JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 1001–1007
0021-9193/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 182, No. 4

Cysteine-Scanning Mutagenesis of the Periplasmic Loop
Regions of PomA, a Putative Channel Component of the
Sodium-Driven Flagellar Motor in Vibrio alginolyticus
YUKAKO ASAI, TOMOKAZU SHOJI, IKURO KAWAGISHI, AND MICHIO HOMMA*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan
Received 13 September 1999/Accepted 16 November 1999

The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in
Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of protondriven motors, have four transmembrane segments and one transmembrane segment, respectively, and arethought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between
membrane segments 1 and 2 (loop1-2) and at positions 167 to 180 between membrane segments 3 and 4(loop3-4). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for thechannel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembranesegment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also
replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutationsin the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the fivesuccessive substitutions from M169C to K173C of loop3-4 impaired motility. In some mutants that retained
substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and Nethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology
predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we
predicted more directly the membrane topology of loop3-4. None of the loop1-2 residues were labeled, suggestingthat the environments around the two loops are very different. A few of the mutations were characterized
further. The structure and function of the loop regions are discussed.
(2). On the basis of homologies to MotA and MotB of protondriven motors, it is believed that the four transmembrane regions of PomA and the one transmembrane region of PomB
form a complex and function as a sodium channel (2, 40). An
Asp residue in the transmembrane region of Escherichia coli
MotB has been proposed to act as the donor in H⫹ transfer toa recipient near the cytoplasmic side of the protein (38). ThisAsp residue is conserved in PomB and may be involved in Na⫹
transfer. The C-terminal domains of MotY, PomB, and MotB
have sequence similarities to peptidoglycan-interacting proteins (2, 12). MotX is inferred to be a component of the Na⫹channel of the motor because when MotX of Vibrio parahaemolyticus, which is closely related to V. alginolyticus, is overexpressed in E. coli, growth is retarded in proportion to theexternal Na⫹ concentration. This effect can be suppressed by
the addition of amiloride (29). PomA has the greatest similarity to the H⫹-driven motor component MotA of the photosynthetic bacterium Rhodobacter sphaeroides. We have shown thatthe proton motor component, MotA, of R. sphaeroides cangenerate torque by coupling with the sodium ion flux in placeof PomA of V. alginolyticus (3). This suggests that PomA does
not determine ion selectivity in the motor components.
The rotation of the sodium-driven polar flagella is very fast,over 1,000 revolutions per second in V. alginolyticus (28), andthe speed is very stable (30). When rotation is slowed byphenamil, an amiloride analog, fluctuations in speed becomelarger (31). The fluctuations induced by phenamil are explained by changes in the functional force-generating unit
number in a motor and are attributed to a low dissociation rateof the inhibitor from the force-generating unit. Motility mutants resistant to phenamil, which is thought to interact withthe sodium channel of the flagellar motor, were isolated, andthe phenotype was named Mpar for motility resistant to phena-

The bacterial flagellum is driven by a rotary motor poweredby the electrochemical gradient of a specific ion (either proton
or sodium ion) across the cytoplasmic membrane. Rotational
force is generated by an interaction between elements of therotor and force-generating units in the cytoplasmic membrane(7, 21). Multiple force-generating units are inferred to function
independently because rotational speed changes in a series of
equal increments after the component proteins of the forcegenerating units are induced from regulatable plasmids. Sixteen or eight steps were observed in fully functional H⫹-driven
motors (8, 9). Five to nine steps were observed in Na⫹-driven
motors when the progress of motors was slowed by a photoactivated inhibitor (32).
Sodium-driven flagella have certain advantages for the studyof motor energetics. The sodium motive force can be manipulated more readily than the proton motive force (18, 19) sothat the rotation speed can be controlled by the sodium concentration in the medium (30). Amiloride and its analogs,
which are inhibitors of the epithelial Na⫹ channel (6), inhibit
the rotation of the sodium-driven bacterial flagellar motor in a
fairly specific manner (4, 39). Furthermore, the rotation speedof the sodium-driven flagella of Vibrio alginolyticus can bemeasured easily because each cell has a single flagellum at onepole and the diameter of the sheathed flagellar filament is ca.
30 nm and is greater than that of unsheathed flagella in otherspecies (1, 13).
The sodium-driven motor in V. alginolyticus consists of the
products of at least four genes, pomA, pomB, motX, and motY
* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan. Phone: 81-52-789-2991. Fax: 81-52-789-3001.
E-mail: g44416a@nucc.cc.nagoya-u.ac.jp.
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TABLE 1. Bacterial strains and plasmids
Strain orplasmidV. alginolyticus
VIO5
VIO586
NMB188E. coli DH5␣

Plasmids
pSU41
pSU21
pYA2032
pYA301

Genotype or descriptiona

Reference

VIK4 laf (Pof⫹ Laf⫺)
VIO5 pomA (Laf⫺ Pom⫺)
VIO586 che (Laf⫺ Pom⫺ Che⫺)

34
2
25

F⫺ ␭⫺ recA1 hsdR17 endA1 supE44
thi-1 relA1 gyrA96 ⌬(argFlacZYA)U169 ␾80dlacZ⌬M15

16

kan (Kmr) Plac lacZ␣
cat (Cmr) Plac lacZ␣ (MCS same asthat in pSU41)
pSU21 1.45-kb XbaI-EcoRI
fragment (pomA⫹)
pSU41 0.8-kb BamHI-BamHI
fragment (pomA⫹)

5
5
2
25

a
Cmr, chloramphenicol resistant; Kmr, kanamycin resistant; MCS, multicloning site; Plac, lac promoter.

mil (26). The Mpar mutations have recently been mapped to
PomA or PomB at the residue near the cytoplasmic ends of theputative transmembrane segments (25). The phenamil resistance mutations have been mapped similarly in V. parahaemolyticus (20).
PomA and MotA are predicted to have two short loops inthe periplasm (2, 41). The loop regions may help to maintainthe arrangement of the membrane-spanning helices that form
the ion channel, guide the Na⫹ ion or proton, respectively, into
the channel, or interact with the coupling ions, Na⫹ and H⫹, orwith other proteins, such as PomB or MotB, respectively. In
this study, we investigated the function of these loop regions byperforming systematic cysteine-scanning mutagenesis andcharacterizing the motility of the Cys mutants and the binding
effects of sulfhydryl (SH) reagents on the mutant’s motility.
Moreover, a combination of genetic introduction of Cys andchemical labeling allowed us to obtain topographical information on periplasmic loops of PomA.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The strains and plasmids
used in this work are listed in Table 1. V. alginolyticus cells were cultured at 30°C
in VC medium (0.5% polypeptone, 0.5% yeast extract, 0.4% K2HPO4, 3% NaCl,
0.2% glucose) or VPG medium (1% polypeptone, 0.4% K2HPO4, 3% NaCl,
0.5% glycerol). For compact colony formation, VC medium–1.25% agar plates
were used. For swarm assays, VPG medium–0.25% agar plates were used. Whennecessary, chloramphenicol and kanamycin were added to final concentrations of
2.5 and 100 ␮g/ml, respectively. E. coli cells were cultured at 37°C in LB medium(1% tryptone, 0.5% yeast extract, 0.5% NaCl), and chloramphenicol and kanamycin were added to final concentrations of 25 and 50 ␮g/ml, respectively.
DNA manipulations and sequencing. Routine DNA manipulations were carried out in accordance with standard procedures (36). Restriction endonucleases
and other enzymes for DNA manipulations were purchased from Takara Shuzo(Shiga, Japan) and New England Biolabs (Beverly, Mass.). Nucleotide sequenceswere determined by using the dye terminator cycle sequencing kit (Perkin-ElmerCo.) and an ABI PRISM sequencer (model 377; PE Applied Biosystems).
Cysteine-scanning mutagenesis. Mutagenesis was performed by a two-step
PCR as described previously (25). For the reaction, we used end primers (inwhich restriction enzyme sites [underlined] were added) for the pomA gene,
PomA-B1 (5⬘-GCGGGATCCTGCCGCTCCGGACCTGGATGA-3⬘) andPomA-B2 (5⬘-CTCGGATCCAAGTTACTCGTCAATCTCA-3⬘) or PomA-E2(5⬘-CTCGAATTCAAGTTACTCGTCAATCTCA-3⬘), a pair of the mutant
primers, and pYA2032 as the template. Amplified mutant fragments were digested with BamHI or with BamHI and EcoRI and ligated into the multicloningsite of the plasmid vector pSU41 (5).

Electroporation. Transformation of V. alginolyticus by electroporation was
carried out as described previously (22). The cells were subjected to osmotic
shock and were washed thoroughly with 20 mM MgSO4. Electroporation was
carried out with the Gene Pulser electroporation apparatus (Japan Bio-Rad
Laboratories, Tokyo) at an electric field strength of 5.0 to 7.5 kV/cm.
Measurement of swimming speed. An overnight culture in VC medium was
inoculated into VPG medium at a 100-fold dilution and grown at 30°C toexponential phase. Cells were centrifuged in an Eppendorf tube at 7,000 rpm for
5 min, and the sedimented cells were suspended in Tris motility buffer (TMN50;
50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM glucose, 50 mM NaCl, 250 mM
KCl). Cell suspension was diluted about 100-fold into Tris motility buffer, andmotility of the cells was observed under a dark-field microscope and recorded onvideotape. Swimming speed was determined as described previously (17). When
the Na⫹ concentration was varied, the KCl concentration was changed to holdthe total salt concentration constant. For example, TMN300 contains 300 mM
NaCl and no KCl in Tris motility buffer, TMN50 contains 50 mM NaCl and 250
mM KCl, and TMK300 contains no NaCl and 300 mM KCl.
Detection of PomA proteins. Cell suspensions were mixed with one-fifth volume of sodium dodecyl sulfate (SDS) loading buffer (0.2 M Tris-HCl [pH 6.8],
37.5% glycerol, 6% SDS, 0.004% BPB) and 1/10 volume of 2-mercaptoethanoland boiled for 5 min (40). Proteins in the samples were separated by SDSpolyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred
to a polyvinylidenedifluoride (PVDF) membrane (Millipore Japan, Tokyo) byusing a wet blotting apparatus (Biocraft, Tokyo, Japan). The anti-PomA antibody(PomA91), which was generated as described previously (40), was the primary
antibody, and alkaline phosphatase (AP)-conjugated goat anti-rabbit immunoglobulin G antibody was the secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). The method for detection of the second antibody wasdescribed previously (33).
Labeling with and detection of biotin maleimide. Cell suspensions were prepared as for the above procedure for measurements of swimming speed. For
labeling, the cells suspended in TMN50 were preincubated with 10 mM dithiothreitol (DTT) at room temperature for 30 min and harvested by centrifugation.
The pellet was resuspended in TMN50 and incubated with 0.2 mM biotin maleimide (biotin-PE-maleimide; Dojin Corp., Kumamoto, Japan) for 10 min. The
cells, washed once with TMN50, were solubilized and immunoprecipitated withthe anti-PomA (PomA1312) antibody, as described previously (40). The immunoprecipitates were separated by SDS-PAGE and transferred to a PVDF membrane as described above. Biotinylated proteins were detected with streptavidinconjugated horseradish peroxidase and chemiluminescence (Amersham Corp.).

RESULTS
Cysteine-scanning mutagenesis of the PomA extracellularloop regions. Mutations were introduced into the loop1-2 andloop3-4 segments of PomA, defined as the regions from V21 to
L36 and from S167 to A180, respectively, by a PCR method(Fig. 1). The mutations were confirmed by sequencing one or
the other DNA strand of the pomA region. Sixteen mutations
were made in loop1-2, and 14 mutations were made in loop3-4.
In some cases, a silent mutation or a mutation in the noncoding
region was also present; however, those mutations did notaffect the motility phenotype. In addition to the periplasmic
loop regions, Cys substitutions were generated in the fourthtransmembrane segment (T186C) and in the predicted cytoplasmic regions (D71C, D148C, and D202C). The four mutants are thought to be the references against which theperiplasmic mutants can be compared. Furthermore, the cytoplasmic charged residues might have an important role for thechannel function.
The mutant plasmids were introduced into the pomA mutant
strain (VIO586), and the motility of the transformed cells was
examined by measuring swarm expansion rates in soft agar
plates with or without dithionitrobenzoic acid (DTNB) (Fig. 2;
Table 2). It is known that DTNB, an SH-modifying reagent, isunable to permeate membranes and causes no significant damage to cellular functions. None of the substitutions in loop1-2
abolished swarming ability, although G23C, S25C, and D31C
seemed to impair swarming. DTNB reduced the swarm rate ofmutants containing the G23C, G24C, F29C, D31C, and T34C
substitutions. The swarming ability of the F29C mutant wasalmost completely inhibited by DTNB. On the other hand,several Cys replacements in loop3-4 severely impaired swarming. The M179C mutant did not form any swarm, and theVOL. 182, 2000

MUTAGENESIS OF PomA LOOP REGIONS

1003

FIG. 1. Model of the membrane topology of PomA. Positions replaced by Cys are shown as large circles. The reactivities with biotin maleimide are indicated by
the following symbols: solid black circles, strong reactivity; striped circles, quite weak reactivity; open large circles, no reactivity. Relative swarm sizes in the mutants
are indicated by shaded circles. Where no shaded circle is given, swarm size was similar to that of the wild type and was not affected by DTNB. Inhibition or lack of
inhibition by DTNB is indicated by ⫹ or ⫺ symbols, respectively, in the circles.

P172C mutant produced significant swarms only after prolonged incubation. In the presence of DTNB, swarm expansion
rates were reduced in the mutants M169C, D170C, K173C,I175C, G176C, and G180C. The I175C mutant seemed to be
more affected by the SH-modifying reagent than the others.
The T186C substitution, which alters the putative fourthtransmembrane segment, eliminated swarming. Residue T186
of PomA corresponds to T209 of E. coli MotA, which is aconserved polar residue that might form part of the proton
channel (37). The mutations D71C, D148C, and D202C, which
are located in regions predicted to be the cytoplasm, did notaffect swarming even in the presence of DTNB.
Effects of the Cys substitutions on the swimming speed ofcells. NMB188 (PomA⫺ Che⫺) cells, which have a smoothswimming mutation so swimming speed can be measured moreeasily (25), were transformed with the mutant plasmids. The
transformed cells harvested in mid-log phase were measured inbuffer containing 300 mM Na⫹ with no SH-modifying reagent,
500 ␮M DTNB, or 200 ␮M N-ethylmaleimide (NEM). In contrast to DTNB, NEM is able to permeate membranes and thereaction is irreversible. The effects of the mutations on swimming and swarming were correlated well with each other, witha few exceptions (Fig. 2; Table 2). Such exceptions are notsurprising because swarming ability is affected by growth andchemotactic behavior. Swarming was severely impaired by the
F29C substitution in the presence of DTNB, but the swimming
speed was not. Correspondingly, we found that Cys-29 is modified slowly by the SH-modifying reagents (Fig. 3). In most of
the mutants, swimming speed was dropped immediately after
the addition of the SH-modifying reagents, but this was nottrue of F29C cells, which slowed over the course of about 10
min after the addition of the SH-modifying reagents. Motility
was restored to its original level in this mutant by the additionof a reducing agent, DTT. In contrast, cells of the I175C
mutant were stopped immediately and completely by 500 ␮M
DTNB or 200 ␮M NEM, and their swimming speeds weredecreased with the concentration of the reagents (Fig. 4). The
slow reaction of Cys-29 is consistent with the following result:the loop1-2 region was not labeled by biotin maleimide, whosemolecular weight is larger than that of DTNB or NEM.
Detection of the mutant PomA proteins. The levels of PomA
proteins were determined for pomA cells (NMB188) transformed with plasmids containing the mutant pomA genes that
conferred impaired motility. Total cell proteins were separated
by SDS-PAGE, and immunoblotting was performed by using
an antibody raised against PomA peptides. We could detect alevel of Cys-substituted PomA proteins comparable to that ofwild-type PomA (data not shown). Of all the mutants tested,
there was no significant difference in the amount of PomA in
any mutant although the electrophoretic mobilities wereslightly different for some mutant proteins (Fig. 5). These
minor changes in mobility and amount were not correlatedwith the severity of the effects of the mutations on motility.
Labeling with biotin maleimide. Generally, maleimide isthought not to bind to thiol groups buried in the hydrophobic
interior of the membrane. To determine which residue is located at each boundary between a periplasmic loop and atransmembrane region, biotin maleimide was reacted against
the Cys-substituted PomA proteins. The results of labeling andimmunoblotting are shown in Fig. 5. Wild-type PomA

1004

ASAI ET AL.

J. BACTERIOL.
TABLE 2. Swarm size and swimming speed of mutants
MutantFIG. 2. Swarm profiles of the Cys mutants. Fresh colonies were inoculated intriplicate into 0.25% agar–VPG plates containing kanamycin without (a) andwith (b) 500 ␮M DTNB and incubated at 30°C for 4 h. The mutations areindicated. The unlabeled nonswarming colonies contain only the vector plasmid(pSU41). pYA301 is a plasmid expressing the wild-type PomA.

(pYA301) was not biotinylated because it has no Cys. T186C,
which is predicted to be in the interior of TM4, was also notlabeled. In loop3-4, the residues from N168C to A178C werelabeled, suggesting that these are exposed to the aqueous
phase. P177C was reproducibly more weakly labeled than theothers. On the other hand, biotin maleimide bound to S167C
quite weakly and did not bind to M179C and A180C at all. It
is possible that S167 and M179 exist at the boundaries between
TM3 and periplasmic loop3-4 and between loop3-4 and TM4,
respectively. We could not detect biotinylated PomA in any
loop1-2 substitutions (Fig. 5a). The residues of loop1-2 may not
be exposed to the periplasm. Three Cys substitutions in thecytoplasmic region were also biotinylated to the same extent
with periplasmic mutants. This result shows that biotin maleimide can penetrate the membrane of V. alginolyticus.
Effect of SH-modifying reagents at different sodium ion concentrations. The extracellular loop regions might interact withNa⫹ as it enters the PomA channel. Therefore, the effect ofNa⫹ on the reaction rates of SH-modifying reagents was investigated with the M169C and D170C mutants (Fig. 6). The
time course of inhibition of the D170C mutant by DTNB
differed depending on whether the Na⫹ concentration was low
or high (Fig. 6c). The M169C mutant did not show any such
effect. The inhibition of the D170C and M169C mutantsreached a maximum at 300 mM NaCl in 5 and 15 min, respectively. Inhibition was relieved by the addition of 1 mM DTT,

None
V21C
L22C
G23C
G24C
S25C
I26C
G27C
M28C
F29C
V30C
D31C
V32C
T33C
S34C
I35C
L36C
S167C
N168C
M169C
D170C
D171C
P172C
K173C
A174C
I175C
G176C
P177C
A178C
M179C
A180C
T186C
D71C
D148C
D202C

Swarm sizea

Swimming speed (␮m/s)b

No addition⫹DTNB

No addition

⫹DTNB

⫹NEM

⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫾
⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹

⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫾
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫾
⫹
⫹
⫾
⫾
⫹⫹⫹
⫾
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹

82
76
67
45
68
43
77
73
75
62
82
32
81
64
58
73
73
77
79
48
52
20
NDc
25
76
22
40
34
50
0
71
0
82
64
77

82
76
69
31
49
39
74
72
73
50
79
17
80
57
57
76
74
69
78
16
26
17
ND
24
72
0
25
30
23
⫺
72
⫺
84
61
75

77
77
67
25
37
33
74
72
74
48
80
16
78
48
43
75
69
60
75
5
37
12
ND
28
59
0
17
34
13
⫺
62
⫺
73
42
68

a
Swarm size was classified into five types (⫹⫹⫹, ⫹⫹, ⫹, ⫾, ⫺) on the basisof the results shown in Fig. 2. The mutants whose swarm ability was detected byprolonged incubation are indicated by ⫾.
b
The average swimming speeds were obtained by measuring at least 20 swimming tracks of cells generated from the integrated video images.
cND, not determined. In the P172C mutant, the motile fraction was too small
for the swimming speed to be measured.

and swimming speed was restored to the original level. The
Na⫹ flow may affect the reactivity of Cys-170 or may change
the environment around the Cys residue.
DISCUSSION
Cysteine or alanine scanning is known as a useful method forthe functional analysis of various ion channels and transporters(14, 23, 27, 35). Applying this method to investigate the flagellar motor was expected to give us information not only about
functional residues but also about the structural changes
around each residue from the binding effects of SH-modifying
reagents. Thirty-four different site-directed pomA mutations
were made in this study. Systematic substitutions by cysteine
were made in the two periplasmic loop regions (loop1-2 andloop3-4), in the fourth transmembrane segment (TM4), and inparts of Asp residues in the cytoplasmic region.
Of the 34 mutants, only two were nonmotile, M179C and
T186C. Nine mutants showed subnormal motility. These mutant PomA proteins were detected in levels comparable to thatof the wild-type protein. By random mutagenesis, only theVOL. 182, 2000

MUTAGENESIS OF PomA LOOP REGIONS

1005

FIG. 3. Time course of reaction of the F29C mutant with the SH-modifying
reagent DTNB. Cells from a 1-ml culture of strain NMB188 containing a plasmid
expressing the F29C protein were harvested in late logarithmic phase and suspended in 200 ␮l of TMN50. This suspension was diluted 10-fold by TMN50 with(■) or without (䊐) 500 ␮M DTNB. After various times, the diluted suspension
was diluted 50-fold more into TMN300 buffer, and the swimming speed wasmeasured within 1 min after the dilution. At 25 min, DTT was added to a 1 mM
final concentration, and the swimming speed was measured after 5 min.

mutation of G176 to the charged residue of Arg or Glu has
been isolated in the periplasmic loop regions (24). T186 ispredicted to be in a transmembrane segment (TM4), and thisThr residue is conserved among MotA proteins of various
species (2). In E. coli MotA, the T209W substitution abolishedmotility. This result was interpreted to mean that this residue
faces the inside of the ion channel (37). M179 is predicted to beat the periplasmic end of TM4. This methionine residue is notconserved in any MotA protein but appears always to be a
nonpolar residue, for example, Ile in E. coli. Many substitutions in loop3-4 had significant effects on motility. The fivesuccessive Cys substitutions from M169C to K173C impaired
motility. This effect may be related to the observation that the

FIG. 4. Effect of varying the concentration of the SH-modifying reagents.
NMB188 cells containing a plasmid expressing the I175C protein were harvested
as described in the legend to Fig. 3. The suspension was diluted 10-fold into
TMN50 containing the indicated concentrations of the SH-modifying reagent
DTNB (䊐) or NEM (F). At various times, the cell suspension was diluted 50-foldmore with TMN300, and the swimming speed was measured within 1 min after
the dilution.

FIG. 5. Labeling PomA with biotin maleimide. Immunoprecipitated PomA
proteins were separated by SDS-PAGE, transferred to a PVDF membrane, anddetected by chemiluminescence of horseradish peroxidase-conjugated streptavidin (a). PomA was detected with anti-PomA antibody and AP-conjugated secondary antibody (b). pSU41 is the vector plasmid (none). The pYA301 plasmidcarries the wild-type (no Cys) pomA gene.

transmembrane segments TM3 and TM4 are more conservedin MotA proteins of various species (2).
During the course of this study, it was found that the D148Y
mutation conferred a resistant phenotype to phenamil, which isa sodium channel inhibitor, for the motor rotation. D148 issuggested to be one of high-affinity phenamil binding sites (25).
This Asp was changed to various amino acid residues, and the
D148C mutant was phenamil resistant (25).
From a set of E. coli motB missense mutations, extragenic
suppressors were isolated and some of these suppressors altered residues on the periplasmic surface of MotA (15). In thatreport, the authors proposed a model in which mutations affecting residues in or near the putative peptidoglycan binding
region of MotB misalign the stator relative to the rotor. The
suppressors in the MotA periplasmic loop regions may cause acompensating realignment to restore motor function. This proposal suggests that the periplasmic loop regions in MotA or
PomA may be important for the overall structure of the motor
complex.
Among the various thiol group-specific binding reagents, wechose DTNB and NEM. It is known that DTNB is unable topermeate membranes and the reaction is reversible by a reducing agent and, on the other hand, that NEM is able topermeate membranes and the reaction is irreversible. Bothreagents had comparable effects on the motility of every mutant. The motility of the I175C mutant was impaired and further decreased by the SH-modifying reagents. Only mutant
I175C, of those made in this study, lost motility completely
when it was treated with the SH-modifying reagents. This residue seems to be exposed to the reagents, and we speculatethat the ion channel is plugged by the modification. The Cys-

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ASAI ET AL.

J. BACTERIOL.

FIG. 6. Effect of Na⫹ on the reactions of the M169C and D170C mutant proteins with SH-modifying reagents. Cells from a 1-ml culture of the wild-type strain (a)and the M169C (b) and D170C (c) mutants were harvested in late logarithmic phase and suspended in 200 ␮l of TMN50. This suspension (10 ␮l) was diluted 10-foldby TMN300 with (F) or without (E) 10 ␮M DTNB and by sodium-free buffer (TMK300) with (■) or without (䊐) 10 ␮M DTNB. After various times, the diluted
suspension was diluted 50-fold more into TMN300, and the swimming speed was measured within 1 min after the dilution. At 15 min, DTT was added to 1 mM.

175 protein might prove useful in probing channel function bya modification using reagents of different sizes.
We tested whether we could observe the difference in accessibility of the SH-modifying reagents with or without sodium
ions by using the M169C and D170C mutants, whose substituted residues are predicted to exist near the boundary ofTM3. The reaction of DTNB with Cys-170 was slower when theNa⫹ concentration was high (Fig. 6c). In contrast, Na⫹ did notprotect Cys-169 from modification. This result may suggestthat Asp-170 faces towards the channel pore and interacts withNa⫹ or that Na⫹ changes the structure of PomA around Asp170. When this reactivity can be assessed directly by chemical
modification under various conditions, structural changes
around Asp-170 might be clarified.
Generally, maleimide is known not to bind to a thiol in thehydrophobic interior of the membrane, and we can use many
derivatives, i.e., those labeled with isotope, those biotinylated,or those labeled with fluorescein, e.g., to directly detect thebinding. Using biotin maleimide, we tried to determine themembrane topology of PomA, and the boundaries of the transmembrane regions and periplasmic loop3-4 are inferred to bearound S167C for TM3 and A178C for TM4 (Fig. 5). Those
results are consistent with the membrane topology predictedfrom the hydrophobicity profiles (2, 11). It is noteworthy that,in spite the fact that swarming was inhibited by DTNB, theA180C protein was not biotinylated (Fig. 5). Swimming speedof the A180C mutant was not inhibited by DTNB or NEM, andthis is consistent with the fact that A180C PomA protein was
not biotinylated. We speculate that DTNB might require a very
long time or need only a certain condition to bind to Cys-180,
and slight motility inhibition was observed only in the swarming plate. This may suggest that the Ala-180 residue exists inthe membrane and faces toward a hydrophilic channel pore.
This prospect is supported by the fact that Thr-186, whichcorresponds to the conserved polar residue for the channel in
MotA (37), is located on the same phase as Ala-180 in thehelical wheel of TM4. None of the Cys substitutions in loop1-2
was labeled with biotin maleimide. On the other hand, themotility of the F29C and D31C mutants was affected by SHmodifying reagents. We showed that the F29C substitution
mutant reacted slowly with the SH-modifying reagent DTNB
(Fig. 3). It has been shown that fluorescein maleimide canreact with S24C in loop1-2 of E. coli MotA in spheroplasts (41).
Although we tried to label F29C with a prolonged incubation
or with spheroplasts, we could not detect biotinylated PomA
(data not shown). This loop1-2 may be covered with otherproteins, such as the motor proteins PomB, MotX, or MotY.
Alternatively, this loop may be embedded in the pore region of
the channel, as predicted in the loops of many ion channels(10).

VOL. 182, 2000

MUTAGENESIS OF PomA LOOP REGIONSThe substituted residues of periplasmic loop1-2 were notaccessible to the SH-modifying reagents, and few Cys substitutions caused motility impairment. However, in loop3-4, manyCys substitutions impaired motility and loop3-4 seems to be
exposed to the periplasm. The two periplasmic loops may havequite different roles in the channel complex of the motor proteins.
ACKNOWLEDGMENTS
We thank Akihito Yamaguchi for invaluable discussions. We especially thank David F. Blair for critically reading the manuscript.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan(to I.K. and M.H.) and the Japan Society for the Promotion of Science(to Y.A.).
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