracellular
stimuli (14); and E. coli MinC and MinD, which arerequired for proper placement of the division septum(15–17). The precise mechanism of polar localization has
not been clarified yet.
The flagellar genes are well conserved among peritrichously flagellated and polarly flagellated bacteria, andgene regulation and morphogenesis are also similar.
*Present address: Department of Bioscience and Biotechnology,
Faculty of Agriculture, Shinshu University, Kamiina, Nagano399-4598, Japany
To whom correspondence should be addressed. Tel: +81-52-7892991, Fax: +81-52-789-3001, E-mail: g44416a@cc.nagoya-u.ac.jp
Vol. 139, No. 1, 2006

Detailed studies of the regulation of flagellar assembly
have been carried out for E. coli, S. typhimurium (1),
C. crecsentus (18), V. cholerae (3) and V. parahaemolyticus(9, 19). Flagella, which are composed of a filament, a hook
and a basal body, are formed from the proximal end
towards the distal tip. First, the MS ring of the basal
body, which is composed of FliF, is embedded in thecytoplasmic membrane. Next, the switch complex or the
C ring, which is composed of three types of protein:
FliG, FliM, and FliN, is constructed on the cytoplasmicside of the MS ring. A specific apparatus for flagellar
protein transport is thought to be assembled inside theC ring, which is the entrance of the channel for flagellar
proteins (20). Rod proteins are exported by way of thisapparatus and then the rod structure is constructed.
Then, other proteins, such as the hook protein and flagellin, are exported similarly and polymerized into tubular
structures that extend from the rods. Formation of the
MS ring is thought to initiate flagellar assembly, so thenumber and location of the MS ring seem to determine
the number and location of flagella.
In a previous study on P. putida, which possesses polar
flagella, a flhF mutant showed a peritrichously flagellated
phenotype, and flhF overexpression resulted in a multiflagellated phenotype (21). Thus, FlhF seems to determine
flagellar placement and number. It has been shown that
FleN (or MotR), the FlhG homologue in P. aeruginosa,
negatively regulates the flagellar number (22, 23). Thus,
fleN mutants show a multiflagellated phenotype. FleN
binds to a flagellar-gene-specific transcriptional regulator,
FleQ (24), which regulates the transcription of Class II
genes in a four-tiered hierarchy with s54 (25). The
Class II genes encode flagellar structural components
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and regulatory proteins, including FlhF and FleN. The
FleN protein suppresses transcription of the Class II
genes and the fleN gene by itself via FleQ. FlrA, theFleQ homologue in V. cholerae, also regulates transcriptionof the Class II genes (3). Both FleQ and FlrA are classified
as Class II in the four-tiered hierarchy. These facts may
show that the regulatory networks for flagellar biogenesis
in the Vibrio and Pseudomonas species are very similar.
In V. alginolyticus and V. parahaemolyticus, two typesof flagellar systems, polar flagella (Pof) and lateral or peritrichous flagella (Laf), are mainly used for movement inliquid media and on solid surfaces, respectively (9). Polar
flagella are generated from the cell pole and the filament issheathed with a membrane structure that is contiguouswith the outer membrane (26). The lateral flagella are
not sheathed. It has been shown that the energy sources
for the polar and lateral flagellar motors are Na+ and H+
motive force, respectively (27–29). Polar flagella are produced constitutively to facilitate swimming in liquid
media. When cells are transferred onto the surface of asolid medium, they produce lateral flagella, and elongate
their bodies to move and spread on the surface. It has
been proposed that polar flagella act as a dynamometerto sense the outer environment and thereby regulate
lateral flagellar production (30).
In this study, we isolated swarm-deficient mutants fromlateral flagella-defective (Pof+ Laf-) mutants that are thewild type as to polar flagellation. Among the mutants, wefound multiflagellate mutants that had increased numbersof polar flagella. We attempted to determine why the
number of flagella increases only at the polar region andhow the number is regulated in the mutants.
MATERIALS AND METHODS

Bacterial Strains and Growth Conditions—The strains
of V. alginolyticus and E. coli used in this study are listed

in Table 1. V. alginolyticus cells were cultured at 30 C
in VC medium (0.5% Tryptone, 0.5% yeast extract, 0.4%
K2HPO4, 3% NaCl, 0.2% glucose) or VPG medium (1%Tryptone, 0.4% K2HPO4, 3% NaCl, 0.5% glycerol). The
E. coli strain used for DNA manipulations was cultured
at 37 C in LB medium (1% Tryptone, 0.5% yeast extract,
0.5% NaCl). When necessary, the following antibiotics
were added: chloramphenicol (2.5 mg/ml for V. alginolyticus
or 25 mg/ml for E. coli) and ampicillin (50 mg/ml).
Isolation of Swarm-Deficient Mutants—Mutagenesis
was carried out using ethyl methane sulfonate (EMS)
and swarm-deficient mutants were isolated from theparental strains (Pof+ Laf–) on 0.25% agar VC plates as
described previously (34). Swarm-deficient colonies were
picked and streaked onto 1.25% agar VC plates for single
colony isolation. Several colonies derived from each mutant
candidate were streaked onto 0.25% agar VC plates andthen their swarming abilities were confirmed. Flagellawere observed by high intensity dark-field microscopy.
DNA Manipulations and Sequencing—Routine DNA
manipulations were carried out according to standard procedures. Restriction endonucleases and other enzymes for
DNA manipulations were purchased from TaKaRa Shuzo(Japan), TOYOBO (Japan), and New England Biolabs(USA). Nucleotide sequences were determined with a
BigDye Terminator v3.1 Cycle Sequencing kit (Applied
Biosystems) and an ABI PRISM 3100-Avant Genetic
Analyzer (Applied Biosystems).
Plasmid Construction—The plasmids used in theseexperiments are listed in Table 1. To express flhF and flhG
in V. alginolyticus, the genes were inserted into arabinoseinducible vector pBAD33. Plasmids pAK322 and pAK520
carry a single copy of flhF and flhG, respectively. For
coexpression, plasmid pAK721 carries a single copy of each
flhF and flhG.
Transformation of Vibrio Cells—V. alginolyticus cells
were transformed by electroporation as described (36).

Table 1. Bacterial strains and plasmids used in this study.
Strain or plasmid
V. alginolyticus
138-2
VIO5
NMB201KK148
NMB155
E. coli
JM109

Genotype or description
Wild-type
VIK4 (Rif r Pof + Laf-)
VIO5 (Rif r Mpar Pof + Laf-)
VIO5 flhG (Rifr Pof+ Laf- Multi-Pof)
NMB201 (Rif r Mpar Pof + Laf- Multi-Pof)

Reference or source28
3132This study
33

recA1, endA1, gyrA96, thi-, hsdR17, relA1, supE44, l-, D(lac-proAB);
F0 , traD36, proAB, lacI q , DM15

Plasmids
pGEM5Zf(+)
Cloning vector, Ampr, LacZa
Promega Corp
pGEM00207
The 1.8-kb PCR product (flhF locus) in the EcoRV site of pGEM5Zf(+)
This study
pGEM03608
The 1.8-kb flhF fragment in opposite direction to pGEM00207
This study
pGEM00303
The 1.1-kb PCR product (flhG locus) in the EcoRV site of pGEM5Zf(+)
This study
pBAD24
Ampr, PBAD
35
pBAD33Cmr, PBAD
35
pAK322flhF of pGEM03608 in the SacI/SphI sites of pBAD33
This study
pAK520flhG of pGEM00303 in the SalI/SphI sites of pBAD33
This study
pAK711flhF of pGEM00207 and flhG of pGEM00303 in the NcoI/SphI sites of pBAD24
This study
pAK721flhF and flhG of pAK711 in the EcoRV and SphI sites of pBAD33
This study
Ampr, ampicilin-resistant; Cmr, chloramphenicol-resistant; Rif r, rifampicin-resistant; Mpar, motility resistant to phenamil.
J. Biochem.

Polar Flagellar Number

115

The cells were subjected to osmotic shock and then washedthoroughly with 20 mM MgSO4. Electroporation was carried out according to the manufacturer using a Gene Pulser
electroporation apparatus (Japan Bio-Rad Laboratories,
Tokyo) at an electric field strength of 5.0 to 7.5 kV/cm.
Electron Microscopy—Samples were negatively stainedwith 2% potassium phosphotungstate (pH 7.0) and thenobserved under a JEM-1200 EXII electron microscope(JEOL).
Counting Flagella by Dark-Field Microscopy—Overnight
cultures of cells grown in VC medium were diluted 50-foldwith fresh medium with or without 0.02% arabinose andthen incubated for 2.5 h with shaking. Secondary cultures
were then diluted 30-fold with VPG medium with or without 0.02% arabinose. After a further 2 h incubation, flagellawere observed under a high intensity dark-field microscope(Olympus model BHT) equipped with a 100W mercury
lamp (Ushio USH-102).
Detection of Flagellin—Secondary cultures were obtained
as described in the previous section and then induced with
0.02% or 0.2% arabinose. After a further 2 h incubation,
the cultures were centrifuged at 20,000 · g for 5 min at 4 C.
The pellets were resuspended in double-distilled water.
The cell suspensions were mixed with a one-fifth volume
of sodium dodecyl sulfate (SDS) loading buffer (0.2 M
Tris-HCl [pH 6.8], 37.5% glycerol, 6% SDS, 0.004% bromophenol blue) and a 1/20 volume of 2-mercaptoethanol,and then boiled for 5 min. Proteins in the samples wereseparated by SDS–polyacrylamide gel electrophoresis(SDS-PAGE) and then electrophoretically transferred toa polyvinylidenedifluoride (PVDF) membrane (Millipore)using a semi-dry blotting apparatus (Bio-craft, Japan)
according to the manufacturer’s instructions. Immunoblotting was performed with anti-flagellin antibodies as
described (37).
RESULTS

Isolation of Multi-Pof Mutants—Among mutants whoseswarm size was smaller than that of the parent strains,which were lateral flagellum-defective mutants and thewild type as to polar flagella, we found two mutants,
KK148 and NMB155 (Fig. 1A), whose flagellar numbers
were increased. Both mutants could swim well in VC medium although their swimming behavior and speed weredifferent from those of the parent strains. On electron
microscopic observation, while most parent cells had a single flagellum, we confirmed the multiflagellate phenotype
for KK148 and NMB155 cells, which had about 15 and7 flagella per cell, respectively. The flagella were grown
not only from the cell pole but also from around it (Fig.
1B). We also observed that some KK148 and NMB155 cells
had only a few flagella and these flagella had been generated from the cell pole like in the parent cells (data notshown). Therefore, these mutations seem not to be involvedin the regulation of flagellar placement.
Cloning and Sequence Analysis of the flhF and flhG
Genes—In Pseudomonas species, a multiflagellate phenotype has been observed when the flhF gene is overexpressed (21) or when the fleN gene is disrupted (23).
In Vibrio species, such as V. parahaemolyticus, V. cholerae,
and V. vulnificus, whose genome sequences are available (38–40), homologous genes, their organization, andVol. 139, No. 1, 2006

Fig. 1. Phenotypes of multi-Pof mutants KK148 and NMB155.
(A) Swarming ability. 0.5 ml aliquots of overnight cultures werespotted onto 0.25% agar VC plates, followed by incubation at 30 C
for 6h. (B) Electron micrographs of wild-type cells (VIO5) andmutant cells (KK148, and NMB155). Cells were negatively stainedwith potassium phosphotungstate. Bar, 1 mm.

Fig. 2. Schematic representation of the flhF and flhG
locus based on the V. parahaemolyticus BB22 sequence(19). Arrowheads indicate the positions and orientations of theprimers used in this study.

neighboring genes are all similar to those in Pseudomonas.
To clone the flhF and flhG genes of V. alginolyticus, PCR
primers which matched regions conserved among the otherthree Vibrio species were synthesized (Fig. 2). Fragments
amplified from the chromosomal DNA of strain 138–2

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A. Kusumoto et al.

Fig. 3. Alignment of the amino acid sequences of V. alginolyticus FlhF (A) and FlhG (B) with homologues from various
species. Alignment of the sequences was performed using
GENETIX (version 12.1.0; GENETIX Corporation). Abbreviations:
Va, V. alginolyticus; Vc, V. cholerae; Pa, P. aeruginosa; Ec, E. coli;
Ph, Pyrococcus horikoshii; Ta, Thermus aquaticus. White letters inblack boxes denote amino acid residues identical to those of
V. alginolyticus FlhF or FlhG at the aligned positions. Lettersin gray boxes denote amino acid residues identical to those of
E. coli MinD or FtsY at the aligned positions. When the residues
completely matched, the positions are indicated by asterisks. Thickunderlines in (A) indicate (1) the ALLEADV motif, (2) motif I(P-loop), (3) motif II (insertion box domain loop), (4) motif III,and (5) motif IV, (6) the DARGG motif, and (7) the closing loop.
Thick underlines in (B) indicate (1) the P loop, (2) switch I, (3)switch II, and (4) the amphipathic helix. The mutation site in
KK148 is indicated by the closed circle above the sequence of
Va-FlhG.

using these primers were cloned into vector pGEM5Zf(+).
We determined the sequence from the end of flhA, which isthe first gene of the operon and supposed to encode a component of the flagella-specific export apparatus essential
for flagellar assembly, to the beginning of fliA, which

encodes s28. There are non-coding regions of 28 bp between
flhA and flhF, and 14 bp between flhF and flhG. The endof the flhG gene and the beginning of the fliA gene overlap
by 11 bp. The nucleotide sequence has been deposited in
DDBJ under accession No. AB191524.
J. Biochem.

Polar Flagellar Number

Fig. 4. Swarming ability of the wild-type (VIO5) and multiPof strains (KK148 and NMB155) containing plasmid
pAK322 (flhF), pAK520 (flhG), pAK721 (flhFflhG), or pBAD33(vector control). 0.5 ml aliquots of overnight cultures werespotted onto 0.25% agar VC plates with chloramphenicol, and
0% (A), 0.02% (B), or 0.2% (C) arabinose, followed by incubation
at 30 C for 5 h.

The predicted sizes of FlhF and FlhG from V. alginolyticus are 57 kDa and 32 kDa, respectively. With the BLAST
search program, the predicted amino acid sequence of FlhF
was fond to be 28.3% similar to that of E. coli FtsY, a signal
recognition particle (SRP) receptor protein (Fig. 3A). FtsY
comprises three domains, two of which are called the N andG domains; the amino-terminal N domain is a four-helix
bundle that is packed tightly against the G domain and the
G domain adopts a clasical GTPase-fold in which there are
four conserved motifs (I–IV) (41–43). The C-terminal G
domain, which contains a GTP-binding motif, exhibitsa high level of homology among proteins similar to FlhF.
The N-terminal N domain, which is a four-helix bundlethat is packed tightly against the G domain, is thought
to play a role in regulation of the nucleotide-binding
state of the protein. In SRP GTPases, it has been reported
that there are conserved regions; motifs I (P-loop), II (insertion box domain loop), III, and IV, the closing loop, and theVol. 139, No. 1, 2006

117
ALLEADV and DARGG motifs (44). Motifs I, II, III, and IV,
which define the GTP binding site (45), are highly conserved in FlhF proteins but the N domain is not conserved.
Homology among the N terminal regions of FlhF proteins
is low. However, a short stretch at the very beginning of the
N terminal region exhibits high similarity. The predicted
amino acid sequence of FlhG is 23.9% similar to thatof E. coli MinD, which serves as a cell division inhibitor(46, 47); this similarity is distributed over the entire protein and the N-terminal region exhibits a high level ofhomology with the ATP-binding motif of MinD (Fig. 3B).
In MinD proteins, it has been reported that there areconserved regions; the P-loop (Walker A motif), switch I,
switch II, and the amphipathic helix. All these conserved
regions, except the amphipathic helix one, are also highly
conserved in FlhG proteins.
The flhF and flhG genes from the two multi-Pof mutants
were cloned by means of the same method and thensequenced. We found no mutation in the KK148 flhF
gene. In the KK148 flhG gene, we found that the CAG
codon for Gln109 was changed to a TAG nonsense codon.
When the mutant flhG of KK148 was expressed in wildtype cells, the swarming ability and flagellar number were
not affected (data not shown). We found no mutation in
the flhF and flhG genes from NMB155.
Effects of flhF and flhG on Swarming Ability—The
effects of flhF and flhG gene expression in the wildtype and multi-Pof mutant cells on swarming ability
were examined (Fig. 4). pAK322, carrying a single copyof flhF, reduced the swarming ability of VIO5 cells upon
induction. KK148 cells expressing flhG recovered theswarming ability on arabinose induction, but the swarming
ability of NMB155 cells was not recovered (Fig. 4).
Arabinose induction of pAK520, carrying the flhG gene
(flhG), reduced the swarming ability of wild-type VIO5
cells. pAK721, carrying both the flhF and flhG genes(flhFflhG), improved the swarming ability of KK148 cells
even without arabinose induction, but did not affect theswarming of NMB155 cells. The flhF gene might containsome promoter activity.
Effects of flhF and flhG Expression on Flagellar
Number—The polar flagella of V. alginolyticus can easily
be observed on high intensity dark-field microscopy, so wedetermined the number of polar flagella per cell (Fig. 5).
The number of flagella on most VIO5 cells was one(Fig. 5A). The number of flagella on VIO5 cells containing
pAK322, carrying flhF, was increased when the flhF transgenes were induced (Fig. 5B). Although 77% of non-induced
cells possessed a single polar flagellum, 33% of flhFinduced cells possessed 2 to 4 polar flagella and only 48%of them possessed a single polar flagellum. When KK148
or NMB155 was used as the host, an increase in the number of polar flagella on flhF expression was not observed(Fig. 5,F and J).
When flhG was expressed in VIO5 and KK148 cellswith pAK520, the number of polar flagella was decreased(Fig. 5, C and G). Although 82% of non-induced VIO5 cells
possessed a single polar flagellum, 52% of flhG-induced
VIO5 cells did not possess any polar flagella and only
47% of them possessed a single polar flagellum. Although
63% of non-induced KK148 cells possessed more than
4 polar flagella, 25% of flhG-induced KK148 cells possesseda single polar flagellum, 21% of them had 2 to 4 polar

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A. Kusumoto et al.

Fig. 5. Number of polar flagella per plasmid-bearing cell. Cells
of VIO5, KK148, and NMB155 containing a plasmid, pAK322 (flhF),
pAK520 (flhG), pAK721 (flhFflhG), or pBAD33 (vector control), fromovernight cultures were re-cultured in VC medium at 30 C for 2.5 h
and then diluted 1:30 with VPG medium with (filled bars) or without(open bars) 0.02% arabinose. After 2 h incubation at 30 C, the cells

flhF
flhG
flhFflhG
Fig. 6. Detection of flagellin. Western blotting analysis of thewild-type (VIO5) and multi-Pof strains (KK148 and NMB155) lacking (A) and carrying (B) a plasmid, pAK322 (flhF), pAK520 (flhG),
pAK721 (flhFflhG), or pBAD33 (vector control), was performed
using polyclonal anti-flagellin antibodies. Cells cultured overnight(cf. legend to Fig. 5) were re-cultured in VC medium with 0, 0.02
or 0.2% arabinose at 30 C for 2.5 h, and then diluted 1:30 with VPG
medium with 0, 0.02 or 0.2% arabinose. After 2 h incubation at
30 C, the cell cultures were centrifuged, and the cell fractions werediluted to an optical density at 660 nm of 0.5 for all the strains in(A), and of 0.5 for VIO5, 0.2 for KK148, and 0.3 for NMB155 in (B).
10 ml of each sample was subjected to SDS-PAGE. Proteins wereseparated by SDS-PAGE and immunoblotted using anti-flagellin
antibodies.

were observed under a high intensity dark-field microscope. The
flagellar number per cell was determined and classified into one
of four categories, no flagella, one flagellum, two-four flagella,
and more than five flagella per cell. The experiments were independently performed twice and the graphs were made constructed withthe average data.

flagella, and only 25% of them had more than 4 polar
flagella.
Taken together, our findings imply that FlhG is anegative regulator of the polar flagellar number andthat the swarming ability of KK148 cells is reduced by
the multi-flagellation caused by the nonsense mutationin the flhG gene. In contrast, flhG expression in
NMB155 did not result in a significant decrease in thenumber of polar flagella (Fig. 5K). This implies that
NMB155 exhibits its polar multiflagellate phenotype
because of a mutation in some gene other than flhG. In all
cell lines carrying pAK721, which contains both the flhF andflhG genes, the number of flagella was decreased even without arabinose induction (Fig. 5, D, H, and L). The flhF genemight contain some promoter activity as mentioned above.
Amount of Flagellin in Cells—We investigated theamount of flagellin in the multiflagellate mutants bymeans of Western analysis using polyclonal anti-flagellin
antibodies. A larger amount in flagellin than that of theparent strains was detected in the multi-Pof cells, and theamount in KK148 cells was much greater than that in
NMB155 ones (Fig. 6A). Cells containing the various plasmids carrying flhF and/or flhG were also examined as tothe amounts of flagellin in the cells. The transgenes were
induced with 0.02% or 0.2% arabinose. When pAK322, carrying the flhF gene, was introduced into VIO5, no significant increase in the amount of flagellin was seen (Fig. 6B).
The expression of flhG decreased the amount of flagellinpresent in all strains (Fig. 6B). This is consistent with thedecrease in the number of flagella induced by flhG gene
expression (Fig. 5, C, G and K). The FlhG protein may
J. Biochem.

Polar Flagellar Number
suppress flagellin synthesis. Coexpression of flhF and flhG
dramatically decreased the amount of flagellin in all
strains.
DISCUSSION

We isolated multi-Pof mutants, KK148 and NMB155, by
screening for low swarming ability. The swarm sizes of thetwo strains were very small but their swimming abilities
were not severely affected. We found a nonsense mutation,
Gln109 to a stop codon, in the flhG gene of KK148.
However, the mutant gene in the NMB155 strain remains
to be identified. We focused on the flhF and flhG genes
because the homologous gene products in Pseudomonas
are known to regulate flagellar location and number,respectively (21–23). We cloned the flhF and flhG genes
from V. alginolyticus. The FlhF protein exhibted 28.3%
similarity to E. coli SRP receptor protein FtsY. In E. coli,
membrane proteins are translated by membrane-boundribosomes that are targeted to FtsY (41, 42). The Cterminal region of FlhF exhibited especially high similarity
to FtsY. The N-terminal region of FlhF is not conserved in
Vibrio and Pseudomonas except for the very beginning.
This might suggest that the functions of FlhF in Vibrio
and Pseudomonas are slightly different. The C-terminal
region of FtsY is essential for its function and contains a
GTP-binding motif. These results may suggest the possibility that FlhF targets FliF, the MS-ring component, tothe cell pole. In B. subtilis, it has been shown that FlhF is
dispensable for protein secretion (48).
The FlhG protein showed 23.9% similarity to E. coli cell
division inhibitor MinD. MinD functions in the cytoplasm
with its C-terminal helix region attached to the cell’s
inner membrane, and is responsible for recruiting
MinC and MinE to the membrane (49, 50). MinD cooperates with MinC to form an inhibitor of septum initiation,and the MinCD complex is topologically regulated by
MinE (11). These Min proteins show dynamic movementin the cell; MinD is a key component for oscillation with
ATPase activity and dimerizes in the presence of ATP,and the polar zone of MinD starts to disassemble fromthe middle of the cell towards the pole (15). It has beenreported that C-terminal amino acid residues F263, L264,
L267, and F268 are essential for membrane attachmentof E. coli MinD (50). These residues, however, are notconserved in FlhG. It is not known whether the FlhG protein attaches to the inner membrane or whether it forms
a dimer.
Expression of flhG in the wild-type cells reduced theirswarming ability, and decreased the amount of flagellin
and the number of polar flagella. This suggests that FlhG
suppresses flagellin synthesis directly or indirectly, resulting in a decrease in the number of polar flagella. FleN, theFlhG homologue in Pseudomonas, is thought to suppress
transcription of structural components of flagella via FleQ,
which is a s54 (RpoN)-dependent transcriptional activator(24, 25, 51). It was speculated that FleN plays a crucial rolein maintaining a single flagellum by down-regulating theFleQ activity. In V. alginolyticus, FlhG may also suppress
transcription of flagellar components including flagellin
via FleQ homologue FlaK. We have indeed demonstrated
that s54 is involved in the expression of polar flagellar
genes in V. alginolyticus (52). In H. pylori, FlhF and FlhA
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119
have been characterized as functional equivalents tomaster regulators (7). Among Vibrio, Helicobacter, andPseudomonas species, FlhF and FlhG homologs may notcompletely play the same role in flagellar gene regulation
but at least they regulate the expression of the majorityof flagellar genes directly or indirectly like a master regulator. When expression of all flagellar genes is elevated,
does the flagellar number increase? We speculate that
increases in the amounts of flagellar proteins are necessary
for hyper-flagellation but are not sufficient on their own.
V. alginolyticus should have a mechanism for maintaining
a single flagellum; otherwise the flagellar number would
be more variable under different culture conditions.
Expression of flhF in wild-type cells increased the number of polar flagella. FlhF may be involved in the regulationof the flagellar number. However, the intrinsic functionof FlhF in V. alginolyticus remains to be elucidated. In aprevious study of P. putida, which possesses polar flagella,
it was found that FlhF determined the flagellar placement,a flhF mutant had a peritrichously flagellated phenotype, and flhF overexpression resulted in a multiflagellate
phenotype (21). V. alginolyticus FlhF might also be
involved in determination of the site of polar flagella.
In this study, FlhF and FlhG co-expression efficiently
decreased the number of polar flagella and the amountof flagellin. It is likely that FlhG acts with FlhF directly
or indirectly.
FtsY is a component of the prokaryotic SRP receptor andFfh is a prokaryotic SRP that is homologous to SRP54.
Both are homologous GTPase components of the signal
recognition targeting pathway for protein secretion. Theyinteract directly with each other during cotranslationaltargeting of proteins to membranes. The structure of the
GTP-dependent heterodimeric complex was determined
and its coordinated activation was explained by the structure (44, 53). The conformational changes coupled to theformation of an extensive interface may function allosterically to signal formation of the targeting site and translocon. The conserved motifs of the SRP GTPases, I to IV,
which correspond to the residues directly involved innucleotide binding and hydrolysis, are well-conserved in
FlhF although several additional motifs that are restricted
to the SRP GTPases are not conserved. In a structuralsimilarity search, Ffh was found to resemble MinD (54).
This structural similarity suggests heterodimer formationof FlhF and FlhG in a similar manner to that of Ffh and
FtsY. From the structural similarity, we can speculate that
FlhF and FlhG form a heterodimeric complex. From theresults of this study we infer that the FlhG protein interacts with the FlhF protein either directly or indirectly. We
are currently trying to obtain direct evidence of the interaction between FlhF and FlhG by means of biochemical
analysis.
We thank Dr. Shin-Ichi Aizawa for the stimulating discussions
and encouragement. We are also grateful to Drs. Chris Jonesand Gillian Fraser for critically reading the manuscript. This
work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, and Cultureof Japan; the Japan Science and Technology Corporation(to M.H. and to T.Y.); and from the Soft Nano-Machine
Project of the Japan Science and Technology Agency (to T.Y.
and M.H.).

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A. Kusumoto et al.
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