Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Science, 200245Original ArticleMotX and MotY colocalize to the outer membraneM. Okabe, T. Yakushi, M. Kojima and M. Homma

Molecular Microbiology (2002) 46(1), 125–134

MotX and MotY, specific components of thesodium-driven flagellar motor, colocalize to the outermembrane in Vibrio alginolyticus

Mayuko Okabe, Toshiharu Yakushi,* Masaru Kojima
and Michio Homma
Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-Ku, Nagoya, Japan.
Summary
Rotation of the sodium-driven polar flagella of Vibrio
alginolyticus requires four motor proteins: PomA,
PomB, MotX and MotY. MotX and MotY, which are
unique components of the sodium-driven motor of
Vibrio, have been believed to be localized in the inner(cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY
colocalize to the outer membrane. Both proteins,when expressed together, were detected in the outermembrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments,
the N-termini of the primary translation products musthave signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX
and MotY require each other for efficient localization
to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that
describes motor proteins function in the outer membrane for flagellar rotation.
Introduction
Bacterial flagellar motors are the locomotive organelles
that drive the cell by rotating their helical flagellar filament(Berg, 1995; Blair, 1995; Macnab, 1996). In Gramnegative bacteria, the rotor portion of the flagellar basal
body consists of an axial rod and four rings: L, P, MS andC rings. The L, P and MS rings are thought to be embedded in the outer membrane, the peptidoglycan layer, andthe inner (cytoplasmic) membrane respectively (Derosier,1998). It is speculated that the cytoplasmic C ring is surAccepted 5 July, 2002. *For correspondence. E-mail 4juji@
cc.nagoya-u.ac.jp; Tel. (+81) 52 789 2992; Fax (+81) 52 789 3001.

© 2002 Blackwell Publishing Ltd

rounded by 6–16 stator particles (force-generating units)
in the inner membrane (Blair and Berg, 1988; Khan et al.,
1988; Muramoto et al., 1994). The motor is energized bya transmembrane electrochemical potential using either
protons or sodium as the coupling ion (Manson et al.,
1977; Hirota et al., 1981).
Motility (mot) mutants of bacteria have paralysed flagella and are unable to generate the torque required torotate the filament. Two motor genes, motA and motB,
have been identified in Escherichia coli and Salmonella,
whose motors use protons as the coupling ion for therotation of flagella (Dean et al., 1984; Stader et al., 1986).
MotA and MotB are inner membrane proteins and are
believed to convert the electrochemical potential tomechanical energy in concert with the rotor component.
MotA has four transmembrane segments and one large
cytoplasmic segment, which has been proposed to electrostatically interact with FliG (Zhou et al., 1995, 1998a;
Lloyd et al., 1999). MotB has a single N-terminal transmembrane segment where an Asp residue is essential
for proton flow as a part of the torque-generation process(Zhou et al., 1998b). MotB also has a peptidoglycanbinding motif at its C-terminus (Chun and Parkinson,
1988; De Mot and Vanderleyden, 1994). The transmembrane segments of MotA and MotB are thought to forma proton channel (Blair and Berg, 1990; Sharp et al.,1995a, b). MotA is thought to undergo a conformationalchange that is coupled to proton flow to alter its interaction
with FliG, thereby driving rotation (Kojima and Blair,
2001).
Alkalophilic Bacillus firmus and Vibrio species use
sodium as the coupling ion for flagellar rotation (Imaeand Atsumi, 1989; Yorimitsu and Homma, 2001). Vibrio
alginolyticus and Vibrio parahaemolyticus have sodiumdriven polar flagella and proton-driven lateral flagella,
which are used for movement on a surface (Atsumi et al.,
1992; Kawagishi et al., 1995). The sodium-driven motorof V. alginolyticus rotates stably and remarkably fast, up
to 1700 r.p.s., compared with rotation of the proton-driven
motors of E. coli and Salmonella, which has a speed ofaround 300 and 200 r.p.s. respectively (Lowe et al., 1987;
Kudo et al., 1990; Magariyama et al., 1994; Muramoto
et al., 1995). Lateral flagellar motor genes, lafT and lafU

126 M. Okabe, T. Yakushi, M. Kojima and M. Hommaof V. parahaemolyticus, are quite similar to their equivalents motA and motB in other proton motors (McCarterand Wright, 1993). In contrast, four motor genes (pomA,
pomB, motX and motY) have been identified as essential
for rotation of the sodium-driven polar flagellum in Vibrio(Asai et al., 1997; Gosink and Häse, 2000; McCarter,
2001; Okabe et al., 2001).
PomA and PomB are orthologues of MotA and MotB
respectively. Physical interaction between PomA and
PomB has been demonstrated (Yorimitsu et al., 1999).
A purified PomA/PomB complex was shown to havesodium-conducting activity in vitro (Sato and Homma,
2000a). It appears that PomA forms a stable homodimer
and the two subunits function together (Sato and Homma,
2000b). A periplasmic loop region of PomA is considered
important for dimer formation (Yorimitsu et al., 2000).
A hybrid motor that consists of PomB, MotX and MotY
from V. alginolyticus and MotARS from the proton-driven
motor of Rhodobacter spheroides functions as a sodiumdriven motor in Vibrio cells (Asai et al., 1999). In contrast,
MotBRS functions with neither MotARS nor PomA in Vibrio.
Furthermore, the chimeric MomB protein, which contains
the N-terminal transmembrane region of MotBRS and the
C-terminus of PomB, serves in conjunction with MotARS
as a sodium-driven motor in V. alginolyticus (Asai et al.,2000).
MotX and MotY are unique to the sodium-driven flagellar motor of Vibrio. The deduced amino acid sequencesof MotX and MotY show high identity among the Vibrio
species. MotX and MotY have been characterized asmembrane proteins because their sequences predict asingle transmembrane segment. Both proteins are recovered from the membrane fraction when expressed ineither E. coli or Vibrio (McCarter, 1994a; Okabe et al.,2001). The membrane localization of MotY in E. colidepends on MotX, and MotY remains in the soluble fraction unless MotX is coexpressed (McCarter, 1994a). MotX
and MotY mutually stabilize by interaction within the membrane (Okabe et al., 2001). MotY also has a putative
peptidoglycan-binding motif in its C-terminal region, as
does MotB and PomB (McCarter, 1994b; Okunishi et al.,1996). MotX and MotY each have two cysteine residues,
one of which is found in the conserved tetrapeptide CQLV
that is in a similar position from the N-terminus.
In E. coli, there are no homologues of MotX and MotY.
Overproduction of MotX in E. coli is lethal and its lethality
is suppressed by amiloride, a sodium channel blocker(McCarter, 1994a). This harmful effect of MotX does not
require MotY, nor is the production of MotY alone detrimental to E. coli. Therefore, it was suggested that MotX
participates somehow in sodium translocation across theinner membrane. A corollary to this proposed function isthat MotX and/or MotY may modulate the ion specificityof the PomA/PomB complex.

In this paper, we present evidence that MotX and MotY
localize to the outer membrane. Our data require that analternative model for the function of MotX and MotY be
developed.

Results
Membrane localization of MotX and MotY
MotX and MotY were classified as membrane proteinsbecause they both possess a single hydrophobic domainof sufficient length to span the inner membrane (McCarter,
1994a, b). Both proteins are predicted to have a large
C-terminal periplasmic domain, topology that makes
them type II membrane proteins (McCarter, 2001). It wasalso known that MotY can be recovered in the soluble
fraction when expressed in E. coli in the absence of MotX
(McCarter, 1994a). This result could be explained by the
relatively low hydrophobicity of the MotY transmembraneregion (Okabe et al., 2001). On the other hand, thesequence alignment of MotX and MotY of Vibrio species
reveals that the putative transmembrane regions are
not highly conserved (Okabe et al., 2001). That fact
could indicate that the transmembrane domain of theproteins is not particularly important. A prediction of subcellular localization of MotX and MotY using an on-line
programme (http://psort.ims.u-tokyo.ac.jp/) (Nakai andKanehisa, 1991) showed that their most probable destination is the periplasmic space. Also, this predicted that
their putative transmembrane segment may be cleaved
during translocation across the inner membrane.
To determine the subcellular localization of MotY, we
carried out subcellular fractionation such as spheroplasting and membrane separation using sucrose density
gradient centrifugation. E. coli cells expressing either or
combinations of the motor proteins were converted into
spheroplasts and then fractionated to periplasmic, cytoplasmic and membrane fractions. The lac operon repressor, LacI, maltose-binding protein, MBP, and OmpA wereused as the diagnostic marker proteins for the cytoplasm,
periplasm and membranes respectively. As shown in
Fig. 1, we compared the lanes designated as W andothers, and a significant portion of MotY seemed to be
degraded during the fractionation, even if coexpressedwith MotX from pKJ601 and pKJ701. When MotY wasexpressed by itself, however, a relative amount of MotY
was highest in the periplasmic fraction (lanes 10–12).
Even under these conditions, a little MotY could be
detected in the membrane fraction if high amounts of thesample were loaded on SDS-PAGE (data not shown).
When coexpressed with MotX, MotY was detected exclusively in the membrane fraction (Fig. 1, lanes 14–16). The
plasmid pKJ601 (motX+ motY+) complemented motX andmotY mutants of Vibrio (data not shown), which means© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

MotX and MotY colocalize to the outer membrane 127

Fig. 1. Subcellular localization of MotX and MotY. Escherichia coli
strain JM109 cells harbouring plasmid pSU41 (vector), pKJ401(motX+), pKJ502 (motY+), pKJ601 (motX+ motY+) or pKJ701 (motX+
motY+ pomAB+) were converted to spheroplasts. The periplasmic
fraction was separated from spheroplasts by low speed centrifugation(10 000 g, 5 min). The spheroplasts were disrupted by sonication.
Then, cytoplasmic and membrane fractions were prepared by ultracentrifugation. Equivalent amounts of the whole cell (W), periplasmic(P), cytoplasmic (C) and membrane (M) fractions were subjected to
SDS-PAGE, followed by immunoblotting using anti-MotX, MotY,
OmpA, MBP and LacI antisera.

MotX and MotY produced from the plasmid are functional.
The localization of MotX and MotY were not changed
upon coexpression with PomA/PomB (lanes18–20).
Figure 1 suggests that the previously observed soluble
form of MotY (McCarter, 1994a) resides in the periplasm,
not in the cytoplasm of E. coli. MotX, on the other hand,
is found in the membrane fraction despite the presenceor absence of the other motor proteins.
As coexpressed MotX and MotY were both detected in
the membrane fraction, the inner versus outer membrane
localization of the two proteins was determined by densitygradient centrifugation (Fig. 2). V. alginolyticus NMB191(DpomAB) harbouring plasmid pKJ701 (motX+ motY+
pomAB+) was analysed for localization of MotX, MotY andPomA/PomB. The plasmid pKJ701 complemented the
motX, motY or DpomAB mutation of Vibrio, indicating thatfunctional MotX, MotY and PomA/PomB proteins aresynthesized from the artificial motY-motX-pomAB operon(data not shown). As shown in Fig. 2A, MotX and MotY
colocalized in the higher density fraction, in which an
OmpA-like protein could also be detected. In contrast,
PomA and PomB were detected in the lower density fraction. This result clearly indicates that MotX and MotY
localize to a different membrane than PomA/PomB in V.
alginolyticus.
The inner versus outer membrane localization of MotX
and MotY was also determined in E. coli because methods for separation of the inner and outer membranes of
V. alginolyticus are not as well established as in E. coli.
Also, the antisera against diagnostic inner and outer membrane proteins are not available. Membranes of strain
JM109 harbouring plasmid pKJ601 (motX+ motY+) werefractionated by sucrose density gradient centrifugation(Fig. 2B). The relative amounts of the inner membraneprotein SecG (Nishiyama et al., 1993), the outer mem© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

brane protein OmpA (Klose et al., 1988), and MotX andMotY were determined by immunoblotting. MotX andMotY were contained in the same membrane fraction as
OmpA (Fig. 2B). A negligible amount of MotX and MotY
was detected in the inner membrane fraction, where SecG
was present. When E. coli cells expressing PomA, PomB,
MotX and MotY were fractionated, most of MotX andMotY were again found in the outer membrane fraction,
whereas PomA and PomB were present in the inner membrane fraction (Fig. 2C). A very little part of MotX could be
detected in the inner membrane fraction, indicating the

Fig. 2. Membrane localization of motor proteins. Membrane preparations were analysed by sucrose density gradient centrifugation. The
gradient was divided into 21 or 24 fractions from the bottom. MotX,
MotY, PomA, PomB, OmpA and SecG in each fraction were detectedby immunoblotting using the appropriate antiserum. The membrane
was prepared from Vibrio alginolyticus strain NMB191 (DpomAB)
harbouring plasmid pKJ701 (motX + motY + pomAB +) (A), E. coli
JM109 cells harbouring plasmid pKJ601 (motX + motY +) (B) and E.
coli JM109 harbouring plasmid pKJ701 (motX + motY + pomAB +) (C).
In B and C, OmpA and SecG were detected in fractions 3–8 and
11–16 respectively.

128 M. Okabe, T. Yakushi, M. Kojima and M. Homma
MotX may slightly change its localization upon coexpression with PomA/PomB.
Signal peptide cleavage of MotX and MotY
If MotX and MotY localize to the outer membrane, theymust have an N-terminal signal sequence (Pugsley,1993). MotX and MotY were partially purified from V. alginolyticus as described in Experimental procedures. Their
N-terminal amino acid sequences were analysed by
Edman degradation. The N-terminal sequence of MotX
was determined to be NVADV, indicating that the precursor was cleaved between Ala21 and Asn22. The N-terminusof MotY was determined to be VMGKR, indicating that theprocessed form of MotY begins with Val22. Additionally, Nterminus of MotX protein prepared from E. coli expression
system had the identical sequence with that from V. alginolyticus. This result confirmed that the same processing
reaction of MotX takes place within the two bacteria.
Altering the membrane localization of MotX and MotY
As described above, when expressed alone, most of MotY
seemed to be in the periplasm as a soluble protein. On
the other hand, when MotX and MotY were coexpressed,
the both localized to the outer membrane (Fig. 2). We
speculate that properties of MotY were altered by MotX.
Figure 1 shows that, without MotX, most of MotY is recovered in the periplasmic fraction, however, some could bein the membrane fraction (data not shown). Membrane
fraction of strain JM109 harbouring plasmid pKJ401(motX+) or pKJ502 (motY+) was fractionated by sucrose
density gradient centrifugation (Fig. 3). Comparing
Figs. 2B and C, and 3A, MotX seems to be detected in a
strange fraction, in which it does not correspond witheither OmpA or SecG, in the absence of MotY. This result
leads to three possibilities that MotX is present in (i) neither membrane (ii) the both membranes (iii) an intermediate membrane other than the inner and the outermembranes. In contrast, MotY was detected exclusively
in the outer membrane fraction even in the absence of
MotX (Fig. 3B).
MotX and MotY colocalize to the outer membrane
Previous studies and Fig. 1 showed that MotY is targetedto the membrane in the presence of MotX (McCarter,
1994b; Okabe et al., 2001). As shown in Figs 1 and 3A,
MotX expressed by itself is in the membrane fraction,
but its distribution corresponded with neither SecG nor
OmpA. However, MotX coexpressed with MotY mostly
localizes to the outer membrane (Figs 2B and C). These
results indicate that both MotX and MotY are required for
localization of either one to the outer membrane.

Fig. 3. Membrane localization of MotX and MotY solely expressed.
Membranes were prepared from E. coli JM109 cells harbouring either
plasmid pKJ401 (motX +, A) or pKJ502 (motY +, B) and analysed bysucrose density gradient centrifugation. The gradient was divided into
23 fractions from the bottom. MotX, MotY, OmpA and SecG in eachfraction, which was separated by SDS-PAGE, were detected byimmunoblotting. OmpA and SecG were detected in fractions 4–9 and
10–15 respectively.

To elucidate the biochemical properties of MotX and
MotY, we attempted to solubilize them with several detergents. Membrane fractions of strain NMB94 (motX )expressing MotX (Fig. 4A) or of strain VIO542 (motY )expressing MotY (Fig. 4B) were used. Like E. coli, thelatter strain mostly produces soluble MotY, but a little
fraction of MotY is recovered in the membrane fraction,and the membrane-bound MotY can be not released bywashing with buffer not containing any detergents (datanot shown). MotX could not be solubilized any of thefollowing four detergents: Triton X-100, b-octylglucoside,sucrose monocaprate and CHAPS. On the other hand,
MotY was solubilized by all four detergents (Fig. 4). Membrane fractions from VIO542 cells expressing both MotX
and MotY (Fig. 4C) and from NMB191 (DpomAB) expressing MotX, MotY, PomA and PomB (Fig. 4D) from a plasmid
were also treated with detergents. Under these conditions,
MotY was not solubilized. MotY did not become solubilized upon coexpression with MotX. This property was notchanged by the simultaneous expression of PomA/PomB.
These results may indicate that the membrane-binding
manner of MotY would be different in strains having andlacking MotX. Also, a physical interaction between MotX
and MotY exists.
Discussion
The sodium-driven polar flagellum of Vibrio is completely
paralysed by motX or motY mutation, suggesting that
MotX and MotY proteins are essential for the sodiumdriven motor. Mutations of pomA or pomB confer the same© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

MotX and MotY colocalize to the outer membrane 129

Fig. 4. Solubilization of MotX and MotY with several detergents.
Membranes from V. alginolyticus NMB94 (motX94) cells harbouring
plasmid pKJ401 (motX +, A), VIO542 (motY542) cells harbouring plasmid pIO6 (motY +, B), plasmid pKJ601 (motX + motY +, C), and
NMB191 (DpomAB) cells harbouring plasmid pKJ701 (motX + motY +pomAB +, D) were solubilized with 2.5% each of Triton X-100 (TX), boctylglucoside (OG), sucrose monocaprate (SC) or CHAPS (CH). The
supernatant (s) and precipitate (p) were prepared by ultracentrifugation (200 000 g, 10 min) and subjected to SDS-PAGE and immunoblotting using anti-MotX antiserum (A, and upper panels of C and D)
and anti-MotY antiserum (B, and lower panels of C and D).

polar-flagellar-motility phenotype, as PomA and PomB
proteins form a complex to conduct sodium ions. On theother hand, the function of MotX and MotY is obscure,which have been believed to be located in the inner membrane. In this paper, we concluded that MotX and MotY
colocalize to the outer membrane from the following
observations: (i) MotX and MotY did not have theirputative transmembrane segments, suggesting they arecleaved during translocation across the inner membrane;(ii) They localized to the outer membrane in E. coli,
whereas PomA/PomB were in the inner membrane. In theVibrio cell, they were detected in the fraction different fromthat of PomA/PomB, which may suggest their location in
the outer membrane; (iii) The outer membrane localizations of MotX and MotY were dependent on each other.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

Furthermore, MotY could not be solubilized in the presence of MotX, supporting their interaction in the outermembrane. The L-ring component, FlgH, is a flagellar
protein located in the outer membrane (Macnab, 1996).
This protein is thought to participate in the structure offlagellum, bushing for rotating rod in the outer membrane,rather than in the motor rotation. Therefore, we suggestthat MotX and MotY are the first example of outer membrane motor proteins.
The putative transmembrane region of each protein,
MotX or MotY, exhibits relatively low similarity among theVibrio species (Okabe et al., 2001). This implies that thisregion may not have important roles in structure and function. Such assumptions are reasonable because we demonstrated that the hydrophobic N-terminal sequence of
MotX and MotY was not present in the mature protein. It
can be speculated that the peptide was cut during translocation across the inner membrane, probably in a Sec
machinery-dependent manner (Pugsley, 1993). We conclude that the predicted transmembrane regions are
signal sequences for export. The protein translocation
mechanism in V. alginolyticus seems to be equivalent to
the E. coli Sec system, because several Sec factors of V.
alginolyticus show high similarity to those of E. coli andalso can function in E. coli (Tokuda et al., 1990; Kunioka
et al., 1998; Nishiyama et al., 1998). In this study, MotX
proteins from the two expression systems of E. coli and
V. alginolyticus had the identical N-terminus, suggesting
functionally homologous enzymes are relevant to the processing of secretory proteins in the organisms. Fromthese lines of evidence, we speculate that primarily translated forms of MotX and MotY are translocated across theinner membrane and signal sequences are cleaved toconvert the mature proteins and localize to the outer membrane of V. alginolyticus. MotX and MotY homologues
have not been reported from any other bacteria thanVibrio species, including alkalophilic Bacillus species thathave sodium-driven flagella. That is reasonable, because
Bacilli do not have an outer membrane. Rhizobium meliloti
requires a periplasmic motor protein MotC for the protondriven flagellar rotation (Platzer et al., 1997). This protein,however, has significant sequence homology with neither
MotX nor MotY.
The secondary-structures of MotX and MotY from V.
alginolyticus were predicted by the method of Chou andFasman (Chou and Fasman, 1974). This method showedthat MotX is more a-helical than MotY, which exhibits
an extensive b-strand structure (data not shown). Outer
membrane proteins often form a b-barrel structure in
their membrane-spanning domains (Pugsley, 1993;
Koebnik et al., 2000). A three-dimensional structure
prediction program, 3D-PSSM based on the SCOP
(http://www.sbq.bio.ic.ac.uk/~3dpssm/) (Kelley et al.,2000), showed that MotY has similarities to OmpA trans-

130 M. Okabe, T. Yakushi, M. Kojima and M. Homma

Fig. 5. A hypothetical model for the sodium-driven motor. The sodiumdriven polar flagellum of Vibrio is sheathed. The energy source forflagellar rotation is provided by an electrochemical gradient acrossthe inner membrane. MotX and MotY colocalize to the outer membrane and may participate in conduction of sodium ions in concert
with PomA/PomB. From the predicted structure, MotX seems not tohave enough b-sheet to span the outer membrane (A), but may alter
its conformation upon interaction with MotY (B). IM, inner membrane;
PG, peptidoglycan; OM, outer membrane.

membrane domain with a relatively high E-value (8.26E05). As this domain of OmpA (1–171 residues), of which
the structure has been determined, loses its C–terminal
peptidoglycan association region (Pautsch and Schulz,
1998), the similarities are restricted in the region embedded in the outer membrane. On the other hand, there
is no candidate similar to MotX with such a high E-value.
Structure prediction may infer that MotY is an integral
outer membrane protein and MotX has too short b-sheet
structure to span the outer membrane (Fig. 5A).
MotY has a peptidoglycan-binding motif in its C-terminal
portion that was proposed to anchor the protein to the
peptidoglycan (McCarter, 1994b; Okunishi et al., 1996),
thus forming a bridge between the outer membrane andthe cell wall (Fig. 5). When MotY is expressed by itself,
most of that remains in the periplasm but the rest moves
exclusively to the outer membrane. This result corroborates the earlier observation that MotY is a membraneprotein that is not stable in the absence of MotX
(McCarter, 1994a; Okabe et al., 2001). Noteworthily, incontrast with MotX, a limited portion of MotY itself is able
to precisely localize to the outer membrane in the absenceof MotX (Fig. 3B). On the other hand, most of MotX seems
to associate with neither membrane, but rather is detected
in intermediate fractions between the inner and outermembranes, when expressed by itself (Fig. 3A). From thisresult, three possible behaviours of MotX without MotY
are speculated as follows. First, MotX associates withneither membrane, but rather with some large molecule,such as protein complex and the peptidoglycan. Second,
MotX associates with the both membranes, some are in
the outer and others are in the inner membrane. Finally,

the third possibility is an intermediate membrane. Further
experiments may help to understand the ambiguous localization of MotX expressed by itself. Whatever may happen
in the absence of MotY, MotX coexpressed with MotY
localizes to the outer membrane and seems to interact
therein. Also, both MotX and MotY seem to be mutuallyrequired for their precise outer membrane localization. In
such situation, the both proteins are present, MotX may
be embedded in the outer membrane (Fig. 5B).
Although an interaction between MotX and MotY canbe suggested by Fig. 4, a major question remains to beanswered, how might MotX and MotY contribute to motor
function in the outer membrane? One possibility is thatthey make a pore for sodium ions in the outer membrane.
MotX has been reported as a sodium channel that canbe inhibited by amiloride, a sodium channel blocker(McCarter, 1994a). Many outer membrane proteins with ab-barrel structure form pores that allow water, ions andvarious small molecules to pass across the bilayer. The
predicted secondary and tertiary structure of MotY may
be embedded in the outer membrane to form a structure
like porin. Sodium ions may pass through the structure
and may be delivered to other flagellar motor components,
such as PomA and PomB (Fig. 5). Figure 2C suggests
the possible interaction between MotX and PomA/PomB,
supporting such speculations. There are reports pointing
out that MotA and MotB show moderate sequence similarity to ExbB and ExbD, which are the inner membraneproteins functioning in cooperation with TonB and outermembrane receptors to drive active transport of certain
nutrients across the outer membrane (Cascales et al.,
2001; Kojima and Blair, 2001). This similarity implies thatthe electrochemical potential in the inner membrane may
be transferred to MotX and MotY in the outer membrane
by function of PomA/PomB to drive the flagellar rotation
therein.
We have observed that overexpression of MotX suppressed motY mutations (Okabe et al., 2001). MotY mayfunction as a kind of chaperon for MotX, or vice versa.
The polar flagellum of Vibrio is covered by a membranous
sheath that extends from the outer membrane (Furuno
et al., 2000). MotX and MotY could be present in the
sheath and affect its formation coordinate with the rotationof flagellum. The both proteins have been found being in
the outer membrane in this study. The precise roles ofthem are not clear at present. However, they should function in the outer membrane to rotate the sodium-driven
flagellar motor.

Experimental procedures
Bacterial strains and growth conditions
The strains of Vibrio alginolyticus and Escherichia coli used
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

MotX and MotY colocalize to the outer membrane 131
Table 1. Bacterial strains and plasmids.
Strain or plasmid

Genotype or description

Vibrio alginolyticus strains
VIO542

Rifr Laf– motY542

NMB94

Laf– motX94

NMB191

Rifr Laf– DpomAB

Escherichia coli strains
JM109
Plasmids
pSU41
pMO200pMO401
pKJ401
pIO6
pKJ502
pKJ503
pKJ601
pYA303
pKJ701

Reference

Okunishi et al. (1996)
Okabe et al. (2001)
Yorimitsu et al. (1999)

recA1, endA1, gyrA96, thi–, hsdR17, relA1, supE44, l-,D(lac-proAB); F¢, traD36, proAB, lacIq, DM15

Yanisch-Perron et al. (1985)

kan (Kmr) Plac lacZa
pSU21, 1.0 kb EcoRI–EcoRI fragment (motX +)pSU41, 700 bp ClaI–BamHI fragment (motX +)pSU41, 700 bp XbaI–BamHI fragment (motX +)pSU38, 1.0 kb HindIII–XbaI fragment (motY +)pSU41, 1.0 kb SalI–XbaI fragment (motY +)pSU41, 1.0 kb SalI–XbaI fragment (motY-his6+)
pKJ502 (motY +), 700 bp XbaI–BamHI fragment (motX +)pSU41, 1.9 kb SphI–SalI flagment (pomAB +)pKJ601 (motY + motX +), 1.9-kb SphI–SalI fragment (pomAB +)Bartolome et al. (1991)
Okabe et al. (2001)
Okabe et al. (2001)
This work
Okunishi et al. (1996)
This work
This work
This work
Kojima et al. (1999)
This workin this study are listed in Table 1. V. alginolyticus cells were
cultured at 30∞C on 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 strains used for DNA manipulations andmembrane localization experiments were cultured at 37∞C on
Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract,
0.5% NaCl). When necessary, kanamycin was added to a
final concentration of 100 mg ml-1 (for V. alginolyticus) or
50 mg ml-1 (for E. coli ).

inserted between the 3¢-end of the motY open reading frame(ORF) and the XbaI site of the plasmid pKJ402. As thisplasmid complemented the motY mutation of Vibrio, the Cterminal histidine tag does not perturb MotY function (datanot shown). All motor genes of the plasmid constructed here
do not have the native promoter and are under the control ofthe lac promoter–operator. Transformation of Vibrio cells byelectroporation was carried out as described previously(Kawagishi et al., 1994).

Membrane preparation and solubilization
Plasmid construction
DNA manipulations were carried out according to standard
procedures (Sambrook et al., 1989). Plasmids are listed inTable 1. Plasmid pKJ401 carrying motX was constructed bycloning a 700 basepair (bp) XbaI–BamHI fragment of polymerase chain reaction (PCR) products using pMO200 as atemplate. The 5¢-TTAGAT-3¢ sequence, 18 bp upstream fromthe initiation codon of motX, was changed to 5¢-TCTAGA-3¢,
to introduce the XbaI site. Also, the 5¢-GGATCC-3¢ sequence(BamHI site) was introduced just adjacent to the termination
codon of motX. Plasmid pKJ502 was constructed to carry a
1.0 kb motY by introducing SalI (5¢-GTCGAC-3¢) and XbaI
(5¢-TCTAGA-3¢) sites at 66 bp upstream of the initiation
codon and just adjacent to the termination codon of motY
respectively. A 700 bp XbaI–BamHI fragment (containing
motX) of pKJ401 was inserted into the corresponding sitesof pKJ502 (carrying motY) to construct pKJ601, which carries
an artificial motY-motX operon. A 1.9 kb BamHI–SacI fragment (pomAB+) of pYA303 was inserted in the same restriction sites of pKJ601; the resultant plasmid pKJ701 carries a
synthetic motY-motX-pomAB operon. Plasmid pKJ503 carried the gene encoding C-terminally hexahistidine-tagged
MotY (MotY-His6) in pSU41 by PCR using synthetic oligonucleotide primers. The nucleotide sequence of 5¢-CATCACC
ATCACCATCACTAATCTAGA-3¢ (XbaI site is underlined) was© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

Vibrio cells harbouring plasmids were cultured at 30∞C on
VPG medium. Cells were harvested and washed twice with
25 mM Tris-HCl, 10 mM MgCl2, 300 mM NaCl, pH 7.5 andstored at -80∞C until used. The frozen cells were thawed andresuspended (0.2 g ml-1, wet weight) in 20 mM Tris-HCl,
5 mM MgSO4, 0.5 mM phenylmethylsulphonyl fluoride(PMSF), 10 mg ml-1 of DNase I, pH 8.0. Membrane vesicles
were prepared by subjecting the suspension to a single passage through a French press (5501-M Ohtake Works) at
500 kg cm-2 at 4∞C. Undisrupted cells were removed by lowspeed centrifugation (10 000 g for 5 min at 4∞C), and themembrane fraction was recovered from the supernatant by
ultracentrifugation at 100 000 g for 2 h. The membrane pellet
was suspended in 20 mM Tris-HCl, 0.5 mM PMSF, 20% (w/v) glycerol, pH 8.0, homogenized, and stored at -80∞C until
used. The membrane preparations (10 mg ml-1) were resuspended in 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM PMSF,
10% (w/v) glycerol, 5 mM imidazole, pH 8.0, and 2.5% (w/v)
each of the following detergents: Triton X-100, b-octylglucoside (Dojin), sucrose monocaprate (Dojin), and 3-[3-cholamidopropyl-dimethylammonio] propanesulphonic acid (CHAPS)(Dojin). It was then incubated at 4∞C for 10 min and centrifuged for 10 min at 200 000 g to separate the solubilized
supernatant and the insoluble precipitate. Protein concentrations were determined by the BCA protein assay kit (Pierce).

132 M. Okabe, T. Yakushi, M. Kojima and M. Homma
Spheroplast formation
Conversion of E. coli cells into spheroplasts was performed
as described previously (Osborn et al., 1972). Briefly, E. coli
strain JM109 cells harbouring each plasmid were harvested
and resuspended in 20 mM Tris-HCl, 0.75 M sucrose,
100 mM NaCl (pH 7.5). A twofold volume of 1.5 mM EDTA,
100 mM NaCl (pH 8.0) was gradually added, following theaddition of lysozyme and DNase I, to convert to spheroplasts.
The spheroplast suspension was centrifuged (10 000 g for
5 min) to separate periplasmic fraction from the spheroplasts.
The precipitated spheroplasts were resuspended in an
appropriate buffer and disrupted by sonication. Cytoplasmic
and membrane fractions were prepared by ultracentrifugation(200 000 g, 10 min). Spheroplasting of V. alginolyticus was
performed according to the method described previously(Nakamaru et al., 1999). The efficiency of spheroplast formation was determined from the fraction of round cells observedby phase-contrast microscopy.

Membrane localization of MotX and MotY
Escherichia coli strain JM109 harbouring plasmid pKJ401,
pKJ502, pKJ601 or pKJ701 was grown on Luria–Bertani
medium at 37∞C to the mid-exponential phase. The expression of motor proteins was induced for 1 h by addition of
1 mM isopropyl-b-D-thiogalactopyranoside (IPTG). V. alginolyticus strain NMB191 harbouring plasmid pKJ701 wasgrown on VC medium and harvested in the mid-logarithmic
phase. The cells were harvested and converted to spheroplasts. The spheroplast suspension was sonicated, and thetotal membrane fraction was obtained by centrifugation(200 000 g, 10 min) after the removal of unbroken spheroplasts. The total membranes were resuspended in 50 mM
Tris-HCl (pH 7.5) containing 1 mM EDTA. It was then applied
to a 35–55% (w/w) stepwise sucrose gradient containing
50 mM Tris-HCl (pH 7.5) and 1 mM EDTA. After centrifugation at 80 000 g for 12 h at 4∞C, the gradient was divided into
21–24 fractions from the bottom to the top. OmpA (Yamada
et al., 1989), SecG (Nishiyama et al., 1993), maltose binding
protein (MBP) (New England Biolabs), LacI (Abo et al.,2000), MotX (Okabe et al., 2001), MotY (Okabe et al., 2001),
PomA (Yorimitsu et al., 1999) and PomB (Yorimitsu et al.,1999) proteins in each fraction were detected by sodium
dodecyl sulphate–polyacrylamide gel electrophoresis (SDSPAGE) followed by immunoblotting with the respective
antisera.

Preparation of MotX and MotY and determination of theirN-terminal amino acid sequences
MotX was prepared from both E. coli strain JM109 harbouring
plasmid pMO401 and V. alginolyticus strain NMB94 harbouring the plasmid. Membranes prepared as described above
were suspended in buffer A (20 mM Tris-HCl, 150 mM NaCl,
10% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 mM imidazole,
pH 8.0) at 10 mg membrane protein per ml. The membranes
were pretreated with 2.5% Triton X-100 on ice for 10 min, andthe Triton-insoluble materials were recovered by ultracentrifugation (200 000 g, 10 min). The precipitates were resus-

pended in buffer A containing 3% (w/v) SDS in a tenth volumeof the initial solution and treated at 80∞C for 10 min. The SDSsolubilized material contained MotX. MotY was purified fromthe membrane fraction of the strain NMB94 harbouring plasmid pKJ503, which encodes C-terminally histidine-tagged
MotY. The membranes (10 mg ml-1 protein) were treated with
2.5% Triton X-100 in buffer B (20 mM Tris-HCl, 150 mM NaCl,
10% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 mM imidazole,pH 7.5). Triton-insoluble material was removed by ultracentrifugation (100 000 g, 30 min), and the supernatant was
passed over Ni-NTA agarose. Bound His-tagged MotY waseluted with buffer B containing 0.05% Triton X-100 and
200 mM imidazole. Partially purified MotX and MotY wereseparated from impurities by SDS-PAGE and electrophoretictransfer to polyvinylidene difluoride membrane (Millipore) andstained with Coomassie blue R250. The bands corresponding to MotX and MotY were excised, and their N-terminal
amino acid sequences were determined by Aproscience(Tokushima), based on Edman degradation. Five N-terminal
amino acid sequences of MotX from both E. coli and V.
alginolyticus expression systems were identical as describedin Results.

Acknowledgements
We thank Professor Hajime Tokuda for the kind donation ofanti-OmpA and SecG antisera, and Professor Tatsuhiko Abo
for anti-LacI antiserum. We acknowledge Professors Taiji
Nakae and Ikuro Kawagishi for stimulating discussion. We
also thank Ms. Noriko Ui and Emiko Ichikawa for their technical support, and Mr Atsushi Hijikata for his critical suggestions at the beginning of this study. Finally, we give our
gratitude to Professors Rüdiger Schmitt and Michael D. Manson for their review and critique of this manuscript. This work
was supported by grants-in-aid for scientific research fromthe ministry of Education, Science and Culture of Japan (toT.Y. and M.H.).

References
Abo, T., Inada, T., Ogawa, K., and Aiba, H. (2000) SsrAmediated tagging and proteolysis of LacI and its role in theregulation of lac operon. EMBO J 19: 3762–3769.
Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi, I., andHomma, M. (1997) Putative channel components for the
fast-rotating sodium-driven flagellar motor of a marine bacterium. J Bacteriol 179: 5104–5110.
Asai, Y., Kawagishi, I., Sockett, E., and Homma, M. (1999)
Hybrid motor with the H+-and Na+-driven components canrotate Vibrio polar flagella by using sodium ions. J Bacteriol
181: 6322–6338.
Asai, Y., Sockett, R.E., Kawagishi, I., and Homma, M. (2000)
Coupling ion specificity of chimeras between H+- andNa+-driven motor proteins, MotB and PomB. Vibrio polar
flagella. EMBO J 19: 3639–3648.
Atsumi, T., McCarter, L., and Imae, Y. (1992) Polar andlateral flagellar motors of marine Vibrio are driven by different ion-motive forces. Nature 355: 182–184.
Bartolome, B., Jubete, Y., Martinez, E., and de la Cruz, F.
(1991) Construction and properties of a family of© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

MotX and MotY colocalize to the outer membrane 133
pACY184-derived cloning vectors compatible with pBR322
and its derivatives. Gene 102: 75–78.
Berg, H.C. (1995) Torque generation by the flagellar rotary
motor. Biophys J 68: S163–S167.
Blair, D.F. (1995) How bacteria sense and swim. Annu Rev
Microbiol 49: 489–522.
Blair, D.F., and Berg, H.C. (1988) Restoration of torque in
defective flagellar motors. Science 242: 1678–1681.
Blair, D.F., and Berg, H.C. (1990) The MotA protein of E. coli
is a proton-conducting component of the flagellar motor.
Cell 60: 439–449.
Cascales, E., Lloubes, R., and Sturgis, J.N. (2001) The TolQTolR proteins energize TolA and share homologies with theflagellar motor proteins MotA-MotB. Mol Microbiol 42: 795–
807.
Chou, P., and Fasman, G. (1974) Prediction of protein conformation. Biochemistry 13: 222–245.
Chun, S.Y., and Parkinson, J.S. (1988) Bacterial motility:
membrane topology of the Escherichia coli MotB protein.
Science 239: 276–278.
De Mot, R., and Vanderleyden, J. (1994) The C-terminal
sequence conservation between OmpA-related outermembrane proteins and MotB suggests a common function
in both Gram-positive and Gram-negative bacteria, possibly in the interaction of these domains with peptidoglycan.
Mol Microbiol 12: 333–334.
Dean, G.D., Macnab, R.M., Stader, J., Matsumura, P., and
Burks, C. (1984) Gene sequence and predicted amino acidsequence of the motA protein, a membrane-associatedprotein required for flagellar rotation in Escherichia coli. J
Bacteriol 159: 991–999.
Derosier, D.J. (1998) The turn of the screw: the bacterial
flagellar motor. Cell 93: 17–20.
Furuno, M., Sato, K., Kawagishi, I., and Homma, M. (2000)
Characterization of a flagellar sheath component, PF60,
and its structural gene in marine Vibrio. J Biochem (Tokyo)
127: 29–36.
Gosink, K.K., and Häse, C.C. (2000) Requirements for conversion of the Na+-driven flagellar motor of Vibrio cholerae
to the H+-driven motor of Escherichia coli. J Bacteriol 182:
4234–4240.
Hirota, N., Kitada, M., and Imae, Y. (1981) Flagellar motorsof alkalophilic Bacillus are powered by an electrochemical
potential gradient of Na+. FEBS Lett 132: 278–280.
Imae, Y., and Atsumi, T. (1989) Na+-driven bacterial flagellar
motors. J Bioenerg Biomemb 21: 705–716.
Kawagishi, I., Okunishi, I., Homma, M., and Imae, Y. (1994)
Removal of the periplasmic DNase before electroporation
enhances efficiency of transformation in a marine bacterium Vibrio alginolyticus. Microbiology 140: 2355–2361.
Kawagishi, I., Maekawa, Y., Atsumi, T., Homma, M., andImae, Y. (1995) Isolation of the polar and lateral flagellumdefective mutants in Vibrio alginolyticus and identificationof their flagellar driving energy sources. J Bacteriol 177:
5158–5160.
Kelley, L., MacCallum, R., and Sternberg, M. (2000)
Enhanced genome annotation using structural profiles in
the program 3D-PSSM. J Mol Biol 299: 499–520.
Khan, S., Dapice, M., and Reese, T.S. (1988) Effects of mot
gene expression on the structure of the flagellar motor. J
Mol Biol 202: 575–584.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134

Klose, M., MacIntyre, S., Schwarz, H., and Henning, U.
(1988) The influence of amino substitutions within themature part of an Escherichia coli outer membrane protein(OmpA) on assembly of the polypeptide into its membrane.
J Biol Chem 263: 13297–13302.
Koebnik, R., Locher, K.P., and Van Gelder, P. (2000) Structure and function of bacterial outer membrane proteins:barrels in a nutshell. Mol Microbiol 37: 239–253.
Kojima, S., and Blair, D.F. (2001) Conformational change inthe stator of the bacterial flagellar motor. Biochemistry 40:
13041–13050.
Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I., and Homma,
M. (1999) Na+-driven flagellar motor resistant to phenamil,
an amiloride analog, caused by mutations of putative channel components. J Mol Biol 285: 1537–1547.
Kudo, S., Magariyama, Y., and Aizawa, S.-I. (1990) Abruptchanges in flagellar rotation observed by laser dark-fieldmicroscopy. Nature 346: 677–680.
Kunioka, E., Matsuyama, S., and Tokuda, H. (1998) Cloning
and expression of the secA gene of a marine bacterium,
Vibrio alginolyticus, and analysis of its function in Escherichia coli. Gene 216: 303–309.
Lloyd, S.A., Whitby, F.G., Blair, D.F., and Hill, C.P. (1999)
Structure of the C-terminal domain of FliG, a componentof the rotor in the bacterial flagellar motor. Nature 400:
472–475.
Lowe, G., Meister, M., and Berg, H.C. (1987) Rapid rotationof flagellar bundles in swimming bacteria. Nature 325:
637–640.
Macnab, R. (1996) Flagela and motility. In Eschericia coli and
Salmonella. Neidhardt, F.C. (chief-ed.). Washington, DC:
American Society for Microbiology, pp. 123–145.
Magariyama, Y., Sugiyama, S., Muramoto, K., Maekawa, Y.,
Kawagishi, I., Imae, Y., and Kudo, S. (1994) Very fast
flagellar rotation. Nature 381: 752.
Manson, M., Tedesco, P., Berg, H., Harold, F., and Van derDrift, C. (1977) A protonmotive force drives bacterial flagella. Proc Natl Acad Sci USA 74: 3060–3064.
McCarter, L.L. (1994a) MotX, the channel component of
the sodium-type flagellar motor. J Bacteriol 176: 5988–
5998.
McCarter, L.L. (1994b) MotY, a component of the sodiumtype flagellar motor. J Bacteriol 176: 4219–4225.
McCarter, L.L. (2001) Polar flagellar motility of the Vibrionaceae. Microbiol Mol Biol Rev 65: 445–462.
McCarter, L., and Wright, M. (1993) Identification of genes
encoding components of the swarmer cell flagellar motor
and propeller and a sigma factor controlling differentiationof Vibrio parahaemolyticus. J Bacteriol 175: 3361–3371.
Muramoto, K., Sugiyama, S., Cragoe, E.J., and Imae, Y.
(1994) Successive inactivation of the force-generating
units of sodium-driven bacterial flagellar motors by a photoreactive amiloride analog. J Biol Chem 269: 3374–3380.
Muramoto, K., Kawagishi, I., Kudo, S., Magariyama, Y., Imae,
Y., and Homma, M. (1995) High-speed rotation and speed
stability of sodium-driven flagellar motor in Vibrio alginolyticus. J Mol Biol 251: 50–58.
Nakai, K., and Kanehisa, M. (1991) Expert system for predicting protein localization sites in Gram-negative bacteria.
PROTEINS: Structure, Function, and Genetics 11: 95–
110.

134 M. Okabe, T. Yakushi, M. Kojima and M. Homma
Nakamaru, Y., Takahashi, Y., Unemoto, T., and Nakamura,T. (1999) Mechanosensitive channel functions to alleviate
the cell lysis of marine bacterium, Vibrio alginolyticus, byosmotic downshock. FEBS Lett 444: 170–172.
Nishiyama, K., Mizushima, S., and Tokuda, H. (1993) A novel
membrane protein involved in protein translocation acrossthe cytoplasmic membrane of Escherichia coli. EMBO J
12: 3409–3415.
Nishiyama, K., Furuta, M., and Tokuda, H. (1998) Molecularcloning and functional characterization of SecE of a marine
bacterium, Vibrio alginolyticus. Biochem Biophys Res
Comm 251: 894–897.
Okabe, M., Yakushi, T., Asai, Y., and Homma, M. (2001)
Cloning and characterization of motX, a Vibrio alginolyticus
sodium-driven flagellar motor gene. J Biochem 130: 879–
884.
Okunishi, I., Kawagishi, I., and Homma, M. (1996) Cloning
and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus. J Bacteriol 178: 2409–2415.
Osborn, M.J., Gander, J.E., Parisi, E., and Carson, J. (1972)
Mechinism of assembly of the outer membrane of
Salmonella typhimurium. Isolation and characterization ofcytoplasmic and outer membrane. J Biol Chem 247: 3962–
3972.
Pautsch, A., and Schulz, G.E. (1998) Structure of the outermembrane protein A transmembrane domain. Nat Struct
Biol 5: 1013–1037.
Platzer, J., Sterr, W., Hausmann, M., and Schmitt, R. (1997)
Three genes of a motility operon and their role in flagellar
rotary speed variation in Rhizobium meliloti. J Bacteriol
179: 6391–6399.
Pugsley, A.P. (1993) The complete general secretory
pathway in Gram-negative bacteria. Microbiol Rev 57: 50–
108.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (eds). (1989)
Molecular Cloning: a Laboratory Manual, 2nd edn. Cold
Spring Harbor, New York: Cold Spring Harbor Laboratory
Press.
Sato, K., and Homma, M. (2000a) Functional reconstitutionof the Na+-driven polar flagellar motor component of Vibrio
alginolyticus. J Biol Chem 275: 5718–5722.
Sato, K., and Homma, M. (2000b) Multimeric structure of
PomA, the Na+-driven polar flagellar motor component ofVibrio alginolyticus. J Biol Chem 275: 20223–20228.
Sharp, L.L., Zhou, J.D., and Blair, D.F. (1995a) Features of
MotA proton channel structure revealed by tryptophan-scanning mutagenesis. Proc Natl Acad Sci USA 92: 7946–
7950.
Sharp, L.L., Zhou, J.D., and Blair, D.F. (1995b) Tryptophanscanning mutagenesis of MotB, an integral membraneprotein essential for flagellar rotation in Escherichia coli.
Biochemistry 34: 9166–9171.
Stader, J., Matsumura, P., Vacante, D., Dean, G.E., andMacnab, R.M. (1986) Nucleotide sequence of the Escherichia coli MotB gene and site-limited incorporation of its
product into the cytoplasmic membrane. J Bacteriol 166:
244–252.
Tokuda, H., Kim, Y., and Mizushima, S. (1990) In vitro proteintranslocation into inverted membrane vesicles preparedfrom Vibrio alginolyticus is stimulated by the electrochemical potential of Na+ in the presence of Escherichia coli
SecA. FEBS Lett 264: 10–12.
Yamada, H., Matsuyama, S., Tokuda, H., and Mizushima, S.
(1989) A high concentration of SecA allows proton motive
force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles. J Biol Chem
264: 18577–18581.
Yanisch-Perron, C., Vieira, J., and Messing, J. (1985)
Improved M13 phage cloning vectors and host strains:
nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119.
Yorimitsu, T., and Homma, M. (2001) Na+-driven flagellar
motor of Vibrio. Biochim Biophys Acta 1505: 82–93.
Yorimitsu, T., Sato, K., Asai, Y., Kawagishi, I., and Homma,
M. (1999) Functional interaction between PomA and
PomB, the Na+-driven flagellar motor components ofVibrio
alginolyticus. J Bacteriol 181: 5103–5106.
Yorimitsu, T., Sato, K., Asai, Y., and Homma, M. (2000)
Intermolecular cross-linking between the periplasmic
loop3-4 regions of PomA, a component of the Na+-driven
flagellar motor of Vibrio alginolyticus. J Biol Chem 275:
31387–31391.
Zhou, J.D., Fazzio, R.T., and Blair, D.F. (1995) Membrane
topology of the MotA protein of Escherichia coli. J Mol Biol
251: 237–242.
Zhou, J., Lloyd, S.A., and Blair, D.F. (1998a) Electrostaticinteractions between rotor and stator in the bacterial
flagellar motor. Proc Natl Acad Sci USA 95: 6436–6441.
Zhou, J., Sharp, L.L., Tang, H.L., Lloyd, S.A., Billings, S.,
Braun, T.F., and Blair, D.F. (1998b) Function of protonatable residues in the flagellar motor of Escherichia coli: a
critical role for Asp 32 of MotB. J Bacteriol 180: 2729–
2735.

© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134