2nd Revised Manuscript # JBRP-00058

Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor
gene

Mayuko Okabe, Toshiharu Yakushi, Yukako Asai#, and Michio Homma*

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

Running title: a motX gene encoding a component of the sodium-driven motor*Corresponding author

Tel: 81-52-789-2991
Fax: 81-52-789-3001

E-mail: g44416a@cc.nagoya-u.ac.jp#

Present address: Lab. of Sensory Neuroscience, The Rockefeller University,

1230 York Ave. #314, New York, New York 10021-6399, USA1

Summary

Four motor proteins, MotX, MotY, PomA and PomB, have been identified as
constituents of the Na+-driven flagellum of Vibrio species. In this study, the complete motX
gene was cloned from Vibrio alginolyticus and was shown to complement three mot mutations,motX94, motX115 and motX119, as well as a V. parahaemolyticus motX mutant. The motX94
mutant contains a frameshift at Val86 of MotX, while the motX115 and motX119 mutations aresubstitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX wasoverexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased,
and vice versa, suggesting that MotX and MotY stabilize mutually by interacting at themembrane level. When a plasmid containing the motX gene was introduced into motY mutants
NMB117 (motY117) and VIO542 (motY542), the mutations were suppressed. In contrast, motY
could not recover any swarm-defective motX mutants studied. Considering the above evidence,
we propose that MotX is more directly involved than MotY with the mechanical functioning of
the Na+-type flagellar motor, and that MotY may stabilize MotX to support its interaction withother Mot proteins.

2Introduction

The bacterial flagellum is the only rotary machine for movement in the biological
kingdom. This machine is driven by a specific ion flow which is dependent upon theelectrochemical potential (1-3). Protons and sodium ions are the two known coupling ions, andare used for fueling to separate types of such motors. The motor itself is embedded in thecytoplasmic membrane at the base of the flagellar structure, and is composed of a stator and arotor. In Gram-negative bacteria, the rotor is composed of more than ten protein components,
forming a rod and four ring structures: the L, P, MS, and C rings. It is speculated that the stator
complexes (the force-generating units) are arranged around the base of the flagellum where theyinteract with the rotor protein FliG, a component of the C ring (4, 5).
In a proton-driven motor, the force-generating unit is composed of MotA and MotB (6, 7).
MotA has four transmembrane segments and one large cytoplasmic loop (8). MotB has a single
N-terminal transmembrane segment, in which an important Asp residue is speculated to convey
the protons (9), and a peptidoglycan-binding motif at the C-terminus (10, 11). The four MotA
and one MotB transmembrane segments, predicted to produce α-helical structures, are thought toform an ion channel (12-14). One of the most prominent hypotheses is that H+ flow through thischannel induces structural changes in MotA which result in alteration of the force-generating
unit’s interaction with the rotor component FliG, thereby generating rotary power (15, 16).
In a sodium-driven motor, the force-generating unit is thought to be composed of PomA,
PomB, MotX, and MotY (17-21). PomA and PomB are homologous to MotA and MotB,
respectively. PomA is required to stabilize PomB, and direct interaction between PomA and
PomB to form a complex with stoichiometry 2:1 has been demonstrated (22, 23). PomA and
PomB have been purified from Vibrio alginolyticus, and when reconstituted into
proteoliposomes, the PomA/B complex catalyzed sodium influx while PomA alone did not (23).
3

PomA appears to stably dimerize to form an ion channel with PomB, and both halves of the
PomA homodimer function cooperatively to conduct sodium ions (24). We proposed that the
periplasmic loop of PomA is important for dimer formation and function (25, 26). The
heteromultimeric complex is thought to act as a sodium channel.
A hybrid motor consisting of the proton-type MotA and sodium-type subunits PomB,
MotX, and MotY can work as a sodium-driven motor in Vibrio cells (27). Furthermore,
although the proton-type MotB does not function with either MotA or PomA in Vibrio cells, it
has been shown that PomB can be replaced with the chimeric protein MomB, which is madefrom the N-terminal segment of MotB and the C-terminal segment of PomB (28). PomA and
PomB may not play a primary role in ion selectivity.
MotX and MotY, which are unique components of sodium-driven motors, were first
identified in Vibrio parahaemolyticus (19, 20). Both proteins have a putative single
transmembrane domain, two cysteine residues, and a conserved tetrapeptide CQLV in theputative periplasmic region. The predicted pI is basic for both proteins, 9.8 for MotY and 8.6 forMotX. MotY has a peptidoglycan-binding motif in the C-terminal region which is also observedin both MotB and PomB. It was suggested that MotX forms a sodium-conducting component of
the torque generator, as overexpression of MotX in E. coli kills the cells and this effect can beblocked by amiloride, a known sodium-channel inhibitor. When both MotX and MotY are
produced in E. coli, MotY localizes to the cell membrane in the presence of MotX. However,MotY localizes to the cytoplasm when MotX is not co-expressed. From these observations, it
was inferred that MotX interacts with MotY (19). However, those experiments have been
performed only in E. coli and not in the native host cell. The function of MotX and MotY hasremained unclear, although it is thought that they may be essential for the recognition of sodium
ion by the motor. In this report, we present the cloning of the V. alginolyticus motX gene andcharacterization of the resulting MotX protein.

4Materials and Methods

Bacterial strains, plasmids, and growth conditions. V. alginolyticus VIO542(motY542) is derived from VIO5 (21). V. alginolyticus NMB85 (motY85), NMB94 (motX94),
NMB115 (motX115), NMB117 (motY117) and NMB119 (motX119) were isolated from YM4(29). V. parahaemolyticus LM4170 (lafX313::lux motX118::mini-Mu lac (Tets)) (19) andLM4171 (lafX313::lux motY141::mini-Mu lac (Tets)) (20) were kindly donated by L. McCarter.
Escherichia coli DH5α (F- λ- recA1 hsdR17 endA1 supE44 thi-1 relA1 gyrA96 ∆(argFlacZYA)U169 f 80dlacZ∆M15) (30) was used for all plasmid constructions. The plasmid pCR2.1
was purchased from Invitrogen (CA). The plasmids pSU21 and pSU41 are broad host range
vectors having the replication origin of the plasmid p15A, a multiple cloning site downstream ofthe lactose promoter-operator, and either a chloramphenicol or a kanamycin resistance gene,
respectively (31). V. alginolyticus and V. parahaemolyticus cells were cultured at 30 °C in VC
medium (0.5% polypeptone, 0.5% yeast extract, 0.4% K2HPO4, 3% NaCl, 0.5% glucose) or VPG
medium (1% polypeptone, 0.4% K2HPO4, 3% NaCl, 0.5% glycerol). When necessary,chloramphenicol and kanamycin were added at final concentrations of 2.5 and 100 µg/ml,
respectively. E. coli cells were cultured at 37 °C in LB medium (1% tryptone, 0.5% yeast extract,
0.5% NaCl).
DNA manipulations and sequencing. Routine DNA manipulations were carried out
according to standard procedures (32). Restriction endonucleases and other enzymes for DNA
manipulations were purchased from Takara Shuzo (Kyoto, Japan). Nucleotide sequences were
determined using the dye terminator cycle sequencing kit (Perkin-Elmer Co.) and an ABIPRISM sequencer (model 310 or 377; PE Applied Biosystems).
Transformation of Vibrio cells. Transformation of Vibrio cells was carried out byelectroporation as described previously (33). The cells were subjected to osmotic shock and5washed thoroughly with 20 mM MgSO4. Electroporation was carried out with the Gene Pulser
electroporation apparatus (Japan Bio-Rad Laboratories, Tokyo, Japan) with an electric field of
5.0 to 7.5 kV/cm.
PCR products. Oligonucleotide primers for PCR were synthesized by Sawady
Technology Co. (Tokyo, Japan). PCR amplification was carried out with the TaKaRa Taq kit(Takara Shuzo, Kyoto, Japan) and Thermal Cycler (Perkin-Elmer Japan, Chiba, Japan) as
recommended by the distributors.
Construction of plasmids. The 1-kb PCR product containing motX was cloned into thevector pCR2.1 and the resultant plasmid was named pMO100. The 1-kb EcoRI fragment of
pMO100 was subcloned into the multicloning site of the plasmid vector pSU21 and named
pMO200. The plasmid pMO401 was constructed by cloning a 700-bp ClaI-BamHI fragment of
PCR products into the multicloning site of the vector pSU41. For the reaction, we used pMO200
as the template and VamotX.Cla1 (5'-TCATCGATGTTTGGTTTCTTAGATACC-3') and
VamotX.BamH1 (5'-GACGGATCCGTTGTTTGCCTTAATAGGACG-3') as end primers (therestriction sites are underlined). When we constructed pMO401, we obtained a plasmid
containing three point mutations in motX (A7L, A8G and I62T) which was named pMO400.
Site-directed mutagenesis was performed by a two-step PCR using the end primers and a pair ofmutant primers as described previously (34). The mutant primers used were VamotX.A146VFW(5'-ACCTAAATGTGCGTATTCAT-3') and VamotX.A146VRV (5'ATGAATACGCACATTTAGGT-3') (changed bases are underlined) for pMO406 (A146V).
Detection of the Mot proteins. Overnight cultures of cells in VC medium were diluted
50-fold with VPG medium. At mid-log phase, cells were harvested, resuspended in TMN50 (50mM Tris-HCl [pH 7.5], 5 mM Glucose, 5 mM MgCl2, 50 mM NaCl, 250 mM KCl), andsonicated on ice twice for 10 s, using a Sonifier 250 (Branson Ultrasonics Co., Danbury, CT).
Undisrupted cells were removed by low speed centrifugation (12,000 x g for 5 min at 4 °C), and6

the membrane fraction was recovered from the supernatant by centrifugation at 70,000 x g for 10
min. The membrane pellet was suspended in TMN50 and a sodium dodecyl sulfate (SDS)
loading buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 1% SDS, 0.1% bromophenol blue)
containing β-mercaptoethanol. The suspension was boiled for 5 min and the proteins wereseparated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoreticallytransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Japan, Tokyo) using asemi-dry blotting apparatus (Biocraft, Tokyo, Japan). The PVDF membranes were thenincubated with the first antibody. The bound antibodies were detected with horseradish
peroxidase-conjugated anti-rabbit IgG and the ECL assay system (Amersham Pharmacia
Biotech).
Antibody. An anti-MotX antibody (MotX192) was raised against a synthetic peptide
corresponding to the fragment L192-W211 of the amino acid sequence deduced from the motX
gene. The anti-MotY antibody (MotY264) was raised against a synthetic peptide corresponding
to MotY fragment G264-R283. The anti-MotX and anti-MotY antibodies were obtained from
Sawady Technology (Tokyo, Japan) and Biologica (Nagoya, Japan), respectively.

Results

Cloning of V. alginolyticus motX gene. We previously reported the cloning of thepromoter region of V. alginolyticus motX (18). To clone the entire region of the motX structuralgene, we amplified the region containing it by PCR using an oligonucleotide primer
corresponding to the motX promoter region and mixed oligonucleotide primers corresponding to
the hmp gene, which in V. alginolyticus is adjacent to motX (Fig. 1) (19). The resulting 1 kb
DNA fragment was amplified by PCR and cloned into vector pCR2.1, yielding plasmid pMO100.

7

The fragment was sequenced (Fig. 2A) and the sequence of the chromosomal motX gene wasalso determined directly to verify that of the cloned fragment. As previously described, the motX
promoter region contains both the σ54-consensus and σ28-consensus sequences (18). According
to the ORF, the sequence codes for 212 amino acids. A putative rho-independent transcription
terminator was found at the end of the gene. Thus, motX would be transcribed as a
monocistronic message from the chromosome. The predicted amino acid sequence is 94 % and
85 % identical to the motX gene products of V. parahaemolyticus and V. cholerae, respectively(Fig. 2B). The ORF contains a potential transmembrane region near the amino terminus and a
relatively large periplasmic region at the carboxy-terminus, according to a hydropathy profileand the positive inside rule as reported previously for V. parahaemolyticus (19).
Characterization of V. alginolyticus mot mutants. The pom (or mot) mutants of V.
alginolyticus, which possess non-functional yet intact polar flagella, were isolated from thelateral flagella-deficient strain YM4 (29) after the treatment with ethyl methanesulfonate. Of the
mot mutants, NMB94, NMB115 and NMB119 were unable to swarm when transformed with theplasmid carrying motY, pomA or pomB. However, as shown in Fig. 3A, they were all able toswarm when transformed with plasmid pMO200 carrying the motX gene. Therefore, we
concluded that the above three mutants resulted from mutations in the motX gene. In addition,
pMO200 also restored the swarming capability of the V. parahaemolyticus motX mutant,
LM4170 (Fig. 3B). The motX chromosomal region of the three mutants was sequenced. In
NMB94 (motX94), a two-base deletion (T488 and G489) was found (Fig. 2A), which shifted thereading frame from Cys87 onwards. The NMB115 (motX115) and NMB119 (motX119) mutants
were found to possess substitutions of Ala146 (GCG) to Val (GTG) and of Gln194 (CAG) toamber (TAG), respectively.
Characterization of mutant MotX. The A146V mutant NMB115 appeared not toswarm (Fig. 3), but upon prolonged incubation did exhibit motility a little (data not shown).

8

NMB115 cells in culture medium actually displayed a slow motile phenotype. The plasmid
pMO406, which carries the motX gene with the A146V substitution, was introduced into NMB94.
The transformant, pMO406/NMB94, swarmed comparably to the wild type (data not shown),suggesting that the A146V mutation by itself may not directly affect flagellar function, rather, it
may exert its effects on other factors such as protein stability. As shown in Fig. 4, the MotX
protein expressed in NMB115 was detected in smaller amounts compared to the wild-type. This
may suggest that the mutant protein of A146V MotX is unstable, yet possibly quite functional.
NMB94 harboring pMO400 could swarm equally as well as the wild-type, although pMO400
contains three point mutations in motX (data not shown). The amount of MotX expressed fromplasmid pMO401 was very high, while expression from plasmids pMO406 and pMO400, whichcarry the mutant motX gene, was low (Fig. 4). Those mutations may affect the expression of thegene or the stability of the protein. It is worth noting that the growth rate of cells carrying
pMO401 was not affected.
Interaction between MotX and MotY in vivo. It has been reported that MotY of V.
parahaemolyticus, when produced in E. coli, is localized to the membrane only when expressed
together with MotX, suggesting that the two interact with each other (19). Here, such possible
interaction between MotX and MotY was also examined in V. alginolyticus. MotX was detectedin the membrane fraction of the wild-type strain, but not in the membrane fraction of the motY
mutant, VIO542. However, MotX was detected in the membrane fraction of the motY mutant
harboring the plasmid pIO6, which carries motY (Fig. 5A). We suggest that MotX, expressedfrom the chromosome of VIO542, is stabilized in the membrane by MotY expressed from theplasmid. Symmetrically, MotY was detected in the membrane of the motX mutant NMB94 whenharboring the plasmid pMO401 carrying motX, although it could not be detected when harboring
the vector plasmid (Fig. 5B). These results suggest that MotX and MotY interact and mutuallystabilize each other in the membrane of V. alginolyticus.

9Multicopy motX suppresses motY mutation. As described above, MotX and MotY
help to stabilize each other. If this stabilization is indeed the main function of one of these two
proteins, then one should find that overexression of one should help to overcome a deficiency ofthe other. This appears to be true for MotY. The non-swarming MotY mutants VIO542, whose
mutation site has not been determined and NMB117, which possesses an amber (TAG) mutation
at position Gly32, both recovered swarming capacity when transformed with pMO401, a motXcontaining plasmid.
On the other hand, plasmid-derived MotY was not capable of rescuing motX mutations(data not shown). This suggests, however, that MotY might not be required for mechanicalmotor function per se, but may stabilize or support the structure of MotX in the membrane andpermit normal function. On the other hand, MotX, in addition to its role in stabilizing MotY,
may be more directly involved in the motor function of the V. alginolyticus Na+-type flagella,potentially assisting in torque generation.

Discussion

We cloned the entire region of the motX gene of V. alginolyticus. The predicted aminoacid sequence of V. alginolyticus motX has 94% identity with V. parahaemolyticus motX, and
85% identity with V. cholerae motX. The putative transmembrane regions have relatively low
similarity, as it is the case for MotY among the Vibrio spp. (21). If the transmembrane regions
were contributing to function or channel formation, one would expect them to display highsimilarity. In this case, the transmembrane region may not have a special function other than tosimply anchor the protein in the membrane. We are in the process of trying to exchange the
MotX transmembrane region with that from other membrane proteins.

10By using the cloned gene, we identified three motX mutants, NMB94, NMB115 and
NMB119. Western blot analysis of different mutant proteins showed that NMB115 MotX
A146V protein levels are significantly lower compared to wild-type when both proteins are
expressed from identical plasmids (Fig. 4). When a plasmid containing motX with the NMB115
A146V mutation was introduced back into NMB115 cells, the mutant cells recovered motility
similar to wild-type cells. Taken together, these results suggest that NMB115 may producefunctional protein, but just not in the amount required to produce a complement of functional
flagella. Most likely, the A146V mutation has a negative effect on MotX stability, and a further
look at this mutant may help reveal details about the kinetics of MotX and flagellar turnover.
The motor genes of V. cholerae and V. alginolyticus are homologous, and the polar
flagellum of V. cholerae is sodium driven (35, 36). Deletion strains defective in pomA, pomB,
motX, or motY of V. cholerae, which are non-motile, became weakly motile when E. coli motA
and motB genes are introduced (37). E. coli proton motor components can restore function to a
Vibrio sodium motor lacking MotX and MotY, but this hybrid motor is driven by the protonmotive force rather than by sodium. We therefore propose that MotX and MotY determine theion specificity for the sodium motor.
It has been reported that overexpression of MotX, but not MotY, kills E. coli (19), andthat this was exacerbated by addition of Na+ to the medium. Furthermore, amiloride, which is aknown inhibitor of sodium channels in animal cells as well as Na+-driven flagellar motors,
protected against the deleterious effects of MotX induction. This would suggest that MotX is
involved in sodium metabolism and may be a component of the motor’s sodium channel.
However, in this study, when MotX was over-produced in Vibrio cells, the growth of the cells
was not affected. This discrepancy between the Vibrio and E. coli results may be explained bydifferences in expression levels, by an inhibition in channel formation in Vibrio cells, or that
MotX affects only the sodium channels of E. coli. On the other hand, PomA and PomB wererecently purified as a complex and it was shown that they could catalyze sodium influx inreconstituted proteoliposomes (23). This is not inconsistent with the hypothesis that MotX acts11

to modulate an ion channel composed of PomA and PomB. If this hypothesis were true, thesodium influx allowed by the PomA/B complex should be enhanced by the addition of MotX.
However, MotX and MotY did not co-purify with the PomA/B complex, implying that theirinteraction with the PomA/B complex is weak.
MotX and MotY seem to stabilize each other in V. alginolyticus (Fig.5). It has beenshown that MotY of V. parahaemolyticus is stabilized in the presence of MotX when
overexpressed in E. coli. In addition, MotY was localized in the membrane in the presence of
MotX, and found in the cytoplasm in its absence. However, MotX is stable without MotY in E.
coli, a contrast with Vibrio that may be due to differences in their expression systems.
Alternatively, the level of expression of MotX, which was high enough in E. coli to be detectedby Coomassie blue staining, may affect the stability of MotX. In the present work, MotX wasoverexpressed in Vibrio, but not at a level detectable by Coomassie blue staining.
Here, we also demonstrate that overexpression of MotX could suppress motY mutations(Fig.6), and propose that MotX may interact with the PomA/B complex even in the absence of
MotY. Therefore, the role of MotY might be limited to anchoring or stabilizing MotX. The
putative transmembrane region of MotX is reasonably hydrophobic and predicted to form an αhelical structure. In contrast, the putative transmembrane region of MotY, as suggested
previously (20), is not as hydrophobic and unlikely to form an α-helix. MotY by itself can berecovered as a cytoplasmic protein, without MotX (19), and because of this we strongly suspectthat MotY is not a membrane-embedded protein, even though a transmembrane region waspredicted from the sequence (20, 21). A reliable biochemical study will be necessary toabsolutely determine the localization of MotX and MotY, and is sure to help elucidate their true
functions.

12

Acknowledgments

This work was supported in part by grants-in-aid for scientific research from the Ministryof Education, Science and Culture of Japan (to M.H. and T.Y.), and the Japan Society for the
Promotion of Science (to Y.A.).

13

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36. Kojima, S., Yamamoto, K., Kawagishi, I., and Homma, M. (1999) The polar flagella motorof Vibrio cholerae is driven by an Na+ motive force. J. Bacteriol. 181, 1927-1930
37. Gosink, K. K. and Hase, 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

17

Figure legends

Fig. 1. (A) Map of the chromosomal region flanking the motX gene. The arrows show
the PCR primers used to clone motX. (B) Restriction maps of the plasmids. The white boxes
represent the vector part of pSU21 and pSU41, and the arrows indicate the direction oftranscription from the lac promoter. The inserted fragments are indicated by solid lines.
Abbreviations: B, BamHI; H, HindIII; E, EcoRI; Sp, SphI; X, XhoI; C, ClaI; EV, EcoRV.

Fig. 2. (A) The nucleotide and deduced amino acid sequences of V. alginolyticus motX.
The putative Shine-Dalgarno sequence is underlined. The σ54- and σ28-consensus sequences areindicated by double underlines. Chavrons indicate inverted repeats. The C-terminal sequencecorresponding to the synthetic peptide used for antibody production is enclosed by a box. The
sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession
number (AB070443). (B) Alignment of MotX of V. alginolyticus, V. parahaemolyticus and V.
cholerae . The putative transmembrane region (TM) is enclosed by a box. Va, V. alginolyticus;
Vp, V. parahaemolyticus; Vc, V. cholerae.

Fig. 3. Swarming abilities of various motX mutants harboring V. alginolyticus motX.
Fresh colonies of NMB94, NMB115, NMB119 (A), and LM4170 (B) harboring pSU41 (vector)
or pMO200 (motX) were inoculated in triplicate on 0.3% agar-VC plates containing
chloramphenicol and incubated at 30 ºC for 6 h (A) and 9 h (B).

Fig. 4. Detection of MotX. The membrane fractions from cells were prepared as
described in Materials and Methods. Thirty micrograms of protein (except for pMO401 (wt)which contained 10 µg protein) from the membrane fraction was subjected to SDS-PAGE andanalyzed by immuno-blotting using anti-MotX antibody. From right to left, YM4; NMB115;
18

NMB94 transformed with pSU41 (vector), with pMO401 (wt), with pMO400 (A7L, A8G, I62T),
and with pMO406 (A146V).

Fig. 5. Stability of MotX and MotY. (A) The membranes of NMB94 and VIO542,
harboring pSU41 (vector) or pIO6 (motY), were prepared and subjected to SDS-PAGE andimmuno-blotting using anti-MotX antibody. (B) The membranes of NMB94 and VIO542,
harboring pSU41 (vector) or pMO401 (motX), were prepared and subjected to SDS-PAGE andimmuno-blotting using anti-MotY antibody.

Fig. 6. Suppression of the swarm-defective motY mutations by MotX. Fresh colonies of
VIO542 and NMB117 harboring pSU41 (vector) or pMO401 (motX) were inoculated intriplicate on 0.25% agar-VPG plates containing kanamycin and incubated at 30 °C for 24 h.

19

(A)
purA

motX

hmp

Va purA.1

Vp hmp-mix.1

(B)
E
H HC

pMO100pMO200

C

H EV

H EV

Sp X

E

Sp X B

pMO401200 bpFig. 1. Okabe et al.

A
1

purA -->
HindIII>>>>>>>>>>><<<<<<<<<<<<<AAGCTTAATTGATAGTCGTTAAAGATTGCAAAAGCCGACCTTCTGGTCGGCTTTTTTGTA
A *

60

61

TCTGAACAAAAAACGGCAGTGATTCTCATATGGCAAATTTTTGCCGCTGAGTCTAAGTCA

120

121

HindIIIGAGGCAAAAATTGTCTAAAGCTTAGCTGGTTTTTGCCGATAAGGTTATCAAGCAGAGGAG

180

181

RBS
motX -->
ATCCTTCATCGATCTTTGGTTTCTTAGATACCTTAAAGAGTACGTTTATGAAGTTACGAA
M K L R T

240

241

CAGTGGCTGCTTCACTGTTATTGACCTTGGTAGCTACCACAGCGAAAGCCAATGTTGCTG
V A A S L L L T L V A T T A K A N V A D

300

301

HindIII
ATGTGGGGGCGCCAGTTCCTATCTACACCGAAGCAGAGCTGATAAAGCTTATTGAGCAAA
V G A P V P I Y T E A E L I K L I E Q N

360

361

ATAAGCACTTACAACGTGTGCGCGCTGATAACTGCCAGCTGGTGGAGGATATCGTTGCTCK H L Q R V R A D N C Q L V E D I V A R

420

421

GTGCGACTCGAATTAACTTGCCCGCTTATGAATTTCTGTACGGCGACATGTTGGCGTGGG
A T R I N L P A Y E F L Y G D M L A W G

480

481

∆ (motX94)↑
GTGTGTGTGTCGAGCAAGATGTCGAACTGGGCCTTTACTACATCGAAAATGCAGCACATC
V C V E Q D V E L G L Y Y I E N A A H Q

540

541

AGGGATTACCTGCCGCGCTAGAGCAGATTGGTCGATACTATTCGCGTGGCACTTTAGTGC
G L P A A L E Q I G R Y Y S R G T L V Q

600

601

AGCAAGATAAAGAACGAGCAATTCCTTACTTGCGAGAAGCCGCCTCAATGGGAAACCTAAQ D K E R A I P Y L R E A A S M G N L N

660

661

T (motX115)↑
ATGCGCGTATTCATTTAGCCGAATTGCTCCTGCGTGATTACGGCAGCCCACTTGATTACG
A R I H L A E L L L R D Y G S P L D Y E

720

721

AAGATGCGTATCGTTGGTTATATAACTCTGTCACAGCAGATAAGCGGCAGCATAAGCGTA
D A Y R W L Y N S V T A D K R Q H K R I

780

781

T (motX119)↑
TCACTGTATTGAGAAATGGCCTTGAGCAGCGCATGCCACAAAACGTTATTGCTCGAGCTAT V L R N G L E Q R M P Q N V I A R A K

840

841

>>>>>>>>>>AACGGCGAGATACCTTTTGGTAACGTCCTATTAAGGCAAACAACTTCATACAACAAGGCC
R R D T F W *

900

901

>>>
<<<<<<<<<<<<<<<<<
CACATAATGTGGGCTTTGTTGTTTTCCCGAAGAGTAATCTTGAGCGATTAAACGACTTTG

960

961

TGTGGACCGAAGCACTCATAATG

B

983

TM
Va MKLRTVAASLLLTLVATTAKANVADVGAPVPIYTEAELIKLIEQNKHLQRVRADNCQLVE
Vp ------------M-S---VR-S-------------------------------------Vc ------------AWHSSYNP-Y---L-E-----S---------E----E--K-----I--

60
60
60

Va DIVARATRINLPAYEFLYGDMLAWGVCVEQDVELGLYYIENAAHQGLPAALEQIGRYYSR 120
Vp --------------------------------------M----Q---------------- 120
Vc ------Q--S--S---------------D---------M--------------L------ 120
Va GTLVQQDKERAIPYLREAASMGNLNARIHLAELLLRDYGSPLDYEDAYRWLYNSVTADKR 180
Vp G---------------------------------------------------------Q- 180
Vc -------------------A--------Q-----------------------------Q- 180
Va QHKRITVLRNGLEQRMPQNVIARAKRRDTFW 211
Vp Q--------R---R-----I--------M-- 211
Vc T-N---L--Q-------E-I-------E--- 211

Fig. 2. Okabe et al.

(B)NMB119Fig. 3. Okabe et al.

RS313
(WT)

LM4170ot
X+

NMB115

m

NMB94

ve
ct
or
m
ot
X+
ve
ct
or
m
ot
X+
ve
ct
or
m
ot
X+

YM4
(WT)

ve
ct
or

(A)

5
4

YM

11

B

M

N

94

B

M

N

MotX

Fig. 4. Okabe et al.

pM
O
pM 40
O 6
40
pM 0
O
40
pS 1
U
41

(B)

ve
c
m tor
ot
Y+
ve
c
m tor
ot
Y+

ve
c
m tor
ot
X+
ve
c
m tor
ot
X+

(A)

MotX
NMB94 VIO542(motX) (motY)

MotY
VIO542(motY)

Fig. 5. Okabe et al.

NMB94(motX)

vector
VIO542motX+

vector
NMB117
motX+

Fig. 6. Okabe et al.