- Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control., Yoshitani K, Ishii E, Taniguchi K, Sugimoto H, Shiro Y, Akiyama Y, Kato A, Utsumi R, Eguchi Y, Bioscience, biotechnology, and biochemistry, Bioscience, biotechnology, and biochemistry, 83(4), 684 - 694, Apr. 2019 , Refereed
- Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases, Kinoshita-Kikuta E, Kinoshita E, Eguchi Y, Yanagihara S, Edahiro K, Inoue Y, Taniguchi M, Yoshida M, Yamamoto K, Takahashi H, Sawasaki T, Utsumi R, Koike T, PLoS One, PLoS One, 10(7), e0132598, 2015 , Refereed
- Angucycline antibiotic waldiomycin recognizes common structural motif conserved in bacterial histidine kinases, Yoko Eguchi, Toshihide Okajima, Naoya Tochio, Yoichi Inukai, Riko Shimizu, Shuhei Ueda, Shoko Shinya, Takanori Kigawa, Tamo Fukamizo, Masayuki Igarashi, Ryutaro Utsumi, JOURNAL OF ANTIBIOTICS, JOURNAL OF ANTIBIOTICS, 70(3), 251 - 258, Mar. 2017 , Refereed
Summary:Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, Walk, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coll. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.
- Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis, Akinori Kato, Shuhei Ueda, Taku Oshima, Yoichi Inukai, Toshihide Okajima, Masayuki Igarashi, Yoko Eguchi, Ryutaro Utsumi, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 63(4), 212 - 221, 2017 , Refereed
Summary:The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC(50)s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.
- Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii, Shunpei Miwa, Eri Kihira, Akinori Yoshioka, Kaoru Nakasone, Sho Okamoto, Masaki Hatano, Masayuki Igarashi, Yoko Eguchi, Akinori Kato, Natsuko Ichikawa, Mitsuo Sekine, Nobuyuki Fujita, Yu Kanesaki, Hirofumi Yoshikawa, Ryutaro Utsumi, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 198(11), 1604 - 1609, Jun. 2016 , Refereed
Summary:Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii. However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii.
IMPORTANCE
A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii. These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.
- Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE, Emiko Kinoshita-Kikuta, Eiji Kinoshita, Yoko Eguchi, Tohru Koike, PLOS ONE, PLOS ONE, 11(2), e0148294, Feb. 2016 , Refereed
Summary:Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the.-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.
- Study on in vivo effects of bacterial histidine kinase inhibitor, Waldiomycin, in Bacillus subtilis and Staphylococcus aureus, Md. Fakhruzzaman, Yoichi Inukai, Yohei Yanagida, Hirokazu Kino, Masayuki Igarashi, Yoko Eguchi, Ryutaro Utsumi, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 61(5), 177 - 184, 2015 , Refereed
Summary:Two-component signal transduction systems (TCSs) represent one of the primary means by which bacteria sense and respond to changes in their environment, both intra-and extracellular. The highly conserved WalK (histidine kinase)/WalR (response regulator) TCS is essential for cell wall metabolism of low G+C Gram-positive bacteria and acts as a master regulatory system in controlling and coordinating cell wall metabolism with cell division. Waldiomycin, a WalK inhibitor, has been discovered by screening metabolites from actinomycetes and belongs to the family of angucycline antibiotics. In the present study, we have shown that waldiomycin inhibited autophosphorylation of WalK histidine kinases in vitro from Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans at half-maximal inhibitory concentrations of 10.2, 8.8, 9.2, and 25.8 mu M, respectively. Quantitative RTPCR studies of WaIR regulon genes have suggested that waldiomycin repressed the WalK/WaIR system in B. subtilis and S. aureus cells. Morphology of waldiomycin-treated S. aureus cells displayed increased aggregation instead of proper cellular dissemination. Furthermore, autolysis profiles of S. aureus cells revealed that waldiomycin-treated cells were highly resistant to Triton X-100- and lysostaphin-induced lysis. These phenotypes are consistent with those of cells starved for the WalK/WaIR system, indicating that waldiomycin inhibited the autophosphorylation activity of WalK in cells. We have also confirmed that waldiomycin inhibits WalK autophosphorylation in vivo by actually observing the phosphorylated WalK ratio in cells using Phos-tag SDS-PAGE. The results of our current study strongly suggest that waldiomycin targets WalK histidine kinases and inhibits the WaIR regulon genes expression, thereby affecting both cell wall metabolism and cell division.
- Alkali Metals in Addition to Acidic pH Activate the EvgS Histidine Kinase Sensor in Escherichia coli, Yoko Eguchi, Ryutaro Utsumi, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 196(17), 3140 - 3149, Sep. 2014 , Refereed
Summary:Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na+, K+) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception.
- Mechanism of Activation of PhoQ/PhoP Two-Component Signal Transduction by SafA, an Auxiliary Protein of PhoQ Histidine Kinase in Escherichia coli, Eiji Ishii, Yoko Eguchi, Ryutaro Utsumi, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77(4), 814 - 819, Apr. 2013 , Refereed
Summary:The PhoQ/PhoP two-component signal transduction system in Escherichia coli is activated by SafA, a small membrane protein that modifies the PhoQ histidine kinase. The SafA C-terminal domain (41-65 aa) interacts directly with the sensory domain of PhoQ at the periplasm. We used in vitro and in vivo strategies to elucidate the way SafA modifies the PhoQ/PhoP phosphorelay system. First, the enzymatic activities of membranes from cells overexpressing PhoQ and cells expressing both PhoQ and SafA were compared in vitro. Increased autophosphorylation of PhoQ was observed in the presence of SafA, but it did not increase the dephosphorylation of phospho-PhoP by PhoQ. In addition, SafA increased the phospho-PhoP level on the phosphotransfer assay. We confirmed that induction of SafA results in an accumulation of phospho-PhoP in vivo by the Phos-tag system. Our results suggest that the accumulation of phospho-PhoP is linked to activation of PhoQ autophosphorylation by SafA.
- Isolation and Characterization of Signermycin B, an Antibiotic That Targets the Dimerization Domain of Histidine Kinase WalK, Takafumi Watanabe, Masayuki Igarashi, Toshihide Okajima, Eiji Ishii, Hirokazu Kino, Masaki Hatano, Ryuichi Sawa, Maya Umekita, Tomoyuki Kimura, Sho Okamoto, Yoko Eguchi, Yuzuru Akamatsu, Ryutaro Utsumi, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 56(7), 3657 - 3663, Jul. 2012 , Refereed
Summary:The WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000 Streptomyces extracts. The chemical structure (C23H35NO4; molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 mu g/ml (8 mu M) to 6.25 mu g/ml (16 mu M) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 mu M. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains of Bacillus subtilis and competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. In Bacillus subtilis and Staphylococcus aureus, signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system.
- The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli, Yoko Eguchi, Eiji Ishii, Masatake Yamane, Ryutaro Utsumi, MOLECULAR MICROBIOLOGY, MOLECULAR MICROBIOLOGY, 85(2), 299 - 313, Jul. 2012 , Refereed
Summary:Sensor histidine kinases of two-component signal transduction systems (TCSs) respond to various environmental signals and transduce the external stimuli across the cell membrane to their cognate response regulators. Recently, membrane proteins that modulate sensory systems have been discovered. Among such proteins is SafA, which activates the PhoQ/PhoP TCS by direct interaction with the sensor PhoQ. SafA is directly induced by the EvgS/EvgA TCS, thus connecting the two TCSs, EvgS/EvgA and PhoQ/PhoP. We investigated how SafA interacted with PhoQ. Bacterial two-hybrid and reporter assays revealed that the C-terminal region (4165 aa) of SafA activated PhoQ at the periplasm. Adding synthetic SafA(4165) peptide to the cell culture also activated PhoQ/PhoP. Furthermore, direct interaction between SafA(4165) and the sensor domain of PhoQ was observed by means of surface plasmon resonance. NMR spectroscopy of 15N-labelled PhoQ sensor domain confirmed that SafA and Mg2+ provoked a different conformational change of PhoQ. Site-directed mutagenesis studies revealed that R53, within SafA(4165), was important for the activation of PhoQ, and D179 of the PhoQ sensor domain was required for its activation by SafA. SafA activated PhoQ by a different mechanism from cationic antimicrobial peptides and acidic pH, and independent of divalent cations and MgrB.
- Development of an Antivirulence Drug against Streptococcus mutans: Repression of Biofilm Formation, Acid Tolerance, and Competence by a Histidine Kinase Inhibitor, Walkmycin C, Yoko Eguchi, Norihiro Kubo, Hiroko Matsunaga, Masayuki Igarashi, Ryutaro Utsumi, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 55(4), 1475 - 1484, Apr. 2011 , Refereed
Summary:Two-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential of Streptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors of S. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK in Bacillus subtilis, inhibited the in vitro autophosphorylation activity of three purified S. mutans HKs, i.e., VicK, CiaH, and LiaS. Although S. mutans does not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 mu g/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes of S. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs.
- Regulation of Acid Resistance by Connectors of Two-Component Signal Transduction Systems in Escherichia coli, Yoko Eguchi, Eiji Ishii, Kensuke Hata, Ryutaro Utsumi, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 193(5), 1222 - 1228, Mar. 2011 , Refereed
Summary:Two-component signal transduction systems (TCSs), utilized extensively by bacteria and archaea, are involved in the rapid adaptation of the organisms to fluctuating environments. A typical TCS transduces the signal by a phosphorelay between the sensor histidine kinase and its cognate response regulator. Recently, small-sized proteins that link TCSs have been reported and are called "connectors." Their physiological roles, however, have remained elusive. SafA (sensor associating factor A) (formerly B1500), a small (65-amino-acid [65-aa]) membrane protein, is among such connectors and links Escherichia coli TCSs EvgS/EvgA and PhoQ/PhoP. Since the activation of the EvgS/EvgA system induces acid resistance, we examined whether the SafA-activated PhoQ/PhoP system is also involved in the acid resistance induced by EvgS/EvgA. Using a constitutively active evgS1 mutant for the activation of EvgS/EvgA, we found that SafA, PhoQ, and PhoP all contributed to the acid resistance phenotype. Moreover, EvgS/EvgA activation resulted in the accumulation of cellular RpoS in the exponential-phase cells in a SafA-, PhoQ-, and PhoP-dependent manner. This RpoS accumulation was caused by another connector, IraM, expression of which was induced by the activation of the PhoQ/PhoP system, thus preventing RpoS degradation by trapping response regulator RssB. Acid resistance assays demonstrated that IraM also participated in the EvgS/EvgA-induced acid resistance. Therefore, we propose a model of a signal transduction cascade proceeding from EvgS/EvgA to PhoQ/PhoP and then to RssB (connected by SafA and IraM) and discuss its contribution to the acid resistance phenotype.
- Two-component signal transduction as potential drug targets in pathogenic bacteria, Yasuhiro Gotoh, Yoko Eguchi, Takafumi Watanabe, Sho Okamoto, Akihiro Doi, Ryutaro Utsumi, CURRENT OPINION IN MICROBIOLOGY, CURRENT OPINION IN MICROBIOLOGY, 13(2), 232 - 239, Apr. 2010 , Refereed
Summary:Gene clusters contributing to processes such as cell growth and pathogenicity are often controlled by two-component signal transduction systems (TCSs). Specific inhibitors against TCS systems work differently from conventional antibiotics, and developing them into new drugs that are effective against various drug-resistant bacteria may be possible. Furthermore, inhibitors of TCSs that control virulence factors may reduce virulence without killing the pathogenic bacteria. Previous TCS inhibitors targeting the kinase domain of the histidine kinase sensor suffered from poor selectivity. Recent TCS inhibitors, however, target the sensory domains of the sensors blocking the quorum sensing system, or target the essential response regulator. These new targets are introduced, together with several specific TCSs that have the potential to serve as effective drug targets.
- Molecular Mechanism of Transcriptional Cascade Initiated by the EvgS/EvgA System in Escherichia coli K-12, Junji Itou, Yoko Eguchi, Ryutaro Utsumi, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(4), 870 - 878, Apr. 2009 , Refereed
Summary:Using an EvgS-active mutant (evgSI) in combination with gene deletions, we clarified the molecular mechanism of the transcriptional cascade of acid resistance and multidrug resistance genes initiated by the EvgS/EvgA two-component system in Escherichia coli, followed by sequential induction of the transcriptional regulators, YdeO and GadE. Overexpression of EvgA, the response regulator of the EvgS/EvgA system, is known to induce the expression of a number of acid resistance and multidrug resistance genes, in which the EvgA-YdeO-GadE circuit is involved, but the role of the sensor EvgS in this circuit has remained unsolved. Our results suggest that the transcriptional cascade initiated by the EvgS/EvgA system in fact functions for acid and drug resistance in E. coli.
- Role of a serine residue (S278) in the pore-facing region of the housefly L-glutamate-gated chloride channel in determining sensitivity to noncompetitive antagonists, K. Hirata, C. Ishida, Y. Eguchi, K. Sakai, F. Ozoe, Y. Ozoe, K. Matsuda, INSECT MOLECULAR BIOLOGY, INSECT MOLECULAR BIOLOGY, 17(4), 341 - 350, Aug. 2008 , Refereed
Summary:gamma-Hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin are noncompetitive antagonists (NCAs) of L-glutamate-gated chloride channels (GluCls), yet their potencies are weaker than those on gamma-aminobutyric acid receptors (GABARs). The A302S mutation of Drosophila RDL (resistant to dieldrin) GABAR confers NCA resistance, and housefly GluCls (MdGluCls) possess S278 as the residue corresponding to the A302. Thus, the effects of S278A mutation on the NCA actions on MdGluCls were investigated. The S278A mutation resulted in enhanced blocking by NCAs of the MdGluCl response to 30 mu M L-glutamate. However, such actions of gamma-HCH and picrotoxinin, but not of fipronil, on the S278A mutant were reduced with 200 mu M L-glutamate. Further increases in the L-glutamate concentration led to potentiation by NCAs of the mutant response to L-glutamate.
- B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli, Yoko Eguchi, Junji Itou, Masatake Yamane, Ryo Demizu, Fumiyuki Yamato, Ario Okada, Hirotada Mori, Akinori Kato, Ryutaro Utsumi, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(47), 18712 - 18717, Nov. 2007 , Refereed
Summary:Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/ EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.
- Transcriptional regulation of the Na+-NADMquinone oxidoreductase gene, nqr, in Vibrio anguillarum, a fish pathogen, in the stationary phase, Erina Fujiwara-Nagata, Yoko Eguchi, Ryutaro Utsumi, Mitsuru Eguchi, FISHERIES SCIENCE, FISHERIES SCIENCE, 73(2), 348 - 355, Apr. 2007 , Refereed
Summary:Vibrio anguillarum kills various kinds of fish over a range of salinities from sea water to fresh water, and causes serious damage to aquaculture systems. In this study, the transcriptional regulation of the Na+-NADH:quinone oxidoreductase (Na+-NQR) operon in V. anguillarum from the logarithmic to stationary phases was investigated. Cloning of the Na+-NQR operon revealed a 7 kb nucleotide sequence composed of six open reading frames with amino acid sequence identity of more than 80% with other Vibrio species. Two promoters, nqrP1 and nqrP2, were identified in the region upstream of the nqrA gene using an S1 nuclease assay. The nqrP1 promoter was constitutively activated throughout the logarithmic to stationary phases and possessed -10 (5'-TAGACT-3') and -35 (5'-ATGGCA-3') sequences, which were similar to the consensus sequence of Escherichia coli. On the other hand, the nqrP2 promoter was activated only at the stationary phase and had only a -10 (5'-CATACT-3') and not a -35 sequence. These results suggest that nqrP2, which works specifically in the stationary phase, contributes to starvation-survival in V. anguillarum.
- Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes, Y. Eguchi, M. Ihara, E. Ochi, Y. Shibata, K. Matsuda, S. Fushiki, H. Sugama, Y. Hamasaki, H. Niwa, M. Wada, F. Ozoe, Y. Ozoe, INSECT MOLECULAR BIOLOGY, INSECT MOLECULAR BIOLOGY, 15(6), 773 - 783, Dec. 2006 , Refereed
Summary:Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 mu M and the MdRdl homo-oligomers with an EC(50) value of 101 mu M, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.
- A novel mechanism for connecting bacterial two-component signal-transduction systems, Y Eguchi, R Utsumi, TRENDS IN BIOCHEMICAL SCIENCES, TRENDS IN BIOCHEMICAL SCIENCES, 30(2), 70 - 72, Feb. 2005 , Refereed
Summary:Bacteria have many two-component signal-transduction systems (TCSs) that respond to specific environmental signals by altering the phosphorylated state of a response regulator. Although these systems are presumed to form an intricate signal network, the detailed mechanism of how they interact with each other remains largely unexplained. In a recent study of Salmonella, two TCSs have been discovered to be connected by a protein that protects a response regulator from dephosphorylation promoted by its cognate sensor kinase. This novel mechanism might provide an answer to some of,the linkages found between other TCSs.
- Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems of Escherichia coli, Y Eguchi, T Okada, S Minagawa, T Oshima, H Mori, K Yamamoto, A Ishihama, R Utsumi, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 186(10), 3006 - 3014, May 2004 , Refereed
Summary:Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB. This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes. These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E. coli and also identified the genes that required the two systems for enhanced expression. This is one example of the intricate signal transduction networks that are posited to exist in E. coli.
- Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli, Y Eguchi, T Oshima, H Mori, R Aono, K Yamamoto, A Ishihama, R Utsumi, MICROBIOLOGY-SGM, MICROBIOLOGY-SGM, 149, 2819 - 2828, Oct. 2003 , Refereed
Summary:A constitutively active mutant of histidine kinase sensor EvgS was found to confer multi-drug resistance (MDR) to an acrA-deficient Escherichia coli, indicating the relationship between the two-component system EvgAS and the expression of the MDR system. The observed MDR also depended on an outer-membrane channel, TolC. Microarray and S1 mapping assays indicated that, in the presence of this constitutive mutant EvgS, the level of transcription increased for some MDR genes, including the drug efflux genes emrKY, yhiUV, acrAB, mdfA and tolC. Transcription in vitro of emrK increased by the addition of phosphorylated EvgA. Transcription activation of tolC by the activated EvgS was, however, dependent on both EvgAS and PhoPQ (Mg2+-responsive two-component system), in agreement with the presence of the binding site (PhoP box) for the regulator PhoP in the tolC promoter region. Transcription in vitro of yhiUV also appears to require an as-yet-unidentified additional transcriptional factor besides EvgA. Taken together we propose that the expression of the MDR system is under a complex regulatory network, including the phosphorylated EvgA serving as the master regulator.
- Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli, S Minagawa, H Ogasawara, A Kato, K Yamamoto, Y Eguchi, T Oshima, H Mori, A Ishihama, R Utsumi, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 185(13), 3696 - 3702, Jul. 2003 , Refereed
Summary:Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg2+ stimulon that respond to the availability of external Mg2+ in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 MM MgCl2, WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl2. The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, SI nuclease assays of 26 promoters were performed to verify six new Mg2+ stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg2+ stimulon in E. coli.
- PROTECTIVE ACTIVITY OF ANTIEXOTOXIN A MONOCLONAL-ANTIBODY AGAINST MICE INFECTED WITH TOXIN-PRODUCING PSEUDOMONAS-AERUGINOSA, T KOHZUKI, Y EGUCHI, M KATO, K IRIE, H OHTSUKA, A HIGUCHI, H NOGUCHI, JOURNAL OF INFECTIOUS DISEASES, JOURNAL OF INFECTIOUS DISEASES, 167(1), 119 - 125, Jan. 1993 , Refereed
Summary:The neutralizing and protective effect of murine monoclonal antibody (MAb) 3C7 (IgG1) against Pseudomonas aeruginosa exotoxin A was examined in an experimental mouse model of infection with exotoxin A-producing strains. Treatment with MAb 3C7 blocked the reduction of functional elongation factor 2 (EF-2) in the liver of mice but could not clear the bacteria. Administration of gentamicin caused bacteria to be cleared but did not block reduction of hepatic EF-2 level. Treatment with either MAb 3C7 or gentamicin individually did not prolong time to death, however, the combined therapy with both MAb 3C7 and gentamicin cleared bacteria and blocked the reduction of hepatic EF-2 level, resulting in a significant increase in the survival rate of mice. These results suggest that anti-exotoxin A MAbs show effectiveness against pseudomonal infection caused by exotoxin A-producing strains.
