Microbiology (2008), 154, 1390–1399

DOI 10.1099/mic.0.2007/012641-0

Collaboration of FlhF and FlhG to regulate polarflagella number and localization in Vibrio
alginolyticus
Akiko Kusumoto,1 Akari Shinohara,1,2 Hiroyuki Terashima,1 Seiji Kojima,1,2
Toshiharu Yakushi1,23 and Michio Homma1,2
Correspondence

1Michio Homma

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan

g44416a@cc.nagoya-u.ac.jp

2

Received 23 August 2007
Revised24 January 2008

Accepted 26 January 2008

Soft Nano-Machine Project, CREST, JST, Japan

Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated
bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number ofpolar flagella is positively regulated by FlhF and negatively regulated by FlhG. We now showthat DflhF cells are non-flagellated as are most DflhFG cells; however, some of the DflhFG cells
have several flagella at lateral positions. We found that FlhF–GFP was localized at the flagellatedpole, and its polar localization was seen more intensely in DflhFG cells. On the other hand,most of the FlhG–GFP was diffused throughout the cytoplasm, although some was localized at
the pole. To investigate the FlhF–FlhG interaction, immunoprecipitation was performed by using
an anti-FlhF antibody, and FlhG co-precipitated with FlhF. From these results we propose amodel in which FlhF localization at the pole determines polar location and production of aflagellum, FlhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FlhG
negatively regulates flagellar number in V. alginolyticus cells.

INTRODUCTION
Many motile bacteria have flagella that rotate by means of a
motor embedded in the cytoplasmic membrane and createa driving force by rotating a helical filament, like a screw,
connected to the motor. The number and localization offlagella are different among species. Escherichia coli,
Salmonella typhimurium (Macnab, 1996) and Bacillus
subtilis (Kearns & Losick, 2003) have peritrichous flagella.
Vibrio cholerae (Prouty et al., 2001), Caulobacter crescentus(Brun et al., 1994) and Pseudomonas aeruginosa (Tsuda &Iino, 1983) have a single polar flagellum. Vibrio fischeri(Millikan & Ruby, 2004), Helicobacter pylori (Niehus et al.,2004) and Pseudomonas putida (Harwood et al., 1989) havemultiple flagella at the pole. Vibrio alginolyticus and Vibrio
parahaemolyticus have both a single polar flagellum andperitrichous flagella (McCarter, 2001). The flagella are notessential, and cells must expend significant energy for theirmaintenance. Bacteria minimize the cost of producing
flagella, and it seems that one strategy involves the precise
regulation of flagellar number.
The flagellar genes are conserved among peritrichously
flagellated and polarly flagellated bacteria, and gene
regulation and morphogenesis are also similar among
3Present address: Department of Bioscience and Biotechnology,
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa,
Nagano 399-4598, Japan.

1390

various species (Macnab, 1996; Wu & Newton, 1997;
Prouty et al., 2001; McCarter, 2001). The flagella, which arecomposed of a filament, hook and basal body, are formed
from the proximal end towards the distal tip. During
flagellar morphogenesis, first the MS ring of the basal body,which is composed of FliF, is established on thecytoplasmic membrane (Kubori et al., 1992). Next, theswitch complex or the C ring, which is composed of threeproteins, FliG, FliM and FliN, is assembled under the MS
ring. Then, the specific apparatus for protein export isassembled inside the C ring to form the entrance of thechannel for flagellar proteins (Kubori et al., 1997; Macnab,2004). The rod proteins are exported by way of thisapparatus and the rod structure is constructed. Then, theother proteins, such as the hook protein and flagellin areexported similarly and polymerized into tubular structuresthat extend from the rod. Formation of the MS ring isthought to initiate the flagellar assembly, so the number
and location of MS-ring complexes are likely to determine
the number and location of flagella.
The flagellar genes are hierarchically expressed under strict
control. In Vibrio and Pseudomonas, they are classified into
at least three classes: early genes (master regulator);
intermediate genes, the expression of which depends ons54 (MS ring, hook, basal body, switch, export apparatus,transcriptional regulators including FlaL, FlaM, FlhF and
FlhG, che proteins, s28 and flagellin); and late genes, the2007/012641 G 2008 SGM Printed in Great Britain

Polar-flagellar number and localization

expression of which depends on s28 (flagellins, motor
proteins and FlgM) (Dasgupta et al., 2003; McCarter, 2001;
Prouty et al., 2001). The intermediate class is further
divided into two classes: the FlaK-dependent genes(homologues are FleQ in P. aeruginosa and FlrA in V.
cholerae) that encode the MS ring, switch, export
apparatus, FlaL, FlaM, FlhF, FlhG and s28; and the
FlaLM-dependent genes (homologues are FleSR in P.
aeruginosa and FlrBC in V. cholerae) that encode hookprotein, rod proteins, LP-ring proteins and flagellin. Thesefour gene classes are called class I (early genes), class II(FlaK-dependent intermediate genes), class III (FlaLMdependent intermediate genes) and class IV (late genes).
In V. cholerae, P. aeruginosa and P. putida, overexpressionof FlhF results in an increased number of polar flagella, andan flhF gene disruption gives a reduced number andaberrant placement of flagella in V. cholerae, P. aeruginosaand P. putida (Correa et al., 2005; Murray & Kazmierczak,
2006; Pandza et al., 2000). Moreover, FlhF has beenreported to increase the expression of class III genes in V.
cholerae (Correa et al., 2005). Therefore, it has beensuggested that FlhF increases the number of flagella bypromoting the expression of flagellar genes. However, it isunclear how FlhF determines the location of the flagella. In
V. cholerae and P. aeruginosa, overexpression of FlhG
results in a reduced number of flagella and gives a nonflagellated phenotype, and an flhG gene disruption gives a
hyperflagellated phenotype (Correa et al., 2005; Dasgupta
et al., 2000). In Pseudomonas, it has been shown that the
FleN protein (the homologue is FlhG in Pseudomonas sp.)binds to the flagellar-gene-specific transcriptional regulator, FleQ (Dasgupta & Ramphal, 2001), which regulates
the transcription of class II genes with s54 (Dasgupta et al.,2002). The FleN protein represses transcription of class II
genes and of the fleN gene by itself via FleQ. The FleQ
homologue in V. cholerae, FlrA, also regulates transcriptionof class II genes (Prouty et al., 2001).
We have previously reported a multi-polar flagellar mutantof V. alginolyticus, KK148, and we identified the mutation
responsible, which is a nonsense mutation in the flhG gene
(Kusumoto et al., 2006). We cloned the upstream gene,
flhF, and the flhG gene. These genes are unique to polarflagellated bacteria, although FlhF and FlhG have similarity
with the E. coli signal recognition particle (SRP) receptor
FtsY and the E. coli cell division inhibitor MinD,
respectively (Kusumoto et al., 2006). Overexpression of
FlhF results in an increased number of polar flagella; on theother hand, overexpression of FlhG results in a reduced
number of polar flagella and gives a non-flagellated
phenotype. These results are consistent with those ofearlier studies with V. cholerae and Pseudomonas (Correa
et al., 2005; Dasgupta et al., 2000; Murray & Kazmierczak,
2006; Pandza et al., 2000). Moreover, co-expression of FlhF
and FlhG reduces the number of polar flagella moresignificantly than expression of FlhG alone, implying that
FlhG works together with FlhF to regulate the number ofpolar flagella (Kusumoto et al., 2006).
http://mic.sgmjournals.org

In this study, we show that the polar localization of FlhF is
reduced by FlhG, independent of other flagellar proteins,
and that FlhF and FlhG interact with each other. We
speculate that the FlhF–FlhG interaction inhibits FlhF fromlocalizing at the pole and that flagellation is thus
suppressed. These results contribute to our understandingof the control of flagellar number and the location of theflagellum in mono-polar flagellar systems.

METHODS
Bacterial strains and growth conditions. V. alginolyticus and E.

coli strains used in this study are listed in Table 1. V. alginolyticus cells
were cultured at 30 uC in VC medium [0.5 % (w/v) Tryptone, 0.5 %(w/v) yeast extract, 0.4 % (w/v) K2HPO4, 3 % (w/v) NaCl, 0.2 % (w/v)
glucose] or in VPG medium [1 % (w/v) Tryptone, 0.4 % (w/v)
K2HPO4, 3 % (w/v) NaCl, 0.5 % (w/v) glycerol]. E. coli cells were
cultured at 37 uC in LB medium [1 % (w/v) Tryptone, 0.5 % (w/v)
yeast extract, 0.5 % (w/v) NaCl]. When necessary, the following
antibiotics were used: chloramphenicol (2.5 mg ml21 for V.
alginolyticus or 25 mg ml21 for E. coli), kanamycin (100 mg ml21)
and ampicillin (50 or 100 mg ml21).
DNA manipulations and sequencing. Routine DNA manipulations

were carried out according to standard procedures. Restriction
endonucleases and other enzymes for DNA manipulations werepurchased from TaKaRa Shuzo, Toyobo, and New England Biolabs.
Nucleotide sequences were determined using the BigDye Terminatorv3.1 Cycle Sequencing kit (Applied Biosystems) and an ABI PRISM
3100-Avant Genetic Analyzer (Applied Biosystems).
Gene disruption. The flhF and flhFG deletion strains LPN1 andLPN2 were generated from VIO5 cells, which are defective for
production of lateral flagella and wild-type with respect to the polar
flagellum, by homologous recombination, as described previously(Terashima et al., 2006). The suicide vectors containing the sacB gene,
pKY704-DflhF-sacB and pKY704-DflhFG-sacB, were used for thisconstruction. First, for homologous recombination of the flhF and
flhFG genes, 1073 bp downstream of the flhG gene, containing the fliA
gene and 313 bp from the beginning of the cheY gene, were cloned
into a cloning vector, pGEM5Zf(+), and sequenced. Using theresultant plasmids and primers based on these sequences, we made inframe deletions in flhF or flhG (DNA encoding 475 aa was deleted
from flhF and DNA encoding 772 aa was deleted from flhFG),
which were integrated into the chromosome of VIO5 cells byhomologous recombination. Next, strains that had undergone
the first recombination were cultured in VC medium without
antibiotics overnight, and then screened for the second recombination based on their sucrose sensitivity. Finally, the flhF and flhFG
deletions in the chromosome were confirmed by PCR. The flhF
deletion and flhFG double deletion strains were named LPN1 andLPN2, respectively.
Transformation of Vibrio cells. V. alginolyticus cells weretransformed by electroporation as described previously (Kawagishi
et al., 1994). The cells were subjected to osmotic shock and washedthoroughly with 20 mM MgSO4. Electroporation was carried out
according to the manufacturer’s instructions using a Gene Pulser
electroporation apparatus (Japan Bio-Rad Laboratories) at an electricfield strength of 5.0–7.5 kV cm21.
High-intensity dark-field microscopy. Flagella were observed using

a dark-field microscope (Olympus model BHT) equipped with a
100 W mercury lamp (Ushio USH-102). An image was recorded
1391

A. Kusumoto and others

Table 1. Bacterial strains and plasmids used in this study
Abbreviations: Ampr, ampicillin resistant; Cmr, chloramphenicol resistant; Rifr, rifampicin resistant; Pof+,
possessing polar flagellum; Laf2, lacking lateral flagella; Multi-Pof, possessing multiple polar flagella.
Strain or plasmid
V. alginolyticus strains
VIO5
KK148
YM14
LPN1
LPN2
E. coli strains
DH5a
BL21(DE3)pLysS
Plasmids
pHFS401
pKY704
pKY704-DflhF-sacB
pKY704-DflhFG-sacB
pET-3dpET-flhF
pGEX-6P-2
pGEX-flhG13
pBAD24
pBAD33
pAK322
pAK520pAK721
pAK325
pAK541

Genotype or description
VIK4 (Rifr Pof+ Laf2)
VIO5 flhG (Rifr Pof+ Laf2 Multi-Pof)
YM4 rpoN (Pof2 Laf2)
VIO5 DflhF (Rifr Pof+ Laf2)
VIO5 DflhFG (Rifr Pof+ Laf2)

Okunishi et al. (1996)
Kusumoto et al. (2006)
Kawagishi et al. (1997)
This study
This study

T7 expression host

Grant et al. (1990)
Studier (1991)

sacB in pSU41
Cmr, suicide vector
1425 bp-deleted flhF fragment in pKY704
2316 bp-deleted flhFG fragment in pKY704
Ampr, T7 expression vector
his6-tev-flhF in pET-3d
Ampr, Ptac, gst
gst-flhG in pGEX-6P-2
Ampr, PBAD
Cmr, PBAD
flhF in pBAD33
flhG in pBAD33
flhFG in pBAD33
flhF-egfp in pBAD33
flhG-egfp in pBAD33

Terashima et al. (2006)
Xu et al. (1994)
This study
This study
Novagen
This study
GE Healthcare
This study
Guzman et al. (1995)
Guzman et al. (1995)
Kusumoto et al. (2006)
Kusumoto et al. (2006)
Kusumoto et al. (2006)
This study
This study

using a charge-coupled device (CCD) camera (Sony model SSCM370) and a DVD video recorder (Panasonic model DMR-E100H).
Electron microscopy. Samples were negatively stained with 2 %(w/v) potassium phosphotungstate (pH 7.4), and then observed witha JEM-1200 EXII electron microscope (JEOL).
Fluorescence microscopy. Vibrio cells bearing a plasmid, pAK325
or pAK541, were cultured overnight in VC medium. The overnight
culture was diluted 1 : 100 in VPG medium containing 0.02 % (w/v)
arabinose and 2.5 mg chloramphenicol ml21, and incubated at 30 uC
for 4 h. Fluorescence microscopy was carried out with a ‘tunnel slide’,
which is a rudimentary flow chamber constructed from a coverslip, amicroscope slide and double-sided tape. Poly-L-lysine (0.1 %, w/v)
was loaded into a tunnel slide, and after 5 min the tunnel slide waswashed with VPG medium. Cultures of the cells were applied byexchanging the medium, and then the tunnel slide was turned upsidedown and incubated for 1 min to adhere cells to the coverslip. The
tunnel slides were washed with TMK buffer (50 mM Tris/HCl,
500 mM KCl, 5 mM glucose, 5 mM MgCl2, pH 7.5) and observed
under a BX-50 microscope (Olympus).

To stain polar flagella, cells were treated with the following
preparation before observation. VPG medium containing antiserum
raised against the polar flagellum (Fukuoka et al., 2005) was applied
to the tunnel slide by exchanging the medium. After 3 min
incubation, the tunnel slide was washed with VPG, and then medium
containing a rhodamine-conjugated anti-rabbit-IgG antibody wasadded. After 3 min incubation, the tunnel slide was washed with
VPG, then observed under a microscope. The images were recorded
1392

Reference or sourceand processed using a digital camera (Hamamatsu Photonics, C474280-12AG) and imaging software (BD Bioscience, IPLab, version 3.9.5
r2 and Adobe Photoshop version 7)
Antibody raised against FlhF and FlhG. FlhF was purified from

BL21 (DE3)/pLysS cells harbouring a plasmid producing hexahistidine (His6)-tagged FlhF (His6–FlhF), pET-flhF. Cells were harvested
by centrifugation, washed with buffer A [10 mM Tris/HCl, 150 mM
NaCl, 0.5 mM PMSF, complete EDTA-free protease inhibitor(Roche), pH 8.0], and then frozen at 280 uC. After thawing, thecells were resuspended in buffer A containing 10 mg DNase I ml21
and 5 mM MgCl2. The suspension was then passed twice through a
French pressure cell at 500 kg cm22. After centrifugation (10 000 g for
10 min), His6–FlhF was present in the pellet as inclusion bodies. The
pellet was resuspended in buffer A containing 4 % (w/v) Triton X100, and then sonicated. The Triton-soluble membrane fraction wasremoved by centrifugation (10 000 g for 10 min). To remove themembrane fraction completely, the Triton-insoluble pellet wassuspended again with buffer A containing Triton X-100, sonicated,
and then centrifuged (10 000 g for 10 min). After the Triton-insolublepellet had been rinsed twice with distilled water, the pellet wassuspended in buffer A containing 8 M urea, and incubated at 30 uC
for 2 h with shaking. The suspension was centrifuged (7000 g for
15 min), and then His6–FlhF was purified from the supernatant with
Ni–NTA resin.
FlhG was purified from DH5a cells harbouring a plasmid producing
glutathione S-transferase (GST)-fused FlhG, pGEX-flhG13. Cells were
harvested by centrifugation, resuspended in buffer C (50 mM Tris/
HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) containing 0.6 mg
Microbiology 154

Polar-flagellar number and localizationlysozyme ml21, and then incubated on ice for 30 min. The cellsuspension was passed twice through a French press at 500 kg cm22.
After removal of undisrupted cells by centrifugation (10 000 g for
10 min), the insoluble fraction was removed by ultracentrifugation(100 000 g for 1 h). GST-fused FlhG in the soluble fraction wasapplied to a GSTrap FF column (GE Healthcare). After removal of the
GST tag by applying PreScission protease (GE Healthcare), FlhG waseluted with buffer D (50 mM Tris, 150 mM NaCl, 1 mM EDTA,
1 mM DTT, pH 8.0).
Purified His6–FlhF and FlhG were separated by SDS-PAGE andstained with Coomassie blue R250. The band corresponding to eachprotein was excised and used to inoculate a rabbit. Rabbit anti-FlhG
antibody was produced by Biogate.
Western blot analysis of flagellar proteins. V. alginolyticus cells

were cultured overnight in VC medium. The overnight culture wasdiluted 1 : 100 in VPG medium, and then incubated at 30 uC for 4 h.
Cells were harvested by centrifugation, and then resuspended indouble-distilled water. The cell suspensions were mixed with a onefifth volume of SDS loading buffer [0.2 M Tris/HCl (pH 6.8), 37.5 %(w/v) glycerol, 6 % (w/v) SDS, 0.004 % (w/v) bromophenol blue] anda one-twentieth volume of 2-mercaptoethanol, and then boiled for
5 min. Proteins in the samples were separated by SDS-PAGE and thenelectrophoretically transferred to a PVDF membrane (Millipore)using a semi-dry blotting apparatus (Bio-craft) according to the
manufacturer’s instructions. Immunoblotting was performed with
anti-FlhF, anti-FlhG, anti-FlgI, anti-MotX, anti-MotY, anti-PomA,
anti-PomB and anti-flagellin antibodies, as described previously(Nambu & Kutsukake, 2000; Yagasaki et al., 2006; Fukuoka et al.,2005; Nishioka et al., 1998).
Immunoprecipitation. An overnight culture of DflhFG cells

harbouring a plasmid, pAK520 (flhG) or pAK721 (flhFG), was diluted
1 : 100 into VPG medium containing 0.2 % (w/v) (for pAK520) or
0.01 % (w/v) (for pAK721) arabinose and 2.5 mg chloramphenicol ml21. After 4 h incubation at 30 uC, the cells were harvested bycentrifugation (10 000 g for 5 min), suspended in buffer E [50 mM
Tris/HCl, 150 mM NaCl, complete protease inhibitor (Roche),pH 7.5] to an OD660 of 10, and sonicated to disrupt the cells.
Undisrupted cells were removed by centrifugation (10 000 g for
5 min). The supernatant was ultracentrifuged (100 000 g for 1 h). The
supernatant from this step was diluted 1 : 5 into buffer E containing
0.1 % (w/v) Triton X-100, and anti-FlhF antibody and protein A–
Sepharose CL-4B (GE Healthcare) were added. After 3 h incubation
at 4 uC, the protein A–Sepharose was washed four times with buffer E
containing 0.1 % (w/v) Triton X-100, and was resuspended in buffer
E containing 3 % (w/v) SDS, and boiled. The protein A–Sepharosebound materials were separated by SDS-PAGE, and FlhF and FlhG
were detected by Western blot analysis. To eliminate the signal fromheavy and light chains of the anti-FlhF antibody used for
immunoprecipitation, the detection of FlhF was carried out with an
ExtraCruz F kit (Santa Cruz).

RESULTS

Fig. 1. Electron micrographs of (a) VIO5 (wild-type; wt), (b) KK148(flhG), (c) LPN1 (DflhF), and (d) LPN2 (DflhFG) cells. Cells were
negatively stained with potassium phosphotungstate. Bars, 1 mm.

showed that the overproduction of FlhF increases thenumber of polar flagella (Kusumoto et al., 2006). The DflhF
strain showed almost no swarming ability on 0.25 % agar
VPG plates after 6 h incubation (Fig. 2a, left). After 10 hincubation, however, swarming colonies of DflhF cells
showed definite expansion, although colonies of YM14
cells (an rpoN mutant that does not produce polar flagella)
did not expand at all (Fig. 2a, right). This indicates that avery low percentage of the population of the DflhF strain
have flagella.
High-intensity dark-field microscopic observation of
DflhFG cells indicated that most (~97.5 %) were non-flagellated, although some of them (~2.5 %) had several
flagella. Electron microscopic observation of the DflhFG
cells also indicated that most were non-flagellated,
although some had several flagella at lateral positions(Fig. 1d). Since the KK148 cells, which have a nonsense
mutation in the flhG gene (Kusumoto et al., 2006), had
multiple flagella at the pole (Fig. 1b), FlhF may be involved
in regulating the flagellar number as well as the placementof the polar flagellum.

Phenotypes of the flhF deletion and the flhFG
double deletion strains

Functions of the flhF and flhG genes

To investigate the function of FlhF, flhF and flhFG deletionstrains were generated by means of homologous recombination. Electron microscopic observation revealed that theparent cells (VIO5) had a single polar flagellum (Fig. 1a),but we could not find any flagella on the DflhF cells(Fig. 1c). This is consistent with the previous study, which

The motility of DflhF (LPN1) and DflhFG (LPN2) cells
expressing the flhF and/or flhG genes on 0.25 % agar VPG
plates was tested (Fig. 2b). The DflhF cells recovered theirmotility after expression of flhF in trans (Fig. 2b), and thesecells also regained a polar flagellum, which was confirmedby high-intensity dark-field microscopy. DflhFG cells

http://mic.sgmjournals.org

1393

A. Kusumoto and others

Fig. 2. Swarming ability of the DflhF and DflhFG cells. Swarming
abilities are shown of the wild-type (wt; VIO5), DflhF (LPN1),
DflhFG (LPN2), flhG mutant (KK148) and rpoN mutant (YM14)
cells (a) without plasmids and (b) with plasmids pAK322 (flhF),
pAK520 (flhG), pAK721 (flhFG) and pBAD33 (vector control).
Aliquots (0.5 ml) of overnight cultures were spotted onto 0.25 %agar VPG plates without arabinose (a, left and right, or b, upper) orwith 0.02 % arabinose (b, lower), followed by incubation at 30 6C
for 6 h (a, left), 10 h (a, right) or 5 h (b).

expressing flhF recovered their motility even in the absenceof arabinose (Fig. 2b), and the cells had multiple flagella at
the cell pole, similar to the flhG mutant (KK148) cells.
When the flhG gene was expressed in DflhFG cells (Fig. 2b),no flagella were produced and motility was reduced to thelevel observed in DflhF cells (data not shown). The
expression of both flhF and flhG genes restored the motilityof DflhFG cells to some extent in the absence of inducer,
and to the same level as that of the wild-type in thepresence of 0.2 % arabinose (Fig. 2b). Most of the cells had
one or several polar flagella at the pole in the presence of
0.2 % arabinose (data not shown).
The amount of flagellar proteins in flhF and flhG
mutants
We evaluated the amount of various flagellar proteins in
the wild-type cells, and in the DflhF, flhG, DflhFG and rpoN
mutant cells by means of Western blot analysis (Fig. 3).
FlhF was not detected in the DflhF, DflhFG or rpoN cells(Fig. 3a), and FlhG was not detected in the flhG, DflhFG or
rpoN cells (Fig. 3b). These results confirmed that the flhF
gene, and the flhF and flhG genes in DflhF and DflhFG cells,
respectively, were disrupted, and that the expression of
1394

Fig. 3. Flagellar proteins in various mutant strains. Cells cultured
overnight were recultured in VPG medium at 30 6C for 4 h. Cells
were harvested by centrifugation and resuspended in water.
Proteins were separated by SDS-PAGE and immunoblotted using
anti-FlhF (a), anti-FlhG (b), anti-Salmonella-FlgI (c), anti-FliG (d),anti-PomA (e), anti-PomB (f), anti-MotX (g), anti-MotY (h) and antiflagellin antibodies (i). Lanes: 1, VIO5 cells (wild-type; wt); 2,
LPN1 cells (DflhF; DF); 3, KK148 cells (flhG; G”); 4, LPN2 cells(DflhFG; DFG); 5, YM14 cells (rpoN; N”).

flhFG genes required s54, the rpoN product. While a similar
amount of FlhG was detected both in the wild-type and the
DflhF cells (Fig. 3b), a larger amount of FlhF was detectedin the flhG mutant (Fig. 3a). In the flhG mutant cells, mostof the flagellar structural proteins (FlgI, PomA, PomB,
MotX, MotY and flagellins), except for FliG, were detectedin larger amounts than in the wild-type cells (Fig. 3c, e, f, g,h, i), which is consistent with the number of flagella on thecells. A similar amount of FliG (intermediate class) was
detected in the wild-type, DflhF and flhG mutant cells, alarger amount was detected in the DflhFG mutant, and asmaller amount was detected in the rpoN mutant (Fig. 3d).
These results imply that the fliEFGHIJ operon, which is in
the intermediate class, is most likely regulated by anadditional mechanism besides s54 (RpoN).
Subcellular localization of FlhF and FlhG
To investigate the localization of FlhF and FlhG, GFP was
fused to the C terminus of each protein. FlhF–GFP and
FlhG–GFP were expressed in the DflhF and flhG mutant
Microbiology 154

Polar-flagellar number and localization

cells, respectively, and polar flagella were visualized bymeans of immunofluorescence staining with an antibody
raised against the polar flagellum (Fig. 4). The flagellated
poles of cells showed the accumulation of FlhF–GFP as
dots (Fig. 4a); on the other hand, flhG mutant cells
producing FlhG–GFP failed to generate flagella (Fig. 4b),
suggesting that FlhF–GFP and FlhG–GFP have the sameability to increase or decrease the number of flagella as theintact FlhF and FlhG. FlhF–GFP was diffused throughout

the cytoplasm, with some localization at one or bothpoles in the DflhF cells (Fig. 4a, c). Most of the flagellated
poles had FlhF–GFP foci (Fig. 4a, c). FlhG was also
diffused throughout the cytoplasm in the flhG mutant cells(Fig. 4b). FlhG–GFP was localized at the pole in ~30 %of the cells (Fig. 4b, e, f). Most of the cells transformed withthe flhG-gfp plasmid were not flagellated, and FlhG–
GFP was not detected when the cells were flagellated(Fig. 4d).
Polar localization of FlhF is affected by FlhG

Fig. 4. Localization of GFP-fused FlhF (F-GFP) and FlhG (GGFP). DflhF (LPN1) cells containing plasmid pAK325 (flhF-egfp)(a, c), and flhG mutant (KK148) cells containing plasmid pAK541(flhG-egfp) (b, d, e, f), were observed by fluorescence microscopy.
Cells cultured overnight were diluted 1 : 100 with VPG medium
containing 0.02 % (w/v) arabinose. After 4 h incubation at 30 6C,
the cells were attached to a poly-L-lysine-coated cover glass. To
identify the flagellated pole, the cells were labelled with anti-polarflagellum antiserum as the first antibody and rhodamine-conjugated anti-rabbit IgG antibody as the second antibody, and thenobserved under a fluorescence microscope. Asterisks indicate
polar-localized F-GFP or G-GFP. Arrowheads indicate flagellated
poles. Bars, 5 mm.
http://mic.sgmjournals.org

The localization of FlhF–GFP and FlhG–GFP induced byarabinose was investigated in the DflhFG and rpoN mutant
cells (Fig. 5). The amount of FlhF–GFP and FlhG–GFP in
the cells did not differ among the strains, as confirmed byWestern blotting (data not shown). FlhF–GFP wasintensely localized at the pole in DflhFG cells (Fig. 5b),while FlhF–GFP was detected throughout the cytoplasm in
addition to the poles in DflhF cells (Fig. 5a). We analysed
the ratio of fluorescent signal in the cytoplasm to that at
the pole in FlhF–GFP-expressing cells (Fig. 5h). The signals
at the pole (denoted as ‘P’) and in the cytoplasm (denoted
as ‘C’) were defined as the highest signal at the pole and themean signal in the central cytoplasmic region of the cell,respectively (Fig. 5g). Fig. 5(h) shows the distribution of
the ratio of P to C (P/C). The P/C of DflhF cells ranged
from 1.8 to 15.3, and the mean±SD P/C was 5.1±3.2. The
P/C of DflhFG cells ranged from 6.2 to 29.3 and themean±SD P/C was 14.7±5.8. These results clearly showthat FlhF–GFP is strongly localized at the pole in DflhFG
cells compared with DflhF cells. Interestingly, the localization of FlhF in rpoN mutant cells, which do not express
most of the flagellar proteins, including FlhF and FlhG, wassimilar to that in DflhF cells (Fig. 5c). The P/C of rpoN
mutant cells ranged from 1.5 to 9.1 and the mean±SD P/C
was 3.9±1.8. This may suggest that other factor(s) whose
expression depends on s54 are involved in the polar
localization of FlhF. The FlhG–GFP signal was diffuse
throughout the cytoplasm, although some FlhG–GFP
formed fluorescent foci in flhG, DflhFG and rpoN
mutant cells (Fig. 5d, e, f). FlhG–GFP localized at ~74 %of the flhG mutant cell poles, ~82 % of the DflhFG cell
poles, and ~72 % of the rpoN mutant cell poles. The
localization of FlhG–GFP in DflhFG cells was similar tothat in the flhG mutant cells as well as that in flhF
mutant cells. These results suggest that the polar localization of FlhF is inhibited by FlhG, while thelocalization of FlhG is not influenced by FlhF (see themodel in Fig. 7).
To test whether the polar localization of FlhF is affected by
the presence of FlhG in the absence of other Vibrio
flagellar-related factors, GFP-fused FlhF or FlhG was
produced with FlhG or FlhF in E. coli DflhDCBA cells,
which lack the master regulators of the flagellar genes anddo not express any flagellar genes, and in flagellated E. coli
cells (RP437), which are wild-type for flagella formation
1395

A. Kusumoto and others

Fig. 5. The effect of FlhG on the polar localization of FlhF. The
localization of FlhF–GFP (F-GFP) (a, b, c) and FlhG-GFP (G-GFP)(d, e, f) in cells was observed. The host strains were as follows: (a)DflhF (DF, LPN1); (b, e) DflhFG (DFG, LPN2); (d) flhG mutant (G”,
KK148); (c, f) rpoN mutant (N”, YM14). Cells cultured overnight
were diluted 1 : 100 with VPG medium containing 0.02 %arabinose. After 4 h incubation at 30 6C, the cells were boundto a poly-L-lysine-coated cover glass. Cells were observed under afluorescence microscope. Bars, 2 mm. Quantitative analysis of theeffect of FlhG on polar localization of FlhF was performed (g, h). (g)Schematic illustration of the quantification procedure. Cells were
scanned for fluorescence intensity along the major axis using theline scan mode of ImageJ analysis software (ImageJ version 1.37).
The fluorescence intensity at the pole (‘P’) for one axis was
obtained by subtracting the background value from the highest
intensity (in arbitrary units) within the cell. The fluorescenceintensity in the cytoplasm (‘C’) for one axis was obtained in thesame way, subtracting the background value from the averageintensity of the cytoplasmic middle region (in arbitrary units) within
the cell. (h) Histogram of the ratio of C to P (P/C) of 51 DflhF(LPN1) (black bars), 53 DflhFG (LPN2) (grey bars), and 58 rpoN
mutant (YM14) (white bars) cells expressing FlhF–GFP.

The FlhF–FlhG interaction
Based on the observation that the polar localization of FlhF
was inhibited by FlhG, we speculated that there is aninteraction between FlhF and FlhG. Therefore, we
attempted to directly demonstrate an FlhF–FlhG interaction. The DflhFG cells expressing flhG or flhFG genes
were fractionated into soluble (cytoplasmic) and insoluble(membrane) fractions. FlhF was detected equally in thecytoplasmic fraction (Fig. 6a, lane 5) and in the insoluble
fraction (Fig. 6a, lane 6) from DflhFG cells expressing both
FlhF and FlhG. FlhG was also detected both in thecytoplasmic fraction (Fig. 6a, lanes 2 and 5) and theinsoluble fraction (Fig. 6a, lanes 3 and 6), with more in
the cytoplasmic fraction. There was no apparent differencein the localization profiles of FlhG when either flhG or
flhFG was co-expressed in DflhFG cells.

(data not shown). FlhF–GFP expression was diffuse
throughout the cytoplasm and a fluorescent dot was
detected at the pole of ~36 % of cells. When co-expressedwith FlhG, the fluorescent dot was detected in only ~2 % ofcells and the fluorescence was diffuse throughout thecytoplasm, showing that FlhF–GFP failed to localize at thepole. Polar localization of FlhF and reduction of FlhF polar
localization was also observed in RP437 cells. These results
support the idea that FlhF can localize to the pole in theabsence of other flagellar-related factors and that FlhF
interacts with FlhG. FlhG–GFP was diffuse throughout thecytoplasm and was not localized at the pole in E. coli
DflhDCBA cells or RP437 cells. Therefore, the polar
localization of FlhG seems to require specific factors
present in Vibrio cells.
1396

As the cytoplasmic fraction was confirmed to contain both
FlhF and FlhG, immunoprecipitation assays using an antiFlhF antibody were performed with the cytoplasmic
fraction (Fig. 6b, lane 2). As a negative control, an
immunoprecipitation assay was performed with thefraction containing only FlhG (Fig. 6b, lane 1). A
significant amount of FlhG was immunoprecipitated withan anti-FlhF antibody from the cytoplasmic fraction
containing FlhF and FlhG (Fig. 6b, lane 2), but not fromthat containing FlhG alone (Fig. 6b, lane 1), indicating aninteraction between FlhF and FlhG.

DISCUSSION
From previous studies in V. cholerae and Pseudomonas, it has
been suggested that FlhF and FlhG regulate the number offlagella by upregulating or downregulating the expression of
Microbiology 154

Polar-flagellar number and localization

Fig. 6. Immunoprecipitation assay of the FlhF–FlhG complex
using the anti-FlhF antibody. (a) DflhFG (LPN2) cells expressing
FlhG alone or both FlhF and FlhG were sonicated and thenultracentrifuged. FlhF and FlhG in the whole-cell lysate (W), thecytoplasmic fraction (supernatant after ultracentrifugation; S) andthe insoluble fraction (pellet after ultracentrifugation; P) wereanalysed by Western blotting using anti-FlhF and anti-FlhG
antibodies. (b) FlhF was immunoprecipitated with anti-FlhF
antibody from the cytoplasmic fraction of the DflhFG cells
expressing both FlhF and FlhG (lane 2), followed by Western
blotting. As a negative control, an immunoprecipitation using antiFlhF antibody with the DflhFG cells expressing only FlhG was also
performed (lane 1). The band from the heavy chain of the anti-FlhF
antibody was detected in lane 1 (indicated by an asterisk).

flagellar genes. In this study, larger amounts of theintermediate class proteins (FlgI and MotY) and the lateclass proteins (PomA, PomB, MotX and flagellins) weredetected in the flhG mutant cells (Fig. 3). The results are
consistent with the earlier reports of negative feedback
regulation of intermediate-class gene expression by FlhG in
V. cholerae (Correa et al., 2005) and by the FlhG homologue(FleN) in Pseudomonas (Dasgupta & Ramphal, 2001). The
lack of FlhG causes overexpression of the intermediate-classgenes, including that of s28, leading to overexpression of thelate-class genes. We speculate that multiple flagellation of
the flhG mutant cells requires the overexpression of flagellar
genes.
FlhF, but not FlhG, seems to be involved in the polar
placement of flagella. In electron microscopic observations,
we were unable to find any flagellated DflhF cells, but did
find some DflhFG cells with multiple peritrichous flagella(Fig. 1c, d). Considering that the flhG-defective strain has
multiple flagella at the pole (Fig. 1b), FlhF appears to be
one of the determinants of the polar placement of the
http://mic.sgmjournals.org

Fig. 7. Working hypothesis for the regulation of the number ofpolar flagella by FlhF and FlhG in V. alginolyticus cells. (a) In a wildtype cell, FlhF is localized at the pole and promotes the assemblyof the MS ring of flagella. FlhG in the cytoplasm inhibits FlhF
localization at the pole by interacting with FlhF, and thus represses
excessive flagellation. Therefore, the number of polar flagella isregulated to be one by FlhF and FlhG. FlhF may need a factor ‘X’ tolocalize at the pole, as discussed in the text. The factor may be
involved in the polar localization of FlhF or in the stability of FlhF at
the pole. (b) When FlhG is overexpressed, almost all of the FlhF
molecules are captured by FlhG. FlhG-captured FlhF does not
localize at the pole, and thus FlhG-overproducing cells fail toproduce a polar flagellum. (c) When FlhG is depleted, most of the
FlhF is localized at the pole, and thus the cells produce multiple
flagella at the pole.

flagellum, which is consistent with results for P. aeruginosaand P. putida (Murray & Kazmierczak, 2006; Pandza et al.,2000). If FlhF collaborates with FlhG, they could be colocalized in the cell, or each protein might affect thesubcellular localization of the other. To verify thecollaboration between FlhF and FlhG, we constructed
GFP-fusion variants of both FlhF and FlhG, and observed
their localization in the cell. FlhF–GFP was localized at theflagellated pole, while most of the FlhG was diffusely
expressed throughout the cytoplasm (Fig. 4). Strikingly,
FlhF–GFP localization was more intense in the DflhFG
cells, suggesting that FlhG may negatively affect the polar
localization of FlhF (Fig. 5b). The effect of FlhG on thepolar localization of FlhF was confirmed in a non1397

A. Kusumoto and others

flagellated E. coli strain (data not shown). This leads to the
hypothesis that FlhF and FlhG interact with each other toregulate flagellar number and placement. We were able todemonstrate an interaction between FlhF and FlhG by coimmunoprecipitation (Fig. 6). From these results, we
propose a working hypothesis for the role of FlhF and
FlhG in controlling polar flagellar number and placement(Fig. 7). FlhF works at the pole to determine the placementof the flagellum and promotes the expression of flagellar
genes (Fig. 7a). FlhG interacts with FlhF and inhibits FlhF
from localizing at the pole, resulting in the suppression offlagellation (Fig. 7a). Overproduction of FlhG reduces thepolar localization of FlhF, such that FlhF diffuses
throughout the cytoplasm, and results in a non-flagellated
phenotype (Fig. 7b). Depletion of FlhG causes strong polar
localization of FlhF, leading to hyperflagellation at the pole(Fig. 7c). Consequently, the number of polar flagella iscontrolled to allow the production of a single flagellum by
the collaboration of FlhF and FlhG. FlhG seems to act not
only as an anti-coactivator of s54, as reported in
Pseudomonas, but also an inhibitor of FlhF localization.
FlhF has a GTP-binding motif and shows similarity to E.
coli SRP receptor FtsY, which is a membrane-associatedreceptor that targets the SRP/ribosome-nascent-chain
complex to the translocon. The dissociation of FtsY from
SRP is regulated by GTP hydrolysis of FtsY and SRP (Shan& Walter, 2005). We speculate that the FlhF–FlhG
interaction is also regulated by GTP hydrolysis by analogy
with FtsY. On the other hand, FlhG has an ATP-binding
motif and shows similarity to E. coli MinD. The MinD
dimer associates with the inner membrane and gathers
MinC, which inhibits FtsZ polymerization, and thus
inhibits the generation of the division plane (Rothfield
et al., 2005; Shapiro et al., 2002). The MinD dimer
dissociates into monomers by interacting with MinE, andmonomeric MinD dissociates from the membrane, hydrolysing ATP. Therefore, it is possible that ATP hydrolysis
mediates the interaction of FlhG with FlhF or with FlaK,
which is the Vibrio homologue of Pseudomonas FleQ, andis similar to MinD.
Localization of FlhG–GFP was different in Vibrio and E.
coli cells. FlhG–GFP diffused completely through thecytoplasm and was not localized at the pole in E. coli cells(data not shown), while FlhG–GFP was localized at thepole in some of the Vibrio cells transformed with the flhGgfp plasmid (Fig. 5d, e). We also observed localization of
FlhG–GFP in Vibrio rpoN (s54) mutant cells (Fig. 5f).
These results may indicate that FlhG is localized at the poleby a certain factor which is unique to Vibrio and whosegene is transcribed independently of s54. Polar localizationof FlhF–GFP was observed in E. coli cells (data not shown)and in the rpoN mutant cells (Fig. 5c). It seems that FlhF by
itself could recognize the pole and determine where thepolar flagellum is generated. FlhF–GFP was not strongly
localized at the pole in rpoN mutant cells (Fig. 5c),although rpoN mutant cells did not express flhG, which isexpressed under the control of s54 (Fig. 3b). This implies
1398

that FlhF might require another factor to allow its
localization at the cell pole. We know that FlhF is a key
player in determining the localization of polar flagella;
however, its mechanism is unclear. An investigation of thefunction of FlhF will lead to a better understanding of themechanism of polar localization of flagella.

ACKNOWLEDGEMENTS
We thank Gillian Fraser for useful discussions and Sachi Tatematsu
for assistance with the anti-FlhG antibody production. This work was
supported in part by grants-in-aid for scientific research from the
Hayashi Memorial Foundation for Female Natural Scientists (toA. K.), from the Ministry of Education, Science, and Culture of Japan,
and from the Strategic International Cooperative Program of theJapan Science and Technology Agency (JST) (to M. H.).

REFERENCES
Brun, Y. V., Marczynski, G. & Shapiro, L. (1994). The expression ofasymmetry during Caulobacter cell differentiation. Annu Rev Biochem
63, 419–450.
Correa, N. E., Peng, F. & Klose, K. E. (2005). Roles of the regulatory

proteins FlhF and FlhG in the Vibrio cholerae flagellar transcription
hierarchy. J Bacteriol 187, 6324–6332.
Dasgupta, N. & Ramphal, R. (2001). Interaction of the antiactivator

FleN with the transcriptional activator FleQ regulates flagellar
number in Pseudomonas aeruginosa. J Bacteriol 183, 6636–6644.
Dasgupta, N., Arora, S. K. & Ramphal, R. (2000). fleN, a gene that

regulates flagellar number in Pseudomonas aeruginosa. J Bacteriol 182,
357–364.
Dasgupta, N., Ferrell, E. P., Kanack, K. J., West, S. E. & Ramphal, R.
(2002). fleQ, the gene encoding the major flagellar regulator of
Pseudomonas aeruginosa, is s70 dependent and is downregulated byVfr, a homolog of Escherichia coli cyclic AMP receptor protein. J
Bacteriol 184, 5240–5250.
Dasgupta, N., Wolfgang, M. C., Goodman, A. L., Arora, S. K., Jyot, J.,
Lory, S. & Ramphal, R. (2003). A four-tiered transcriptional

regulatory circuit controls flagellar biogenesis in Pseudomonas
aeruginosa. Mol Microbiol 50, 809–824.
Fukuoka, H., Yakushi, T., Kusumoto, A. & Homma, M. (2005).

Assembly of motor proteins, PomA and PomB, in the Na+-driven
stator of the flagellar motor. J Mol Biol 351, 707–717.

Grant, S. G., Jessee, J., Bloom, F. R. & Hanahan, D. (1990).

Differential plasmid rescue from transgenic mouse DNAs into
Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci
U S A 87, 4645–4649.
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight

regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121–4130.
Harwood, C. S., Fosnaugh, K. & Dispensa, M. (1989). Flagellation of

Pseudomonas putida and analysis of its motile behavior. J Bacteriol
171, 4063–4066.
Kawagishi, I., Okunishi, I., Homma, M. & Imae, Y. (1994). Removal of

the periplasmic DNase before electroporation enhances efficiency oftransformation in a marine bacterium Vibrio alginolyticus.
Microbiology 140, 2355–2361.
Kawagishi, I., Nakada, M., Nishioka, N. & Homma, M. (1997).

Cloning of a Vibrio alginolyticus rpoN gene that is required for polar
flagellar formation. J Bacteriol 179, 6851–6854.
Microbiology 154

Polar-flagellar number and localization
Kearns, D. B. & Losick, R. (2003). Swarming motility in undomes-

Okunishi, I., Kawagishi, I. & Homma, M. (1996). Cloning andticated Bacillus subtilis. Mol Microbiol 49, 581–590.

characterization of motY, a gene coding for a component of thesodium-driven flagellar motor in Vibrio alginolyticus. J Bacteriol 178,
2409–2415.

Kubori, T., Shimamoto, N., Yamaguchi, S., Namba, K. & Aizawa, S.
(1992). Morphological pathway of flagellar assembly in Salmonella

typhimurium. J Mol Biol 226, 433–446.
Kubori, T., Yamaguchi, S. & Aizawa, S. (1997). Assembly of theswitch complex onto the MS ring complex of Salmonella typhimurium
does not require any other flagellar proteins. J Bacteriol 179,
813–817.
Kusumoto, A., Kamisaka, K., Yakushi, T., Terashima, H., Shinohara, A.
& Homma, M. (2006). Regulation of polar flagellar number by the flhF

and flhG genes in Vibrio alginolyticus. J Biochem 139, 113–121.
Macnab, R. (1996). Flagella and motility, In Escherichia coli andPandza, S., Baetens, M., Park, C. H., Au, T., Keyhan, M. & Matin, A.
(2000). The G-protein FlhF has a role in polar flagellar placement andgeneral stress response induction in Pseudomonas putida. Mol
Microbiol 36, 414–423.
Prouty, M. G., Correa, N. E. & Klose, K. E. (2001). The novel s54- ands28-dependent flagellar gene transcription hierarchy of Vibrio

cholerae. Mol Microbiol 39, 1595–1609.
Rothfield, L., Taghbalout, A. & Shih, Y. L. (2005). Spatial control ofbacterial division-site placement. Nat Rev Microbiol 3, 959–968.

Salmonella, pp. 123–145. Edited by F. C. Neidhardt. Washington, DC:
American Society for Microbiology.

Shan, S. O. & Walter, P. (2005). Co-translational protein targeting by

Macnab, R. M. (2004). Type III flagellar protein export and flagellar

Shapiro, L., McAdams, H. H. & Losick, R. (2002). Generating andassembly. Biochim Biophys Acta 1694, 207–217.
McCarter, L. L. (2001). Polar flagellar motility of the Vibrionaceae.

Microbiol Mol Biol Rev 65, 445–462.
Millikan, D. S. & Ruby, E. G. (2004). Vibrio fischeri flagellin A isessential for normal motility and for symbiotic competence duringinitial squid light organ colonization. J Bacteriol 186, 4315–4325.
Murray, T. S. & Kazmierczak, B. I. (2006). FlhF is required forswimming and swarming in Pseudomonas aeruginosa. J Bacteriol 188,6995–7004.
Nambu, T. & Kutsukake, K. (2000). The Salmonella FlgA protein, aputative periplasmic chaperone essential for flagellar P ringformation. Microbiology 146, 1171–1178.
Niehus, E., Gressmann, H., Ye, F., Schlapbach, R., Dehio, M., Dehio, C.,
Stack, A., Meyer, T. F., Suerbaum, S. & Josenhans, C. (2004). Genome-

wide analysis of transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylori. Mol Microbiol 52, 947–961.
Nishioka, N., Furuno, M., Kawagishi, I. & Homma, M. (1998).

Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament. J Biochem 123,
1169–1173.

http://mic.sgmjournals.org

the signal recognition particle. FEBS Lett 579, 921–926.
exploiting polarity in bacteria. Science 298, 1942–1946.
Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve

an inducible T7 expression system. J Mol Biol 219, 37–44.
Terashima, H., Fukuoka, H., Yakushi, T., Kojima, S. & Homma, M.
(2006). The Vibrio motor proteins, MotX and MotY, are associatedwith the basal body of Na+-driven flagella and required for stator
formation. Mol Microbiol 62, 1170–1180.

Tsuda, M. & Iino, T. (1983). Transductional analysis of the flagellar

genes in Pseudomonas aeruginosa. J Bacteriol 153, 1018–1026.
Wu, J. & Newton, A. (1997). Regulation of the Caulobacter flagellar

gene hierarchy; not just for motility. Mol Microbiol 24, 233–239.
Xu, M., Yamamoto, K., Honda, T. & Ming, X. (1994). Construction andcharacterization of an isogenic mutant of Vibrio parahaemolyticus
having a deletion in the thermostable direct hemolysin-related
hemolysin gene (trh). J Bacteriol 176, 4757–4760.
Yagasaki, J., Okabe, M., Kurebayashi, R., Yakushi, T. & Homma, M.
(2006). Roles of the intramolecular disulfide bridge in MotX and

MotY, the specific proteins for sodium-driven motors in Vibrio spp. J
Bacteriol 188, 5308–5314.
Edited by: P. W. O’Toole

1399