JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 778–784
0021-9193/05/$08.00⫹0 doi:10.1128/JB.187.2.778–784.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 187, No. 2

Deletion Analysis of the Carboxyl-Terminal Region of the PomB
Component of the Vibrio alginolyticus Polar Flagellar Motor
Toshiharu Yakushi,* Naoko Hattori, and Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan
Received 13 July 2004/Accepted 11 October 2004

The stator of the sodium-driven flagellar motor of Vibrio alginolyticus is a membrane protein complex
composed of four PomA and two PomB subunits. PomB has a peptidoglycan-binding motif in the C-terminal
region. In this study, four kinds of PomB deletions in the C terminus were constructed. None of the deletion
proteins restored motility of the ⌬pomB strain. The PomA protein was coisolated with all of the PomB
derivatives under detergent-solubilized conditions. Homotypic disulfide cross-linking of all of the deletion
derivatives through naturally occurring Cys residues was detected. We conclude that the C-terminal region of
PomB is essential for motor function but not for oligomerization of PomB with itself or PomA.
membrane (30), are unique to the sodium-driven motor. The
functions of MotX and MotY are not yet clear.
Gram-negative bacteria, such as E. coli and V. alginolyticus,
have a thin but rigid peptidoglycan layer that maintains cell
integrity. E. coli MotB and V. alginolyticus PomB and MotY
have conserved sequences in their C termini that resemble
motifs present in peptidoglycan-associated proteins, such as
OmpA and Pal (1, 14, 26, 31) (Fig. 1A). Some paralyzed andslow-swimming mutations in motB alter residues in or near thisputative peptidoglycan-binding region (7, 14, 38). Amino acidsubstitutions and deletions in the putative peptidoglycan-binding region might also influence other functions of PomB
(MotB), such as the interactions with PomA and PomB. In thepresent study, a series of C-terminal deletions in PomB wasconstructed and characterized.
Sequence conservation between PomB and its homologs in
the C-terminal region and construction of deletion mutant
proteins. The C-terminal periplasmic region of MotB of E. coli
is thought to be involved in interactions with the cell wall (12),and De Mot and Vanderleyden (14) reported conservation
between sequences in the C-terminal half of MotB and in outermembrane proteins, such as Pal or OmpA of E. coli, that are
known to be associated with peptidoglycan (Fig. 1A). Many
missense mutations that inactivate MotB also affect this conserved region. Alignments of sequences at the C termini of V.
alginolyticus PomB, E. coli MotB, and Vibrio parahaemolyticus
LafU show especially significant conservation. In PomB, whichhas 315 amino acid residues, these highly conserved sequences
include GHTD (from Gly-229 to Asp-232), SNWXLSXXRA(from Ser-243 to Ala-252), VXGM (from Val-272 to Met-275),
and NRR (from Asn-293 to Arg-295). Some of these sequences are also conserved in Pal and OmpA (Fig. 1A).
To study the role of the C-terminal conserved regions of
PomB (hereafter, the C region), we arbitrarily divided it into
subregions C1 (Val-227 to Ala-252), C2 (Leu-270 to Asp-277),
and C3 (Ala-291 to Ile-300). We constructed four pomB deletion mutants, ⌬C1, ⌬C2, ⌬C3, and ⌬C (⌬Phe-196 to Gln-315),
corresponding to the subregions, and the properties of themutated proteins were assessed (Fig. 1B).
DNA manipulations were carried out according to standard
procedures (32). An E. coli strain was used for DNA manipu-

Bacteria can swim by rotating their helical flagellar filaments. The flagellar motor embedded in the cytoplasmic membrane generates torque to turn the flagellum. Energy for rotation is provided by the electrochemical gradient of protons or
sodium ions across the cytoplasmic membrane (4, 20, 25, 27,42). Proton-driven flagellar rotation in Escherichia coli requires
the motor proteins MotA and MotB (35). MotA and MotB are
cytoplasmic membrane proteins with four and one transmembrane (TM) segments, respectively (12, 13, 36, 45). MotA and
MotB form a proton-channel complex and serve as a stator.
Successive conformational changes that accompany proton
conduction drive the rotation of the MS ring/FliG complex,
which comprises the rotor of the flagellar motor (6, 24, 37). A
variety of evidence suggests that 8 to 12 torque generators,
composed of the MotA/MotB complex, are placed in a circulararray around the rotor (5, 8, 22).
Of the sodium-driven motors, the polar flagella of Vibrio
species have been the most extensively studied (27, 42). Mostof the flagellar genes encode proteins that are similar to thoseof the proton-driven flagella. Four membrane proteins, PomA,
PomB, MotX, and MotY, are essential for torque generationof the sodium-driven motor (for reviews, see references 27 and42 and the references therein). PomA and PomB are homologsof MotA and MotB, respectively (1), and function as a sodium
channel complex (33). PomA and PomB each form homodimers and assemble into a heterohexameric complex containing four PomA and two PomB subunits (34, 43). Thus,
PomB associates both with PomA and itself.
MotA and MotB of E. coli support motility of the polar
flagellum of Vibrio cholerae and of Vibrio alginolyticus, andcertain chimeric motors containing components from both theproton-driven and sodium-driven types function in E. coli (2,18). These lines of evidence support the hypothesis that therotation mechanisms of the two types of motors are similar. On
the other hand, MotX and MotY, which localize to the outer* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan. Phone: 81-52-789-2992. Fax: 81-52-789-3001.
E-mail: 4juji@cc.nagoya-u.ac.jp.
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FIG. 1. Sequence alignments of the C-terminal regions and the C-terminal deletion derivatives of PomB. (A) Multiple amino acid alignmentsof V. alginolyticus PomB C-terminal region with PomB homologs and other peptidoglycan-associated proteins. The alignments were constructedby using the Clustal W program (version 1.4). White letters with a black background and black letters with a gray background indicate amino acidresidues conserved in all six proteins and in more than four of the six proteins, respectively. The C region (Phe-196 to Gln-315) and the subregions
C1 (Val-227 to Ala-252), C2 (Leu-270 to Asp-277), and C3 (Ala-291 to Ile-300) are shown above the sequence. VaPomB, V. alginolyticus PomB;
EcMotB, E. coli MotB; EcOmpA, E. coli OmpA; EcPal, E. coli Pal, VpLafU, V. parahaemolyticus LafU; NmRmpM, N. meningitidis RmpM. Aminoacid residues with asterisks are proposed to contact peptidoglycan directly (19). Coiled and careted lines indicate ␣-helices and ␤-sheets,
respectively, as determined for the RmpM protein (19). (B) Schematic diagram of PomB deletion proteins. White areas indicate the N-terminal
cytoplasmic region and the C-terminal periplasmic region. Dark gray indicates the TM segment. Light gray indicates the hexahistidine sequence
attached to the C terminus. The black boxes indicate deleted residues. The three black bars (top) indicate the regions corresponding to the three
peptide sequences used for antibody production. Residue numbering and the predicted molecular weights (MW; in thousands) of the PomB
derivatives are shown at the right. wt, wild type; Ab, antibody; a.a., amino acid.

lations (41). The pom genes on the plasmids constructed in thisstudy were expressed from the lac promoter in pSU41 (3).
Plasmid pNH21 (pomB⌬C) was constructed by PCR with
pSK603 (23) as a template. It contains the sequence 5⬘-(GGT)GCA(CAC)CAT(CAC)CAT(CAC)CAT(TAA)GAGCTC-3⬘ atthe 3⬘ end of the pomB open reading frame. This sequence encodes Gly194-Ala195-His6 followed by a termination codon (theSacI site is underlined). Plasmid pNH22 (pomB⌬C1) was constructed by PCR with pKJ201 (39) as a template and has thesequence 5⬘-(GTA)CGC(GTA)TCA-3⬘at the deleted region.
This sequence codes for Val225-Arg226-Val253-Ser254 (the MluI
site is underlined). Plasmids pNH23 and pNH24 (pomB⌬C2 andpomB⌬C3) were constructed in the same way as pNH22, with
PomB⌬C2[5⬘-(CAA)CGT(ACG)GAG-3⬘(Gln268-Arg269Thr278-Glu279; the BsiWI site is underlined)] and PomB⌬C3 [5⬘(AAT)CGC(ATG)CAA-3⬘ (Asn289-Arg290-Met301-Gln302; theSphI site is underlined)]. The pomB genes containing the various
deletions were transferred to pKJ301, which carries pomA and

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FIG. 2. Expression of PomB derivatives and their cross-linked dimers. The NMB192(⌬pomB)/pMMB206 strain harboring plasmids carrying thepomB derivatives (lanes 1 to 10), or the NMB191(⌬pomAB)/pMMB206 strains harboring plasmids carrying wild-type (wt) pomA and the pomB
derivatives (lanes 11 to 20) were cultivated in liquid VPG500 medium until mid-log phase (optical density at 660 nm of ⬃1.0). When necessary,
IPTG (1 mM final concentration) was added 2 h prior to harvest. Whole-cell extracts prepared as described previously (40) were dissolved in
SDS-PAGE loading buffer containing 5% (vol/vol) 2-mercaptoethanol or 2 mM N-ethylmaleimide and subjected to reducing 12%-acrylamide
SDS-PAGE (A) and nonreducing 14%-acrylamide SDS-PAGE (B and C). In the case of the 14% gel, the run time was prolonged until the 31-kDa
band of the Kaleidoscope protein standards (Bio-Rad Laboratories) reached the bottom of the slab. Immunoblotting was performed by using
anti-PomB serum (PomB93), as described previously (44). Panel C is a prolonged exposure of the gel panel B to enhance the signals. The single
square bracket and the double square bracket indicate the positions corresponding to monomer and dimer forms, respectively, of the wild-type,⌬C1, ⌬C2, and ⌬C3 PomB proteins. Asterisks with an arrow and a double arrowhead indicate the positions of monomer and dimer forms,
respectively, of the ⌬C protein and a degradation product of PomB. The bands corresponding to the ⌬C3 protein and its dimer form are indicatedby an arrow and a double arrowhead, respectively. Monomer and dimer forms of degraded ⌬C1, ⌬C2, and ⌬C3 appeared to be similar in size tothose of ⌬C. The ratio of disulfide-bonded PomB dimer to the monomer increased upon coexpression of PomA (⫹PomA).

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FIG. 3. Swarms of the strains expressing the PomB deletion proteins. (A) Pattern of strains spotted. wt, wild type. (B) Aliquots (1 ␮l)of overnight cultures of V. alginolyticus NMB192 (⌬pomB; left) harboring plasmids carrying the pomB derivatives in VC medium werespotted on soft agar VPG500 plates containing 1 mM IPTG and 0.26%
agar, 100 ␮g of kanamycin/ml, and 2.5 ␮g of chloramphenicol/ml andincubated at 30°C for 8 h. Similarly, V. alginolyticus NMB191(⌬pomAB; right) harboring plasmids carrying wild-type pomA and thepomB derivatives were spotted. (C) V. alginolyticus VIO5 (pom⫹) harboring plasmids carrying the pomB derivatives (left) or wild-type pomAand the pomB derivatives (right).

pomB with a His6 tag (39), to make plasmids carrying both
pomA and the pomB derivatives: pNH31, pomA-pomB⌬C;
pNH32, pomA-pomB⌬C1; pNH33, pomA-pomB⌬C2; andpNH34, pomA-pomB⌬C3.
Stability and cross-link formation of PomB derivatives. To
test whether the deletion variants were stable, the amount of
PomB produced in the presence and absence of coexpressed
PomA was compared for each protein. The study used strains
NMB192 (Rifr, Laf⫺, ⌬pomB) (40) and NMB191 (Rifr, Laf⫺,⌬pomAB) (44), which are derivatives of the lateral-flagellardefective strain of V. alginolyticus, VIO5 (Rifr, Laf⫺) (31).
Plasmids encoding the PomB deletion proteins (pNH21,
pNH22, pNH23, and pNH24) (Fig. 1B) were introduced intothe ⌬pomB strain NMB192 containing the compatible plasmid

NOTES

781

pMMB206 (28), which carries the lacI gene, by electroporation
as described previously (21). Plasmids encoding both wild-type
PomA and the PomB derivatives (pNH31, pNH32, pNH33,
and pNH34) were introduced into the ⌬pomAB strain
NMB191 containing pMMB206. The cells were cultured overnight at 30°C on Vibrio complex (VC) medium (0.5% tryptone,
0.5% yeast extract, 0.4% K2HPO4, 3% [513 mM] NaCl, and
0.2% glucose) containing 100 ␮g of kanamycin/ml and 2.5 ␮gof chloramphenicol/ml and then diluted in fresh VPG500 medium (1% tryptone, 0.4% K2HPO4, 500 mM NaCl, and 0.5%
[wt/vol] glycerol) containing antibiotics.
Similar amounts of PomB derivatives were produced in cellswith and without coexpression of PomA at levels above those
encoded by the chromosomal pomA gene (Fig. 2A). All of thederivatives migrated more slowly than expected from theamino acid sequences during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the ⌬C1, ⌬C2,and ⌬C3 PomB preparations, a ca. 30-kDa band with mobility
similar to that of the ⌬C protein was detected. For ⌬C3, the30-kDa band was predominant rather than the full-length ⌬C3protein, which was detected at low levels. These 30-kDa bands(except the ⌬C band) were not detected in the sample boundto the affinity resin through the C-terminal His tag (see Fig. 4),
suggesting that these bands do not have their normal C termini. All of the mutant PomB proteins were isolated in themembrane fractions (data not shown), and the amounts of the
PomA protein found with the different PomB derivatives were
almost the same (data not shown).
A study of deletion mutants of Salmonella MotB suggested
that some of the residues immediately before position 270,
which corresponds to the portion deleted in ⌬C3, are involvedin maintenance of protein structure (29). The ⌬C3 protein was
unstable: a 30-kDa band, which corresponds to a C-terminallydegraded product, was detected with higher intensity than fulllength ⌬C3. The corresponding MotB deletion protein, ⌬(261–270), is nonfunctional and semistable, and the mutation isrecessive. These properties are consistent with those of the⌬C3 protein.
PomB is unstable in the absence of PomA (44). In thepresent study, however, no apparent differences in the amountof PomB were observed with or without coexpression of
PomA. Such an inconsistency is probably due to differences inthe expression systems; i.e., proteins were expressed constitutively in the previous study, whereas those in the present study
were expressed transiently by the addition of inducer.
Wild-type PomB forms cross-linked dimers via naturally occurring N-terminal cytoplasmic Cys residues, suggesting thatthe functional unit of PomB is a dimer (9, 40, 43). Cross-linked
dimers were detected in all of the PomB proteins subjected tononreducing SDS-PAGE (Fig. 2B and C). The dimer bands of⌬C2 and ⌬C3 were faint, whereas significant proportions of theproteins seemed to be present as monomers (Fig. 2B and C,
lanes 8, 10, 18, and 20). The deletions in the C2 and C3 regions
may result in some defects in the homotypic PomB-PomB
interaction.
The dimer forms of all of the PomB derivatives, including
the wild type, increased with respect to the monomer upon
coexpression of PomA. According to one recent model for thearrangement of transmembrane segments in the (MotA2/
MotB)2 or (PomA2/PomB)2 complex, eight transmembrane

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FIG. 4. Association of PomB deletion proteins with PomA. The membrane preparation and cosedimentation procedures to assess theassociation of the PomB derivatives with PomA were performed essentially as described elsewhere (15). Membranes (M fraction) of V. alginolyticus
VIO5 (pom⫹) expressing the PomB derivatives were solubilized with 2.5% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; S fraction}. The solubilized materials were incubated with Ni-charged nitrilotriacetic acid-agarose, followed by extensive washing (Wfraction). The beads were then treated with SDS-loading buffer (B fraction). All four fractions were subjected to SDS-PAGE, followed byimmunoblotting with anti-PomA and anti-PomB. The W⬘ and B⬘ fractions were loaded in fivefold-higher amounts than W and B, respectively. The
bands derived from PomA and PomB are indicated by gray and black arrows, respectively. The asterisk indicates the stable PomB degradation
product (see text). Neither PomB nor PomA was detected in the bound fraction of wild-type (wt) PomB without the His tag (lane 25). All of the
PomB derivatives with His tags were detected in the bound fraction.

segments of MotA or PomA surround the two transmembranesegments of the MotB or PomB dimer (4, 10). It is thus reasonable that coexpression of PomA might affect the geometry
and/or stability of the PomB dimer.
Motility of cells with PomB derivatives. We investigated theswarming abilities of strains expressing the PomB derivatives
with or without coexpression of wild-type PomA from theplasmid (Fig. 3). None of the PomB derivatives supported
swarming in either case (Fig. 3B), nor did the strains swim inliquid medium (data not shown).
None of the PomB-deletion proteins generated in this study
complemented the motility defect of the ⌬pomB strain, although all were detected and localized to the membrane. The
deleted regions of PomB have high similarity to peptidoglycanassociated proteins and PomB homologs (1, 29) (Fig. 1). Presumably, deletions in the C terminus of PomB impair association with peptidoglycan and prevent proper anchoring and/or
localization of the stator elements around the rotor.
Based on in silico docking of the crystal structure of the
OmpA-like domain of the putative peptidoglycan binding protein RmpM from Neisseria meningitidis, Tyr-127, Arg-135, andArg-197 may be involved in peptidoglycan binding (19) (Fig.
1A). According to a multiple sequence alignment (Fig. 1),
Tyr-127 is not conserved in MotB homologs, and there aregaps in the sequence in its vicinity. On the other hand, Arg-135
and Arg-197 are conserved and correspond to Arg-251 (C1region) and Arg-294 (C3 region) of PomB, respectively. In
MotB of E. coli, amino acid replacements R217W, R258C, andR258H at the corresponding residues result in loss of function(7). Therefore, the C1 and C3 regions may directly participate
in binding to peptidoglycan.
To test whether the deletion proteins inhibit motility in thewild-type strain, the swarming and swimming of VIO5 (pom⫹)strains expressing the PomB derivatives with (pNH31, pNH32,
pNH33 and pNH34) or without (pNH21, pNH22, pNH23 andpNH24) plasmid-encoded PomA were assessed in the presenceof 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) (Fig.
3C). The ⌬C1, ⌬C3, and ⌬C proteins had almost no effect on
the motility of VIO5. When the ⌬C2 protein was overproducedin VIO5, both the swarming ability (Fig. 3C) and the swimming(data not shown) of VIO5 were significantly inhibited, and theinhibition was correlated with the concentration of IPTG (datanot shown). However, upon coexpression of plasmid-encoded
PomA and the ⌬C2 protein (pNH33), the swarming and swimming were only slightly inhibited, indicating that the inhibitory
effect of the ⌬C2 protein is compensated by overexpression of
PomA.
Association of PomB derivatives with PomA. We examined
whether the PomB deletion proteins associate with PomA inwild-type cells. We detected the chromosomally encoded
PomA bound to all of the histidine-tagged PomB derivatives
after detergent solubilization (Fig. 4, lanes 4, 8, 12, 16, and 21).
The amount of ⌬C3 protein was significantly lower, presumably because of degradation of the C-terminal region to which
the His tag was attached (Fig. 4, asterisk). PomA was detectedin the bound fraction of wild-type PomB and all of the PomB
derivatives, indicating that PomA associates with the PomB
derivatives. Similar levels of PomA were detected with ⌬C,⌬C2, and wild-type PomB, suggesting that chromosomally encoded PomA associates with ⌬C and ⌬C2 as well as withwild-type PomB. The amounts of PomA bound to ⌬C1 and⌬C3 were significantly lower than those bound to the wild type,⌬C, and ⌬C2. For ⌬C3 in particular, a faint band of PomA wasdetected only when a fivefold amount of bound material was
loaded onto the gel (lane 21). Essentially the same results wereobtained by using the ⌬pomAB strains expressing plasmidencoded PomA and PomB (data not shown).
The ⌬C protein formed a cross-linked homodimer, dimer
formation increased upon coexpression with PomA, and PomA
associated with ⌬C and wild-type PomB equally well. These
results suggest that the putative peptidoglycan-binding region
is not necessary for oligomerization of PomA and PomB. Thisconclusion is reasonable because the TM segments of PomA
and PomB are close to each other (40), and a similar association was proposed for the TM segments of MotA and MotB in
the proton-driven motor (10). Some proteins associated withpeptidoglycan have been described as dimers (11, 19). Although the peptidoglycan-binding region of N. meningitidis

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NOTES

RmpM is proposed to be responsible for dimerization (19), it
does not appear to be solely responsible for dimerization of
PomB.
While complete deletion of the C-terminal region preserves
the association with PomA or with itself, certain smaller deletions weaken it, indicating a possible linkage between the conformation of the C-terminal region and the TM region. The
amount of PomA associated with the ⌬C1 and ⌬C3 proteins
seems low compared to the wild type, ⌬C, and ⌬C2, suggestingthat the ⌬C1 and ⌬C3 proteins have some defects in the association with PomA (Fig. 4). On the other hand, the amountsof PomB homodimer were specifically reduced for ⌬C2 and⌬C3, although similar levels of homodimer were detected in
the other PomB derivatives (Fig. 2). It is noteworthy that theamount of PomA that coprecipitated with ⌬C and disulfide
bond formation of ⌬C were comparable to those of the wildtype PomB. Garza et al. (16, 17) reported that motB missense
mutations P159I and G164D in or immediately preceding the
C region can be suppressed by mutations in motA or in fliG,
which suggests that mutations in the C region can influence thearrangements of the TM region where MotA is presumably
mounted. The slow-swimming phenotype of G240D substitution (C2 region) in MotB, which was suppressed by mutations
in motA but not in fliG (16, 17), may have resulted in misassociation with MotA. Based on various mutations that affect
the C region (7, 38), the structure of this region may in part
determine the conformation around the TM region of PomB.
The linker region between the C and TM regions (29) may
have a rigid rather than a flexible structure to transmit thestructural change. This assumption is consistent with results
showing that the degradation products of ⌬C1, ⌬C2, and ⌬C3
are similar in size to the intact ⌬C protein, suggesting that thelinker region assumes a conformation that is resistant to proteolytic degradation (Fig. 2 and 4).
We thank Ikuro Kawagishi and Masaru Kojima for stimulating discussions and encouragement. We are grateful to Terry A. Krulwich
and David F. Blair for carefully reading the manuscript.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan;
the Japan Science and Technology Corporation (to M.H. and T.Y.);and from the Soft Nano-Machine Project of the Japan Science andTechnology Agency (to T.Y. and M.H.).
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