Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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Rotary motors (bacterial). Invited

Kojima, S., & Blair, D.F.

McGraw-Hill Yearbook of Science & Technology 2003 page： 363-366 2003

Conformational change in the stator of the bacterial flagellar motor Reviewed

S Kojima, DF Blair

BIOCHEMISTRY Vol. 40 ( 43 ) page： 13041 - 13050 2001.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.

DOI： 10.1021/bi011263o

Web of Science

*Conformational change in the stator of the bacterial flagellar motor. Reviewed

Kojima, S., & Blair, D.F.

Biochemistry Vol. 40 page： 13041-13050 2001

A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor Reviewed

S Kojima, T Shoji, Y Asai, Kawagishi, I, M Homma

JOURNAL OF BACTERIOLOGY Vol. 182 ( 11 ) page： 3314 - 3318 2000.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio alginolyticus. We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.

DOI： 10.1128/JB.182.11.3314-3318.2000

Web of Science

A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor

Kojima S, Shoji T, Asai Y, Kawagishi I, Homma M

JOURNAL OF BACTERIOLOGY Vol. 182 ( 11 ) page： 3314 - 3318 2000.6

A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor.

Kojima, S., Shoji, T., Asai, Y., Kawagishi, I & Homma, M.

Journal of Bacteriology Vol. 182 page： 3314-3318 2000

Random mutagenesis of the pomA gene encoding a putative channel component of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. Reviewed International journal Open Access

Seiji Kojima, Mitsuyo Kuroda, Ikuro Kawagishi, Michio Homma

Microbiology (Reading, England) Vol. 145 page： 1759 - 1767 1999.7

Language：English

PomA and PomB are integral membrane proteins and are essential for the rotation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H(+)-driven motor, they are thought to form the Na(+)-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.

DOI： 10.1099/13500872-145-7-1759

Web of Science

PubMed

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force Reviewed Open Access

S Kojima, K Yamamoto, Kawagishi, I, M Homma

JOURNAL OF BACTERIOLOGY Vol. 181 ( 6 ) page： 1927 - 1930 1999.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated, We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.

DOI： 10.1128/JB.181.6.1927-1930.1999

Web of Science

Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components Reviewed

S Kojima, Y Asai, T Atsumi, Kawagishi, I, M Homma

JOURNAL OF MOLECULAR BIOLOGY Vol. 285 ( 4 ) page： 1537 - 1547 1999.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS LTD

The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpa(r) ((m) under bar otility (r) under bar esistant to (p) under bar hen (a) under bar mil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpa(r) strains, NMB205 and NM8201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Go-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpa(r) phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex.(C) 1999 Academic Press.

DOI： 10.1006/jmbi.1998.2377

Web of Science

PubMed

CiNii Research

*Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations of putative channel components. Reviewed

Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I. & Homma, M.

Journal of Molecular Biology Vol. 285 page： 1537-1547 1999

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force. Reviewed Open Access

Kojima, S., Yamamoto, K., Kawagishi, I. & Homma, M.

Journal of Bacteriology Vol. 181 page： 1927-1930 1999

Random mutagenesis of the pomA gene encoding a putative channel component of the Na+-driven polar flagellar motor of Vibrio alginolyticus. Reviewed

Kojima, S., Kuroda, M., Kawagishi, I. & Homma, M.

Microbiology Vol. 145 page： 1759-1767 1999

Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium Reviewed

Y Asai, S Kojima, H Kato, N Nishioka, Kawagishi, I, M Homma

JOURNAL OF BACTERIOLOGY Vol. 179 ( 16 ) page： 5104 - 5110 1997.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions, Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel, MotX Has proposed to be a component of a sodium channel, Were we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions, These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene. suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors mag have similar structure and function as proton driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.

Web of Science

Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium

Asai Y, Kojima S, Kato H, Nishioka N, Kawagishi I, Homma M

JOURNAL OF BACTERIOLOGY Vol. 179 ( 16 ) page： 5104 - 5110 1997.8

Characterization of polar-flagellar-length mutants in Vibrio alginolyticus Reviewed

M Furuno, T Atsumi, T Yamada, S Kojima, N Nishioka, Kawagishi, I, M Homma

MICROBIOLOGY-UK Vol. 143 ( 5 ) page： 1615 - 1621 1997.5

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof(+) Laf(-)), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5 +/- 0.9 mu m, 2.5 +/- 0.6 mu m and 11.2 +/- 3.6 mu m, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. in the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 mu m and 0.062 mu m, respectively, whereas those of the wild-type flagella were 1.5 mu m and 0.16 mu m. The relative amounts of the various molecular components of the long Pof were different from those of the wildtype or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.

DOI： 10.1099/00221287-143-5-1615

Web of Science

PubMed

Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor. Open Access

Kojima, S, Atsumi, T, Muramoto, K, Kudo, S, Kawagishi, I, Homma, M

Journal of Molecular Biology Vol. 265 ( 3 ) page： 310 - 318 1997.1