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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

DOI： 10.3389/fmicb.2021.645965

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Gram-negative bacteria such as Escherichia coli are surrounded by inner and outer membranes and peptidoglycan in between, protecting the cells from turgor pressure and maintaining cell shape. The Rod complex, which synthesizes peptidoglycan, is composed of various proteins such as a cytoplasmic protein MreB, a transmembrane protein RodZ, and a transpeptidase PBP2. The Rod complex is a highly motile complex that rotates around the long axis of a cell. Previously, we had reported that anionic phospholipids (aPLs; phosphatidylglycerol and cardiolipin) play a role in the localization of MreB. In this study, we identified that cells lacking aPLs slow down Rod complex movement. We also found that at higher temperatures, the speed of movement increased in cells lacking aPLs, suggesting that membrane fluidity is important for movement. Consistent with this idea, Rod complex motion was reduced, and complex formation was disturbed in the cells depleted of FabA or FabB, which are essential for unsaturated fatty acid synthesis. These cells also showed abnormal morphology. Therefore, membrane fluidity is important for maintaining cell shape through the regulation of Rod complex formation and motility.

DOI： 10.3389/fmolb.2020.582660

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

© 2019 the Author(s), licensee AIMS Press. RodZ is required for determination of cell shape in rod-shaped bacterium, such as Escherichia coli. RodZ is a transmembrane protein and forms a supramolecular complex called the Rod complex with other proteins, such as MreB-actin and peptidoglycan synthesis enzymes (for e.g., PBP2). Deletion of the rodZ gene changes the cell shape from rod to round or ovoid. Another supramolecular complex called divisome that controls cell division mainly consists of FtsZ-tubulin. MreB directly interacts with FtsZ and this interaction is critical to trigger a transition from cell elongation to cell division. Recently, we found that RodZ also directly interacts with FtsZ, and RodZ recruits MreB to the divisome. Formation of the division ring, called Z ring, is delayed if RodZ does not interact with FtsZ, indicating that RodZ might facilitate the formation of the Z ring during the cell division process. In this mini-review, we have summarized the roles of RodZ in cell elongation and cell division, especially based on our recent study.

DOI： 10.3934/microbiol.2019.4.358

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Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ-dependent manner. We also found that division-site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division-site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.

DOI： 10.1111/mmi.14217

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Bacterial cells, including Escherichia coli and Bacillus subtilis, continuously elongate and divide. Although the cell width is maintained during cell cycle, the molecular mechanisms involved in its regulation remain unknown. MreB has been implicated to play a role in maintaining cell width. Several point mutations in mreB that affect cell width have been identified. The MreB protein forms clusters or polymers in the cell and moves along annular tracks perpendicular to the long axis. This rotation is coupled with peptidoglycan synthesis. Here, we focused on two MreB mutants, MreBA125V and MreBA174T . Cells producing MreBA125V and MreBA174T were thinner and thicker than WT cells, and MreBA125V and MreBA174T rotated faster and slower than WT MreB, respectively. We observed that the rotation rate correlated with the cell wall synthesis rate. Thus, we conclude that the velocity of MreB rotation also affects cell width, that is, the faster the MreB rotates, the thinner the cell width is.

DOI： 10.1111/gtc.12667

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Language：Japanese  

DOI： 10.2142/biophys.59.100

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

Rod shape of bacterial cells such as Escherichia coli is mainly regulated by a supramolecular complex called elongasome including MreB actin. Deletion of the mreB gene in rod-shaped bacterium E. coli results in round-shaped cells. RodZ was isolated as a determinant of rod shape in E. coli, Caulobacter crescentus and Bacillus subtilis and it has been shown to be an interaction partner and a regulator of assembly of MreB through its cytoplasmic domain. As opposed to functions of the N-terminal cytoplasmic domain of RodZ, functions of the C-terminal periplasmic domain including a disordered region are still unclear. To understand it, we adopted an in vivo photo-cross-linking assay to analyze interaction partners to identify proteins which interact with RodZ via its periplasmic domain, finding that the RodZ self-interacts in the periplasmic disordered domain. Self-interaction of RodZ was affected by MreB actin. Deletion of this region resulted in aberrant cell shape. Our results suggest that MreB binding to the cytoplasmic domain of RodZ causes structural changes in the disordered periplasmic domain of RodZ. We also found that the disordered domain of RodZ contributes to fine-tune rod shape in E. coli.

DOI： 10.1111/gtc.12572

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NLM (Medline)  

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. T he error has not been fixed in the paper.

DOI： 10.1038/s41598-018-23160-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Plant chloroplasts originate from the symbiotic relationship between ancient free-living cyanobacteria and ancestral eukaryotic cells. Since the discovery of the bacterial derivative FtsZ gene-which encodes a tubulin homolog responsible for the formation of the chloroplast inner division ring (Z ring)-in the Arabidopsis genome in 1995, many components of the chloroplast division machinery were successively identified. The knowledge of these components continues to expand
however, the mode of action of the chloroplast dividing system remains unknown (compared to bacterial cell division), owing to the complexities faced in in planta analyses. To date, yeast and bacterial heterologous expression systems have been developed for the reconstitution of Z ring-like structures formed by chloroplast FtsZ. In this review, we especially focus on recent progress of our bacterial system using the model bacterium Escherichia coli to dissect and understand the chloroplast division machinery-an evolutionary hybrid structure composed of both bacterial (inner) and host-derived (outer) components.

DOI： 10.3390/ijms19020544

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Authorship：Corresponding author   Language：English   Publisher：SPRINGER  

DOI： 10.1007/s00294-017-0701-z

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Plant chloroplasts proliferate through binary fission, and the stromal-side molecules that are involved in chloroplast division are bacterial derivatives. As in bacteria, the prokaryotic tubulin homolog FtsZ assembles into a ring-like structure (Z ring) at mid-chloroplast, and this process is followed by constriction. However, the properties of chloroplast FtsZs remain unclarified. Here, we employed Escherichia coli as a novel heterologous system for expressing chloroplast FtsZs and their regulatory components. Fluorescently labelled Arabidopsis FtsZ2 efficiently assembled into long filaments in E. coli cells, and artificial membrane tethering conferred FtsZ2 filaments with the ability to form Z ring-like structures resembling the bacterial Z ring. A negative regulator of chloroplast FtsZ assembly, ARC3, retained its inhibitory effects on FtsZ2 filamentation and Z ring-like structure formation in E. coli cells. Thus, we provide a novel heterologous system by using bacterial cells to study the regulation of the chloroplast divisome. Furthermore, we demonstrated that the FtsZ2-interacting protein ARC6, which is a potential candidate for Z ring tethering to the chloroplast inner envelope membrane, genuinely targeted FtsZ2 to the membrane components and supported its morphological shift from linear filaments to Z ring-like structures in a manner dependent on the C-terminal ARC6-interacting domain of FtsZ2.

DOI： 10.1038/s41598-017-03698-6

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod-shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re-assemble, and MreB-free zones were subsequently observed in the cytoplasmic membrane. These MreB-free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y-shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.

DOI： 10.1111/mmi.13639

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features.
Results: Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics.
Conclusions: Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

DOI： 10.1186/s12915-017-0348-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Bacterial cells show various shapes such as round, rod and spiral. Maintenance of cell shape is vital to various cellular events including cell elongation, division and host infection. A model bacterium Escherichia coli shows rod-shape. Rod-shape consists of central cylinder and polar caps. To make rod shape, E. coli has to elongate to a constant direction and divide at mid-cell. In other words, E. coli has a polarity. Bacterial actin MreB localizes to the cylinder, forms clusters (Fig. 1) and plays an important role to regulate the polarity
cells lacking mreB become round shape because the cells lost the polarity. However, mechanism that MreB regulates the polarity remains unknown. We treated WT cells by A22, an antibiotic that inhibits assembly of MreB. MreB which localizes in the cylinder was diffused in the cytoplasmic membrane and the cytoplasm and the cells became round shape as previously shown (Fig. 1). To examine a role of MreB in regulating polarity, we observed a process of restoration of cell shape from round to rod by removing A22. MreB formed clusters and localized to the cytoplasmic membrane asymmetrically. The MreB-free zones finally became the cell poles in rod shape. We conclude that asymmetric localization of MreB would be required to make the cell polarity.

DOI： 10.1093/jmicro/dfw044

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.3412/jsb.69.557

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

RodZ is important for maintaining the rod shape of Escherichia coli. Loss of RodZ causes conversion of the rod shape to a round shape and a growth rate slower than that of wild-type cells. Suppressor mutations that simultaneously restore both the growth rates and the rod shape were isolated. Most of the suppressor mutations are found in mreB, mrdA, or mrdB. One of the mutations was in the promoter region of zipA, which encodes a crucial component of the cell division machinery. In this study, we investigated the mechanism of the suppression by this mutation. ZipA was slightly but significantly increased in the suppressor cells and led to a delay in cell division. While round-shaped mreB and mrdA mutants lose cell bipolarity, we found that round-shaped rodZ mutants retained cell bipolarity. Therefore, we concluded that a delay in the completion of septation provides extra time to elongate the cell laterally so that the zipA suppressor mutant is able to recover its ovoid or rod shape. The suppression by zipA demonstrates that the regulation of timing of septation potentially contributes to the conversion of morphology in bacterial cells.

DOI： 10.1002/mbo3.116

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to rodZ cells, possibly by changing the requirements of MreB in the process.

DOI： 10.1111/mmi.12148

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Cells lacking rodZ are defective not only in cell shape, but also in cell growth at low temperatures. Cold-sensitive growth was suppressed by a mutation of ispA without recovery from the round shape, and the mutation improved cell growth of the wild-type at low temperatures.

DOI： 10.1111/j.1348-0421.2011.00391.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Bacteriophage P1 has a contractile tail that targets the conserved lipopolysaccharide on the outer membrane surface of the host for initial adsorption. The mechanism by which P1 DNA enters the host cell is not well understood, mainly because the transient molecular interactions between bacteriophage and bacteria have been difficult to study by conventional approaches. Here, we engineered tiny E. coli host cells so that the initial stages of P1-host interactions could be captured in unprecedented detail by cryo-electron tomography. Analysis of three-dimensional reconstructions of frozen-hydrated specimens revealed three predominant configurations: an extended tail stage with DNA present in the phage head, a contracted tail stage with DNA, and a contracted tail stage without DNA. Comparative analysis of various conformations indicated that there is uniform penetration of the inner tail tube into the E. coli periplasm and a significant movement of the baseplate away from the outer membrane during tail contraction. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2011.06.005

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

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.

DOI： 10.1128/JB.01245-10

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Language：Japanese   Publisher：日本微生物生態学会  

DOI： 10.20709/jsmeja.24.2_51

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DOI： 10.4161/cib.2.3.7930

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The bacterium Escherichia coli is rod-shaped, and a unit cell keeps regular dimensions of about 1.5 μm long and 0.5 μm wide. The rod-shaped cell is composed of two parts: a cylinder in the center and caps at both ends. The length of the cylinder corresponds to the length of the rod cell. A recent paper reported the genetic regulation of the cell length by rodZ. RodZ is a membrane protein with bitopic topology that assembles underneath the cell membrane to form helical filaments along the lateral axis of the cell with the bacterial actin MreB. RodZ filaments probably interact with enzymes that contribute to peptidoglycan synthesis. Cells lacking rodZ shorten only along the lateral axis of the cell so that the cells become round-shaped instead of rod-shaped. Such spheroidal cells consist only of caps due to the loss of almost all of the cylinder. In addition, carbon metabolism is remarkably disturbed by the deficiency of RodZ. This suggests that the transport of nutrients at the surface of the cylinder is reduced in rodZ mutant cells. Thus, cell morphology is also critical for proper metabolism for cell proliferation. ©2009 Landes Biosciences.

DOI： 10.4161/cib.2.3.7930

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cell shape is critical for growth, and some genes are involved in bacterial cell morphogenesis. Here, we report a novel gene, rodZ, required for the determination of rod shape in Escherichia coli. Cells lacking rodZ no longer had rod shape but rather were round or oval. These round cells were smaller than known round mutant cells, including mreB and pbpA mutants; both are known to lose rod shape. Morphogenesis from rod cells to round cells and vice versa, caused by depletion and overproduction of RodZ, respectively, revealed that RodZ could regulate the length of the long axis of the cell. RodZ is a membrane protein with bitopic topology such that the N-terminal region including a helix-turn-helix motif is in the cytoplasm, whereas the C-terminal region is exposed in the periplasm. GFP-RodZ forms spirals along the lateral axis of the cell beneath the cell membrane, similar to the MreB bacterial actin. Thus, RodZ may mediate spatial information from cytoskeletal proteins in the cytoplasm to a peptidoglycan synthesis machinery in the periplasm.

DOI： 10.1038/emboj.2008.234

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The bacterial actin homologue FtsA has a conserved C-terminal membrane targeting sequence (MTS). Deletion or point mutations in the MTS, such as W408E, were shown previously to inactivate FtsA function and inhibit cell division. Because FtsA binds to the tubulin-like FtsZ protein that forms the Z ring, it is thought that the MTS of FtsA is required, along with the transmembrane protein ZipA, to assemble the Z ring and anchor it to the cytoplasmic membrane. Here, we show that despite its reduced membrane binding, FtsA-W408E could localize to the Z ring and recruit the late cell division protein FtsI, but was defective in self-interaction and recruitment of FtsN, another late cell division protein. These defects could be suppressed by a mutation that stimulates membrane association of FtsA-W408E, or by expressing a tandem FtsA-W408E. Remarkably, the FtsA MTS could be completely replaced with the transmembrane domain of MalF and remain functional for cell division. We propose that FtsA function in cell division depends on additive effects of membrane binding and self-interaction, and that the specific requirement of an amphipathic helix for tethering FtsA to the membrane can be bypassed.

DOI： 10.1111/j.1365-2958.2007.06085.x

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Language：Japanese   Publisher：日本生物物理学会  

The chemoreceptors of Escherichia coli cluster at a cell pole, a property which is critical for signaling. However, little is known about the mechanism of polar localization. Our recent study demonstrated that the aspartate chemoreceptor (Tar)-GFP fusion protein is inserted into lateral membrane regions and migrates to the pole. Unexpectedly, Tar-GFP was found to be arranged into a coil, which reflects a coil of the Sec protein translocation machinery. The Sec coil appeared distinct from the coil of MreB, an actin-like cytoskeletal protein. These findings shed new light on the spatial organ...

DOI： 10.2142/biophys.48.030

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

In bacteria, the actin-like FtsA protein interacts with the tubulin-like FtsZ protein, helping to assemble the cytokinetic Z ring, anchor it to the cytoplasmic membrane and recruit other essential divisome proteins. FtsA also interacts with itself, but it is not clear whether this self-interaction is required for its full functionality. Here we describe new dominant negative missense mutations in Escherichia coli ftsA that specifically inhibit FtsA homodimerization and simultaneously cause disruption of Z rings. The negative effects of one mutation, M71A, were suppressed by altering levels of certain division proteins or by additional mutations in ftsA that promote increased integrity of the Z ring. Remarkably, when FtsA, FtsA-M71A, and other mutants of FtsA that compromise self-interaction were connected in a tandem repeat, they were at least partially functional and suppressed defects of an ftsZ84(ts) mutation. This gain of function by FtsA tandems further suggested that FtsA monomers cause deleterious interactions with FtsZ and that increased dimerization or oligomerization of FtsA enhances its ability to promote Z-ring integrity. Therefore, we propose that FtsZ assembly is regulated by the extent of FtsA oligomerization.

DOI： 10.1111/j.1365-2958.2007.05998.x

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Language：English   Publisher：CELL PRESS  

Bacteria, like eukaryotic cells, regulate their size by coordinating cell growth and division, growing faster and becoming larger when nutrients are more plentiful. Weart et al. (2007) now identify an enzyme in a glucolipid pathway that inhibits assembly of the key cell division protein FtsZ, but only during high nutrient conditions. Delaying cell division during rapid growth allows bacterial cells to become larger.

DOI： 10.1016/j.cell.2007.07.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

FtsN is the last known essential protein component to be recruited to the Escherichia coli divisome, and has several special properties. Here we report the isolation of suppressor mutants of ftsA that allow viability in the absence of ftsN. Cells producing the FtsA suppressors exhibited a mild cell division deficiency in the absence of FtsN, and no obvious phenotype in its presence. Remarkably, these altered FtsA proteins also could partially suppress a deletion of ftsK or zipA, were less toxic than wild-type FtsA when in excess, and conferred resistance to excess MinC, indicating that they share some properties with the previously isolated FtsA* suppressor mutant, and bypass the need for ftsN by increasing the integrity of the Z ring. TolA, which normally requires FtsN for its recruitment to the divisome, localized proficiently in the suppressed ftsN null strain, strongly suggesting that FtsN does not recruit the Tol-Pal complex directly. Therefore, despite its classification as a core divisome component, FtsN has no unique essential function but instead promotes overall Z ring integrity. The results strongly suggest that FtsA is conformationally flexible, and this flexibility is a key modulator of divisome function at all stages.

DOI： 10.1111/j.1365-2958.2007.05738.x

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

Formation of the FtsZ ring (Z ring) in Escherichia coli is the first step in the assembly of the divisome, a protein machine required for cell division. Although the biochemical functions of most divisome proteins are unknown, several, including ZipA, FtsA and FtsK, have overlapping roles in ensuring that the Z ring assembles at the cytoplasmic membrane, and that it is active. As shown previously, a single amino acid change in FtsA, R286W, also called FtsA*, bypasses the requirement for either ZipA or FtsK in cell division. In this study, the properties of FtsA* were investigated further, with the eventual goal of understanding the molecular mechanism behind the bypass. Compared to wild-type FtsA, the presence of FtsA* resulted in a modest but significant decrease in the mean length of cells in the population, accelerated the reassembly of Z rings, and suppressed the cell-division block caused by excessively high levels of FtsZ. These effects were not mediated by Z-ring remodelling, because FtsA* did not alter the kinetics of FtsZ turnover within the Z ring, as measured by fluorescence recovery after photobleaching. FtsA* was also unable to permit normal cell division at below normal levels of FtsZ, or after thermoinactivation of ftsZ84(ts). However, turnover of FtsA* in the ring was somewhat faster than that of wild-type FtsA, and overexpressed FtsA* did not inhibit cell division as efficiently as wild-type FtsA. Finally, FtsA* interacted more strongly with FtsZ compared with FtsA in a yeast two-hybrid system. These results suggest that FtsA* interacts with FtsZ in a markedly different way compared with FtsA.

DOI： 10.1099/mic.0.2006/001834-0

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In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(112-231), the C terminus of full-length MinC, or the C terminus of MinC(122-131), perturbed MinC function, which may explain why cell division inhibition by MinC(122-231), was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

DOI： 10.1128/JB.00666-06

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar-GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar-GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar-GFP derivative with two cysteine residues in the periplasmic domain was cross-linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar-GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP-SecE and a translocation-defective MalE-GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin-like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli.

DOI： 10.1111/j.1365-2958.2006.05145.x

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S432_3

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

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

DOI： 10.1128/JB.187.22.7647-7654.2005

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S260_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S176_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S290_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Chemotactic adaptation to persisting stimulation involves reversible methylation of the chemoreceptors that form complexes with the histidine kinase CheA at a cell pole. The methyltransferase CheR targets to the C-terminal NWETF sequence of the chemoreceptor. In contrast, localization of the methylesterase CheB is largely unknown, although regulation of its activity via phosphorylation is central to adaptation. In this study, green fluorescent protein was fused to full-length CheB or its various parts: the N-terminal regulatory domain (N), the C-terminal catalytic domain (C) and the linker (L). The full-length and NL fusions and, to a lesser extent, the LC fusion localized to a pole. Deletion of the P2 domain from CheA abolished polar localization of the full-length and NL fusions, but did not affect that of the LC fusion. Pull-down assays demonstrated that the NL fragment, but not the LC fragment, binds to the P2 fragment of CheA. These results indicate that binding of the NL domain to the P2 domain targets CheB to the polar signalling complex. The LC fusion, like the chemoreceptor, partially localized in the absence of CheA, suggesting that the LC domain may interact with its substrate sites, either as part of the protein or as a proteolytic fragment.

DOI： 10.1111/j.1365-2958.2004.04176.x

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Language：English   Publisher：1  

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information.

DOI： 10.1186/1477-3155-2-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Many sensory systems involve multiple steps of signal amplification to produce a significant response. One such mechanism may be the clustering of transmembrane receptors. In bacterial chemotaxis, where a stoichiometric His-Asp phosphorelay from the kinase CheA to the response regulator CheY plays a central role, the chemoreceptors (methyl-accepting chemotaxis proteins) cluster together with CheA and the adaptor CheW, at a pole of a rod-shaped cell. This clustering led to a proposal that signal amplification occurs through an interaction between chemoreceptor homodimers. Here, by using in vivo disulfide crosslinking assays, we examined an interdimer interaction of the aspartate chemoreceptor (Tar). Two cysteine residues were introduced into Tar: one at the subunit interface and the other at the external surface of the dimer. Crosslinked dimers and higher oligomers (especially a deduced hexamer) were detected and their abundance depended on CheA and CheW. The ligand aspartate significantly reduced the amounts of higher oligomers but did not affect the polar localization of Tar-GFP. Thus, the binding of aspartate alters the rate of collisions between Tar dimers in assembled signaling complexes, most likely due to a change in the relative positions or trajectories of the dimers. These collisions could occur within a trimer-of dimers predicted by crystallography, or between such trimers. These results are consistent with the proposal that the interaction of chemoreceptor dimers is involved in signal transduction.

DOI： 10.1073/pnas.0306660101

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S243_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S145_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S145_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S145_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S243_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S106_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S180_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S153_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S153_3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Adaptation to persisting stimulation is required for highly sensitive detection of temporal changes of stimuli, and often involves covalent modification of receptors. Therefore, it is of vital importance to understand how a receptor and its cognate modifying enzyme(s) modulate each other through specific protein-protein interactions. In the chemotaxis of Escherichia coli, adaptation requires methylation of chemoreceptors (e.g. Tar) catalyzed by the CheR methyltransferase. CheR binds to the C-terminal NWETF sequence of a chemoreceptor that is distinct from the methylation sites. However, little is known about how CheR recognizes its methylation sites or how it is distributed in a cell. In this study, we used comparative genomics to demonstrate that the CheR chemotaxis methyltransferase contains three structurally and functionally distinct modules: (i) the catalytic domain common to a methyltransferase superfamily; (ii) the N-terminal domain; and (iii) the beta-subdomain of the catalytic domain, both of which are found exclusively in chemotaxis methyltransferases. The only evolutionary conserved motif specific to CheR is the positively charged face of helix alpha2 in the N-terminal domain. The disulfide cross-linking analysis suggested that this face interacts with the methylation helix of Tar. We also demonstrated that CheR localizes to receptor clusters at cell poles via interaction of the beta-subdomain with the NWETF sequence. Thus, the two chemotaxis-specific modules of CheR interact with distinct regions of the chemoreceptor for targeting to the receptor cluster and for recognition of the substrate sites, respectively.

DOI： 10.1074/jbc.M202001200

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

In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheA-activating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class 11 suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S19_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S19_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S20_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S20_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S138_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S137_4

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

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.

DOI： 10.1046/j.1365-2958.2000.01834.x

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S89_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S53_3

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