Microbiology (1999), 145, 1759–1767

Printed in Great Britain

Random mutagenesis of the pomA geneencoding a putative channel component of
the NaM-driven polar flagellar motorof Vibrio alginolyticus
Seiji Kojima, Mitsuyo Kuroda, Ikuro Kawagishi and Michio Homma
Author for correspondence : Michio Homma. Tel : j81 52 789 2991. Fax : j81 52 789 3001.
e-mail : g44416a!nucc.cc.nagoya-u.ac.jp

Division of Biological
Science, Graduate Schoolof Science, Nagoya
University, Chikusa-ku,
Nagoya 464-8602, Japan

PomA and PomB are integral membrane proteins and are essential for therotation of the NaM-driven polar flagellar motor of Vibrio alginolyticus. On thebasis of their similarity to MotA and MotB, which are the proton-conducting
components of the HM-driven motor, they are thought to form the NaM-channel
complex and to be essential for mechanochemical coupling in the motor. To
investigate PomA function, random mutagenesis of the pomA gene by usinghydroxylamine was carried out. We isolated 37 non-motile mutants (26independent mutations) and most of the mutations were dominant ; thesemutant alleles are able to inhibit the motility of wild-type cells when greatly
overexpressed. The mutant PomA proteins could be detected byimmunoblotting, except for those with deletions or truncations. Many of thedominant mutations were mapped to the putative third or fourthtransmembrane segments, which are the most conserved regions. Somemutations that showed strong dominance were in highly conserved residues.
T186I is the mutation of a polar residue located in a transmembrane segmentthat might be involved in ion translocation. P199L occurred in a residue that isthought to mediate conformational changes essential for torque generation in
MotA. These results suggest that PomA and MotA have very similar structuresand roles, and the basic mechanism for torque generation will be similar in the
proton and sodium motors.

Keywords : flagella, sodium ion, Vibrio, motor, pomA

INTRODUCTION

Bacterial flagella are the organelles responsible formotility. At the base of each filament, a rotary motor isembedded in the cytoplasmic membrane, and bacteria
can swim by rotating their helical flagellar filaments.
The motor is powered by the electrochemical gradient ofa specific ion, H+ or Na+, across the cytoplasmic
membrane (Blair, 1995 ; Imae & Atsumi, 1989). The
study of the flagellar motor has been intensively done in
the H+ motors of Escherichia coli and Salmonella
typhimurium. These studies have focused mainly on fivecore proteins involved in the motor function. In therotor part of the motor, three soluble proteins, FliG,
FliM and FliN, are the components for force generation,.................................................................................................................................................

Abbreviation : TM, transmembrane.
0002-3177 # 1999 SGM

flagellar assembly and controlling the direction of motor
rotation (Yamaguchi et al., 1986 ; Sockett et al., 1992 ;Irikura et al., 1993). Together they form a complex,
called the ‘ switch complex ’ or ‘ C-ring ’ (Francis et al.,1994). Among them, FliG has a direct role in force
generation (Lloyd et al., 1996). The stator part of the
motor, two integral membrane proteins, MotA and
MotB, are the components essential for force generation(Dean et al., 1984 ; Stader et al., 1986 ; Block & Berg,
1984 ; Blair & Berg, 1988). They have four and one
transmembrane segments, respectively (Zhou et al.,1995 ; Chun & Parkinson, 1988), and form a H+conducting channel complex responsible for coupling
ion translocation to force generation (Blair & Berg,1990 ; Stolz & Berg, 1991 ; Sharp et al., 1995a ; Garza etal., 1995, 1996). The MotA\MotB channel complex isbelieved to be anchored to the cell wall by the
peptidoglycan-binding domain of MotB (Chun & Park1759

S. K O J I M A a n d O T H E R S

inson, 1988 ; Blair et al., 1991 ; De Mot & Vanderleyden,
1994).
Intensive mutational analysis of MotA, MotB and FliG
of E. coli revealed some critical residues involved intorque generation. It was shown that most of thedominant mutations occurred in the transmembrane
domains of MotA and MotB (Blair & Berg, 1991 ; Blairet al., 1991). In particular, Asp32 of MotB, a conserved
acidic residue in the membrane segment, was suggested
to be the residue conveying protons (Zhou et al., 1998a).
Other recent mutational analyses indicated that threecharged residues of FliG predicted to be on one surface,
Arg281, Asp288, Asp289, and two cytoplasmic charged
residues of MotA, Arg90 and Glu98, are involved intorque generation (Lloyd & Blair, 1997 ; Zhou & Blair,
1997). It was speculated that these charged residues of
FliG and MotA might engage in electrostatic interactions with each other (Zhou et al., 1998b).
The Na+-driven motor has some advantages for study
compared with the H+-driven type. Sodium-motive force
can be manipulated more easily than proton-motive
force, and amiloride and its analogues work as specificinhibitors of the motor (Sugiyama et al., 1988 ; Atsumi etal., 1990). It was thought that the inhibitor interactedwith the Na+-channel components in the motor. Fourproteins essential for torque generation, PomA, PomB,
MotX and MotY, were recently identified in the polar
flagellar motor of Vibrio alginolyticus (Asai et al., 1997 ;Okunishi et al., 1996). PomA and PomB are homologous
to MotA and MotB, and have four and one transmembrane segments, respectively. Rhodobacter sphaeroides MotA, which is the H+-motor component (Shah &Sockett, 1995), is very similar to PomA and contains thesame number of residues (Asai et al., 1997). Therefore,
PomA and PomB are thought to form a Na+-channel
complex in the motor. Recently, mutations which cause
the resistance to the specific inhibitor, phenamil (anamiloride analogue), were identified in PomA and PomB,
supporting the suggestion that these proteins are Na+motor-specific channel components (Kojima et al.,1999). On the other hand, both MotX and MotY, whichhave been identified in Vibrio parahaemolyticus(McCarter, 1994a, b) and have putative single transmembrane segments, are not homologous to the protontype motor proteins, except that MotY has a peptidoglycan-binding motif in the C-terminal region, as do
MotB and PomB. MotX was also inferred to be the Na+channel component of the motor because overproduction of MotX is lethal to E. coli in proportion to theexternal Na+ concentration and because this lethality isreversed by the addition of amiloride (McCarter, 1994a).
Thus, the H+ motor and the Na+ motor have somesimilar and some distinct aspects. We hypothesize thatthe basic mechanism for torque generation will becommon in the H+-driven and Na+-driven motors. If so,
residues critical for motor function will also be common
between the two types of motors. In this study, we
carried out random mutagenesis of the pomA gene inorder to identify residues responsible for the function of
PomA.
1760

METHODS
Bacterial strains, growth conditions and media. V. algino-

lyticus VIO5 (Laf− PomA+) and VIO586 (Laf− PomA−) wereused as the host strains to express the wild-type and mutant
pomA genes (the Laf phenotype is the presence of lateral
flagella) (Okunishi et al., 1996, Asai et al., 1997). For DNA
manipulation, we used the E. coli strain DH5α (Grant et al.,1990). V. alginolyticus cells were cultured in VC medium(0n5 % polypeptone, 0n5 % yeast extract, 0n4 % K HPO , 3 %% inNaCl, 0n2 % glucose) at 30 mC and E. coli cells were# cultured
LB medium (1 % tryptone, 0n5 % yeast extract, 0n5 % NaCl) at
37 mC. For swarm assays, Vibrio cells were inoculated on
0n25 % agar VPG plates (0n25 % agar in VPG medium
containing 1 % polypeptone, 0n4 % K HPO , 3 % NaCl, 0n5 %,#%w\v, glycerol). When necessary, chloramphenicol
was added−
to a final concentration of 2n5 µg ml " for Vibrio cells and 25 µgml−" for E. coli cells.
Plasmids. For isolation of pomA mutants, the pomA gene in
the BamHI fragment of plasmid pYA301 was subcloned into
pSU21, a chloramphenicol-resistant vector (Bartolome! et al.,1991), and named pMK101. The BamHI fragment in pMK101
was inserted in the opposite orientation to the lac promoter in
pSU21. This BamHI fragment was also cloned in the reverse
orientation and the resultant plasmid was named pMK201.
Isolation of pomA mutants. Plasmid pMK101 was treatedwith 2 M hydroxylamine in 0n1 M potassium phosphate (pH6n0), 1 mM EDTA for 4 h at 50 mC. After the treatment,
plasmids were diluted in 10 mM Tris\HCl (pH 7n5), 1 mM
EDTA, and dialysed overnight to remove hydroxylamine.
Then, plasmids were precipitated with ethanol and transformed into the pomA mutant VIO586. We selected Pom−
(polar flagellar motility defective) transformants from thechloramphenicol-resistant and swarm-deficient ones as described previously (Okunishi et al., 1996). The Pom− phenotype was confirmed by isolating mutant plasmids and retransforming them into VIO586.
Dominance. To assay the dominance of the plasmid-borne
pomA mutants, each plasmid was transformed into the PomA+
strain VIO5. Transformants were inoculated on a 0n25 % agar
VPG plate and incubated for 6 h at 30 mC. The diameters of theswarms of each mutant were measured and their mean
diameter was normalized to the diameter of the swarm formedon the same plate by VIO5\pMK201(PomA+) cells. The
relative swarm size of each mutant was obtained from at least
three independent experiments, and the dominance wasdetermined from the mean value of them. The swarm size of
wild-type cells was 0n92p0n06 cm. The mutants werecategorized into four types ; severely dominant (jjj ;0–20 % of wild-type swarm size), strongly dominant (jj ;
21–45 % of wild-type), slightly dominant (j ; 46–70 % of
wild-type) and recessive (k ; 71–100 % of wild-type).
DNA manipulations and sequencing. Routine DNA manipulations were carried out according to standard procedures(Sambrook et al., 1989). Restriction endonucleases and otherenzymes for DNA manipulations were purchased from Takara
Shuzo and New England Biolabs. The nucleotide sequence
was determined by the dideoxy chain termination method
using the ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction Kit and the ABI PRISM 377 DNA sequencer(Perkin Elmer).
Electroporation. Transformation of Vibrio cells by electroporation was carried out as described previously (Kawagishi
et al., 1994).
Immunoblots of PomA. Immunoblotting was carried out asdescribed previously (Nishioka et al., 1998). Cells wereRandom mutagenesis of the pomA gene
harvested and suspended in distilled water at an OD of 10.
The suspension was electrophoresed through a 10''!
% SDSPAGE gel. For the detection of the mutant PomA proteins, weused an anti-PomA peptide antibody (PomA91), which was
generated against peptides derived from three segments of
PomA with an additional cysteine at the N terminus to
keyhole limpet haemocyanin ; 1, CKKDIALTDERHTQGTGVFRAFGDVAP (in the cytoplasmic domain between TM2
and 3) ; 2, CTTLYGAILSNMVFFPIADKLSLRRDQET (inthe TM4) ; 3, CQDGQNPRVIDSYLKNYLNEGK (in the Cterminal cytoplasmic domain).

mutants (alleles 1 and 3) and type II mutants (alleles 2
and 4). We expected that type II mutants might swim
slowly. However, when each of them was grown inliquid broth and examined under the microscope, only asmall fraction of the cells were swimming, at the same
speed as wild-type cells, and most cells were immotile.
All of the type II mutants showed this phenotype. On theother hand, all of the type I mutants were completely
immotile when grown in broth and observed under the
microscope. We characterized the type I mutants further.

RESULTS

Positions of pomA mutations

Isolation of PomA mutants

The nucleotide changes in all the type I pomA alleles
were determined by sequencing. The results are summarized in Table 1. In six mutants (alleles 3, 7, 23, 24, 26
and 37) we could not find any nucleotide changes in thepomA coding region. These mutations might occur inregions that affect the expression of pomA. Nucleotidechanges in the other mutants were of the type expected
for hydroxylamine mutagenesis (GC to AT transitions).
The mutants were grouped into four categories (Table
1). Deletions or terminations were found in ten mutant
alleles. Single and double amino acid substitutions
were found in 16 and 5 mutants, respectively. Double
mutations were detected mainly in the cytoplasmic
domain, and interestingly, two were in adjacent residues. On the other hand, it is noteworthy that most of
the single mutations were mapped to the putative thirdor fourth transmembrane segments, which are highly
conserved between PomA and MotA of R. sphaeroides
or E. coli (Asai et al., 1997) (Fig. 2). Actually, all of themutated residues in the single mutants, except for
Ser193, are conserved in MotA. Five mutations were
found in residues that also gave a dominant, swarmdeficient phenotype when mutated in motA in E. coli.
These residues are Gly8, Gly154, Thr186 and Pro199(mutations in MotA were G6S, G6D, G176S, T209W
and P222L, respectively). Most of the single mutations
were substitutions of a non-charged residue by a polaror charged one, and others were substitutions byresidues with large side chains.

We first subcloned the pomA gene from the plasmid
pYA301 (pomA+, Kmr) into the vector pSU21 (Cmr),
because transformation of Vibrio cells with the Cmr
vector was more efficient than with the Kmr vector.
Random mutagenesis of the pomA gene was carried out
by treating the resultant plasmid, pMK101 (pomA+,Cmr), with hydroxylamine. The mutagenized plasmids
were introduced into the pomA mutant VIO586, which
has non-motile polar flagella, and each transformant
was inoculated on semisolid agar plates. Approximately
1 % of the Cmr clones obtained were deficient forswarming in this screen, and we isolated 43 mutants bythis procedure. These mutants were classified into twogroups : (I) cells completely impaired in polar flagellar
motility on semisolid agar (Pom− ; 37 clones), and (II)cells that swarm much more slowly than wild-type (6clones). Fig. 1 shows typical swarms of the type I

pMK101 (PomA+)/VIO586

12
pMK101 (mutated)/VIO58634.................................................................................................................................................

Fig. 1. PomA mutant phenotypes. Fresh colonies of the pomA
mutant (VIO586) containing plasmid pMK101 were inoculated
on 0n25 % agar VPG plates containing chloramphenicol andincubated at 30 mC for 4 h. Colonies 1 and 3 are examples oftype I mutants (immotile) and colonies 2 and 4 are theexamples of type II mutants (slow swarming).

Dominance of pomA mutations

Next, we investigated the dominant-negative effects ofthe mutant PomA proteins. All the type I mutant pomA
alleles were introduced into VIO5 (pomA+) cells, andfresh transformants were inoculated on semisolid agar
plates. None of the mutant plasmids caused any
significant reduction in swarm size in this assay (datanot shown). The pomA fragment in pMK101 is inserted
in the opposite orientation to the lac promoter, and so
although the protein is expressed at levels sufficient tocomplement a pomA defect, it is not expected to beoverexpressed (for discussion of an analogous constructexpressing motY, see Okunishi et al., 1996). To determine whether higher-level expression of mutant
pomA alleles in trans from multicopy plasmids in
PomA+ cells might affect the dominance of the mutation,
we reversed the orientation of the wild-type and all the1761

S. K O J I M A a n d O T H E R S

Table 1. Nucleotide and amino acid changes of PomA mutants.................................................................................................................................................................................................................................................................................................................

Nucleotide changes were identified as described in Methods. Dominance was determined by swarm size relative to that of wild-type(see Fig. 4). Loop – is the region between TM1 and TM2 (periplasm), loop – is the region between TM2 and TM3 (cytoplasm), and"##$loop – is the region between TM3 and TM4 (periplasm).  ; not examined.
$%
Allele number/type

Deletion2
Nonsense mutations
11, 15, 19, 25
28
16
4
34
31
Single amino acidsubstitutions
10
27
22
6
36
21
17
33
13
1, 29
32
30
5
14
8
Double amino acidsubstitutions35
918, 2012
No mutation
detected in pomA
3, 7, 23, 24, 26, 37

Base change

Amino acidchangeLocation∆262–472

Frameshiftk

Loop – –TM3
#$

C160T
C412T
C676T
C685T
C694T
C578T\C694T

Q54Termination
Q138Termination
Q226Termination
Q229Termination
R232Termination
S193F\R232Termination

k
k
j
j
k


Loop –#$
Loop –#$
C-terminus
C-terminus
C-terminus
TM4\C-terminus

G22A
G23A
G460A
G461A
G470A
(C345T)\C473T
C484T
G526A
G527A
C557T
C578T
C596T
(C594T)\C596T
G662A
C739T

G8S
G8D
G154R
G154E
G157D
(G115G)\T158I
L162F
G176R
G176E
T186I
S193F
P199L
(F198F)\P199L
G221D
R247C

jj
jj
j
j
jjj
jjj
jjj
jj
jjj
jj
k
jjj

k
j

TM1
TM1
TM3
TM3
TM3
TM3
TM3
Loop –$%
Loop –
$%
TM4
TM4
TM4
TM4
C-terminus
C-terminus

C260T\C406T
C391T\C395T
G404A\G507A
G657A\G658A

A87V\H136Y
L131F\T132M
R135Q\M169I
M219I\D220N

jjj
k
jjj
jj

Loop – \loop –#$#$Loop – \loop –#$#$Loop – \TM3#$
C-terminus\C-terminus

mutant pomA genes, placing them downstream of thelac promoter (the resultant plasmid containing wildtype pomA is named pMK201). These mutant plasmids
were introduced into VIO586 (PomA−) and VIO5
(PomA+) cells and each transformant was inoculated onsemisolid agar plates.
As shown in Fig. 3(a), pomA in pMK201 complemented
the PomA− phenotype of VIO586, indicating thatfunction is normal when the PomA protein is expressedfrom this plasmid. Most of the mutant PomA remained
nonfunctional in this assay. Exceptions were G176R and
S193F, which were slightly motile, suggesting that these
two mutations might be suppressed by overexpression
1762

Dominance

of the proteins. As shown in Fig. 3(b), most of themutant proteins reduced the swarming ability of VIO5
(PomA+) cells when they were overexpressed from theplasmids. To quantify the dominance of each mutant,the swarm assays were repeated, and relative swarm
sizes for at least three measurements were averaged. The
data are summarized in Fig. 4. Deleted or truncated
PomA mutants exhibited recessive or slightly dominantphenotypes. On the other hand, most of the single or
double mutants showed dominant effects. The strengthof dominance was ranked as described in Methods(Table 1). The mutations in the transmembrane segments showed the strongest dominance (jjj orjj), consistent with the results of MotA mutagenesis.

Random mutagenesis of the pomA gene.....................................................................................................

Fig. 2. Amino acid alignments of PomA andsites of mutations isolated in this study.
Mutated residues are marked by symbols.
show sites of nonsense mutations, andarrows show the deletion in the allele 2
mutant. Circles show the sites of single
mutations and paired symbols (=, W,orO) show sites of double mutations. Residues
giving dominant mutations in both PomA
and MotA of E. coli are marked by a black
circle. Abbreviations : VaPomA, V. alginolyticus PomA ; RsMotA, R. sphaeroides MotA ;EcMotA, E. coli MotA. White letters in blackboxes show residues identical to PomA.

(a) Host VIO586 (PomA– )(b) Host VIO5 (PomA+ )(c)
Vector

G8D

S193F

WT

G154R

P199L

D 623–833

G154E

G221D

Q54Termination

G157D

R247C

Q138Termination

T158I

A87V/H136YQ226Termination

L162F

L131F/T132M

Q229Termination G176R R135Q/M169I
R232Termination
G8S

G176E M219I/D220N
T186I

Vector*.................................................................................................................................................................................................................................................................................................................

Fig. 3. Swarming abilities of the PomA mutants. Two fresh colonies of the pomA mutant (VIO586) (a) or the wild-type
Pom+ strain (VIO5) (b) each carrying the mutant pomA genes on a multicopy plasmid were inoculated on 0n25 % agar VPG
plates containing chloramphenicol and incubated at 30 mC for 6 h. The mutations are identified in panel (c). In (a) WT
stands for VIO586/pMK201 and ‘ vector ’ stands for VIO586/pSU21 ; in (b) WT stands for VIO5/pMK201 and ‘ vector ’ stands
for VIO5/pSU21. ‘ Vector* ’ stands for VIO586/pSU21 and is only shown in (b), as a non-motile control.

Expression of mutant PomA

To detect the mutant proteins expressed from theplasmid pMK201, we performed immunoblots to thecell suspensions of the mutants by using a polyclonalanti-PomA peptide antibody (Fig. 5). We could detect
PomA bands of the expected size (25 kDa) in all thestrongly (jj) or severely (jjj) dominant mutants.

We observed some small differences in the amounts ofthe proteins between the mutants. In addition to the
25 kDa band (arrow in Fig. 5), we detected a band at
45 kDa, in both the wild-type and the mutants. The
significance of this band is discussed later. We could notdetect the PomA protein in the wild-type strain itself(vector in Fig. 5a and b), or in recessive (k) or slightly
dominant (j) mutants (for example, G154R in Fig. 5b ;1763

S. K O J I M A a n d O T H E R S

Relative swarm size

1
0·8
0·6
.....................................................................................................

0·4

0N

I
69

G1

54

R

22
I/D

M2

19

M1

13

Q/
35

R1

/H

F/T
31

L1

7V

T

A8

r
W

Ve
c

to

C
47

L

(b)

R2

99
P1

E

I
86
T1

76
G1

R
76

F

I

G1

62
L1

57
G1

58

D
G8

T1

S

T

G8

r
to
W

Ve
c

(a)

D

13

6Y

G8
S
G8
D
G1
54
R
G1
54
E
G1
57
D
G1
58
I
L1
62
F
G1
76
R
G1
76
E
T1
86
I
S1
93
F
P1
99
L
G2
21
D
A8 R247
7V
C
/H
13
L1
6Y
31
F/T
13
R1
2M
35
Q
M2 /M16
19
9I
I/D
22
0N

Wi
ld
Typ
e
D6
Q5
23
4T
-83
3
Q1 ermi
na
38
tio
T
n
Q2 ermi
na
26
tio
T
n
Q2 ermi
na
29
tio
Te
r
n
mi
Q2
na
32
tio
Te
rm
n
ina
tio
n

0

2M

Fig. 4. Dominance of the PomA mutants.
Swarm assays of the mutants, as shown in
Fig. 3(b), were repeated at least three times
and the swarm diameters of each mutant
were normalized to that of the wild-typestrain (VIO5/pMK201). The relative swarm
size obtained from each experiment was
averaged and is indicated on the figure witherror bar.

0·2

45 kDa45 kDa25 kDa(PomA)

25 kDa(PomA).................................................................................................................................................................................................................................................................................................................

Fig. 5. Western-blot analysis of the mutant PomA proteins. PomA was not detected in any of the other mutants (datanot shown). Cells transformed by the plasmids pMK201 were harvested and suspended in distilled water. This suspension
was subjected to SDS-PAGE and immunoblotting with the anti-PomA peptide antibody.

other data not shown), except for R247C and L131F\
T132M. All of the deleted or truncated mutant proteins
could not be detected. These mutant proteins might bedegraded or synthesized in very small amounts. L131F\
T132M, which was detected but not dominant, might
have some defect in assembling into the appropriate
position in the motor.
In some mutants, the PomA bands were found at
positions different from the wild-type PomA. The T158I
protein was observed at a higher position than wildtype, and in the G176E and R135Q\M169I mutants,
both the 25 kDa and 45 kDa bands were at significantly
higher positions. Some structural change caused bythese mutations might affect the mobility of the proteinsin electrophoresis.
DISCUSSION
Isolation of pomA mutations

PomA is the homologue of the MotA protein, which isbelieved to be the channel component essential for1764

rotation of the H+-driven motor. PomA should have
similar functions in the Na+-driven motor. To identify
the residues of PomA essential for torque generation, we
carried out random mutagenesis of the pomA gene byhydroxylamine. We isolated 37 non-motile mutants andidentified 26 independent mutations in the pomA gene.
Most of the mutants with single or double amino acidsubstitutions showed severe or strong dominance, andthese mutant PomA proteins expressed from multicopy
plasmids could be detected by immunoblotting. These
results suggest that the mutant proteins which exhibit
dominance are expressed and inserted into the motor
but do not function properly, that is, they seem to havedefects in torque generation. On the other hand,recessive and weakly dominant mutants could not be
detected by immunoblotting. We also could not detect
the wild-type level of PomA (Fig. 5a, b, lanes 1), somutant proteins might be synthesized in very small
amounts, although they do not significantly affect thetorque generation when expressed in PomA+ cells.
Therefore we mainly discuss the strongly dominant
mutations in the subsequent sections.

Random mutagenesis of the pomA gene.................................................................................................................................................

Fig. 6. Hypothetical transmembrane region of PomA. The
residues altered in strongly and severely dominant mutants areshown by black symbols. The residues altered in slightly
dominant mutants are marked by the residue number withwhite symbols. Dominant double mutants are shown by a pairof squares or triangles. Recessive mutants are not marked.

Localization of the dominant mutations

The dominant mutations were localized to the putative
third or fourth transmembrane segments (Fig. 6), which
are highly conserved between PomA and MotA. Thesetransmembrane segments might be more important forfunction than other regions of the protein. Most of themutations introduced amino acids with charged or large
side-chains. According to the tryptophan-scanning mutagenesis of MotA (Sharp et al., 1995b), helices 3 and 4 of
MotA were more sensitive to bulky Trp substitutions
than were helices 1 and 2, suggesting that helices 3 and4 might be more fully surrounded by other protein
segments that cannot accommodate the Trp side chain.
Therefore, substitutions in TM3 or TM4 of PomA
might affect conformation or interfere with the interaction between PomA and other channel components
such as PomB, MotX and MotY, and consequently
disrupt the arrangement of the channel complex, which
would no longer function properly in the motor.
Several dominant double mutants were isolated andmapped to the cytoplasmic domain. M219I\M220N
were mutated in residues that adjoin each other,suggesting that this region might be involved in protein–protein interaction or be important to protein
conformation. It will be necessary to separate thesemutations to determine which confers the Pom− phenotype, or whether both are needed.
Comparison between PomA and MotA

Most of the dominant mutations isolated in this study
occurred in the residues conserved in MotA proteins.
Especially we found five mutations in residues that also
gave a dominant, swarm-deficient phenotype whenmutated in motA in E. coli. The mutations G8S, G8D(TM1) and P199L (TM4) correspond to G6S, G6D andP222L, respectively, isolated in E. coli MotA (Blair &Berg, 1991). These residues are also highly conserved in
MotA homologues (Fig. 2). They would not have a
direct role in ion translocation but rather a structuralrole. Zhou & Blair (1997) focused on, and investigated,
this proline residue (Pro222 in E. coli MotA) by intensive
mutagenesis : they speculated that Pro222 might function
to mediate conformational changes in MotA that couple
the events occurring on the membrane and cytoplasmic
domain during energy conversion. Pro151 of PomA is
another Pro residue located in the putative third TM
segment and is highly conserved among MotA homologues. Mutations of this residue were not isolated inthis study, but according to the mutational analysis of
the corresponding residue in E. coli MotA (Zhou &Blair, 1997), it would have a role similar to that of
Pro199 in PomA. The G154R mutation was identical toa chromosomal mutation in PomA of VIO586 (Y. Asai,
I. Kawagishi, E. Sockett & M. Homma, unpublished).
This residue corresponds to Gly176 in E. coli MotA, andmutation G176S of MotA severely impaired motility(Blair & Berg, 1991).
There are no charged residues in the TM segments of
PomA, although some polar residues exist there. These
residues might be expected to interact with ions in an ion
channel. Among the polar residues, dominant mutations
were isolated in Thr158 (TM3) and Thr186 (TM4).
Thr186 is highly conserved in MotA homologues, andmight have an important role in ion translocation.
Thr158 is also conserved in MotA of R. sphaeroides,
and T158I mutations exhibited the strongest dominance(Fig. 4). In E. coli MotA, the non-motile dominant
mutation A180V was isolated in the corresponding
residue. So a protein carrying the T158I mutation might
affect the ion translocation or impede the rotation of the
motor, acting as a ‘ brake ’ because of its bulky side
chain. In this study we could not isolate the Na+-motorspecific dominant mutations. This suggests that PomA
has very similar profile to MotA as a channel component. In other words, PomA might mainly constitutethe Na+ channel pore structure, and Na+-selectivity
might not be determined by PomA alone.
Expression of mutant PomA

Some mutations of PomA affected the electrophoretic
mobility of the protein. The T158I, G176E and R135Q\
M169I proteins were detected at a position slightly
above the wild-type protein on gels. This alteredmobility might reflect differences in conformation orother modifications. In addition to the PomA bands(25 kDa), we observed bands at approximately 45 kDa
in both wild-type and mutant PomA. We suggest that
this band might represent a different conformation of
PomA or an interaction of PomA with itself or withother proteins, which persist even in the presence of SDS(T. Yorimitsu, K. Sato, Y. Asai, I. Kawagishi & M.
Homma, unpublished). The 45 kDa bands of G176E andR135Q\M169I were also found at the upper position,
supporting this proposal. In some mutants, the 45 kDabands were weaker. It will be necessary to examine more
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S. K O J I M A a n d O T H E R S

systematically the conditions that affect the appearanceof the 45 kDa band.
We think that the basic mechanism of torque generationin the flagellar motor will be common, independent of
the coupling ion, H+ or Na+. On the other hand, it is notknown how the ion selectivity of the motor is determined, or what is the function of the additional
motor genes, motX and motY, specific for the Na+
motor. To answer these questions, it will be necessary toinvestigate interactions between the motor components.
In the case of Na+-type F F ATPase of Propionigenium modestum, an !H"+-type E. coli (F "δ)\Na+-type P. modestum (F jδ) hybrid ATPase!
showed that the F part is exclusively responsible for therecognition of the!coupling ion (Kaim & Dimroth, 1993,
1994). Such hybrid studies, for example Rhodobacter
MotA expressed in a pomA-deleted Vibrio strain, are
now in progress in our laboratory. We hope that further
analysis of the common structure between the Na+
system and H+ system will lead us to the essential pointsof the torque-generating mechanism.
ACKNOWLEDGEMENTS
We thank Dr David F. Blair for critically reading themanuscript. This work was supported in part by grants-in-aid
for scientific researches (to I. K. and M. H.) from the Ministryof Education, Science and Culture of Japan and the Japan
Society for the Promotion of Science (to S. K.).

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Received 27 December 1998 ; revised 22 March 1998 ; accepted 26 March
1999.

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