Biomedical subjects

I Kawagishi

Publications and source records attributed to I Kawagishi.

At least 19 recordsLinked to original sources

PubMed2000

Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella.

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type VIBRIO: alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in VIBRIO: cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C-terminal PomB. MomB proteins, with the entire transmembrane region derived from the H(+)-type MotB, gave rise to an Na(+) motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na(+) motors with PomA, but were changed for Na(+) or Li(+) specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na(+)- and H(+)-type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.

Amino Acid Sequence↗

PubMed2000

The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction.

In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively. CheR binds to major chemoreceptors through their C-terminal motif NWETF, which is distinct from the methylation sites. In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar. Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553. Substitution of residues N549, E551 and T552 had only a slight or little effect. The defects of the W550A and F553A mutations were suppressed by high- and low-level overproduction of CheR respectively. Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain. In an in vitro assay, CheR bound to the wild-type version but not to the mutant versions. These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR-NWETF complex. On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co-crystal.

Aspartic Acid↗

PubMed2000

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio.

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.

Amino Acid Sequence↗

PubMed2000

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

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.

Amino Acid Sequence↗

PubMed2000

Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus.

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 proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment 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 mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. 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 loop(3-4). None of the loop(1-2) residues were labeled, suggesting that 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.

Amino Acid Sequence↗

PubMed2000

Mutational analysis of ligand recognition by tcp, the citrate chemoreceptor of Salmonella enterica serovar typhimurium.

The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterized chemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably other unidentified residues) are involved in the recognition of citrate.

Amino Acid Sequence↗

PubMed1999

Inversion of thermosensing property of the bacterial receptor Tar by mutations in the second transmembrane region.

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.

Amino Acid Sequence↗

PubMed1999

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

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 Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpar strains, NMB205 and NMB201, 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. Co-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpar 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.

Amiloride↗

PubMed1999

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant.

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.

Aspartic Acid↗

PubMed1999

Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus.

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

Bacterial Proteins↗

PubMed1999

Functional interaction between PomA and PomB, the Na(+)-driven flagellar motor components of Vibrio alginolyticus.

Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to form a sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB. As a result, we could demonstrate that PomA and PomB functionally interact with each other.

Antibodies, Bacterial↗

PubMed1999

Hybrid motor with H(+)- and Na(+)-driven components can rotate Vibrio polar flagella by using sodium ions.

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.

Amiloride↗

PubMed1999

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

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.

Flagella↗

PubMed1998

Intersubunit interaction between transmembrane helices of the bacterial aspartate chemoreceptor homodimer.

The transmembrane domain that connects the extracellular and intracellular domains of cell-surface receptors must play a critical role in signal transduction. Here, we report studies of the interaction between the transmembrane helices (TM1 and TM2) of the Escherichia coli aspartate chemoreceptor (Tar). Tar exists as a homodimer regardless of its state of ligand occupancy. A particular residue substitution in TM1 (A19K) abolishes the signaling ability of Tar. This signaling defect can be suppressed by single residue substitutions in TM2 (W192R, A198E, V201E, and V202L). We have found that these suppressors can be divided into two groups. A198E and V201E (class 1) almost completely suppress the defects caused by A19K, and this suppression occurs between two subunits of the Tar dimer. In contrast, W192R and V202L (class 2) fail to suppress some signaling defects, and their suppression does not occur between subunits. Because disulfide-crosslinking studies predict that residues 198 and 201 point toward residue 19 of the partner subunit, we propose that the class 1 suppressors form an intersubunit salt bridge with Lys-19. Indeed, A19K was suppressed by several additional aspartate or glutamate substitutions on the same face of TM2 occupied by residues 198 and 201. None of these intersubunit salt bridges perturb signaling function, suggesting that the mechanism of transmembrane signal propagation does not involve large displacements (such as extensive rotation) of the TM1 and TM2 helices relative to each other.

Amino Acid Substitution↗

PubMed1998

Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament.

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.

Cytoplasmic Granules↗

PubMed1998

Chemotactic adaptation is altered by changes in the carboxy-terminal sequence conserved among the major methyl-accepting chemoreceptors.

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.

Adaptation, Physiological↗

PubMed1997

Uncoupling of ligand-binding affinity of the bacterial serine chemoreceptor from methylation- and temperature-modulated signaling states.

The Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serine-sensing ability of Tsr-T156C, which has a unique cysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that L-serine protects the receptor from modification. In this study, we demonstrated the correlation between protective effects of various attractants and their potencies to elicit attractant responses. This indirect binding assay was used to monitor the affinity of the receptor for L-serine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are unaffected by changes in the methylation state of the receptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylation levels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that the ligand-binding affinity was unaffected by temperature. These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligand-binding assay system should be applicable to other receptors.

Amides↗

PubMed1997

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

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.

Amiloride↗