25 results on '"Aiba H"'
Search Results
2. A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli.
- Author
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Nagasawa, S., Tokishita, S., Aiba, H., and Mizuno, T.
- Subjects
ESCHERICHIA coli ,CELLULAR signal transduction ,PROTEINS ,CELL membranes ,GENETIC regulation - Abstract
Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gone located in this fragment is expressed on a high copy-number plasmid, Nucleotide sequencing revealed that the putative gene encodes a protein of 102452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP. [ABSTRACT FROM AUTHOR]
- Published
- 1992
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3. ecl family genes: Factors linking starvation and lifespan extension in Schizosaccharomyces pombe.
- Author
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Ohtsuka H, Otsubo Y, Shimasaki T, Yamashita A, and Aiba H
- Subjects
- Longevity genetics, Mechanistic Target of Rapamycin Complex 1 metabolism, Cell Cycle, Gene Expression Regulation, Fungal genetics, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins genetics, Schizosaccharomyces pombe Proteins metabolism
- Abstract
In the fission yeast Schizosaccharomyces pombe, the duration of survival in the stationary phase, termed the chronological lifespan (CLS), is affected by various environmental factors and the corresponding gene activities. The ecl family genes were identified in the genomic region encoding non-coding RNA as positive regulators of CLS in S. pombe, and subsequently shown to encode relatively short proteins. Several studies revealed that ecl family genes respond to various nutritional starvation conditions via different mechanisms, and they are additionally involved in stress resistance, autophagy, sexual differentiation, and cell cycle control. Recent studies reported that Ecl family proteins strongly suppress target of rapamycin complex 1, which is a conserved eukaryotic nutrient-sensing kinase complex that also regulates longevity in a variety of organisms. In this review, we introduce the regulatory mechanisms of Ecl family proteins and discuss their emerging findings., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)
- Published
- 2023
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4. Genes affecting the extension of chronological lifespan in Schizosaccharomyces pombe (fission yeast).
- Author
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Ohtsuka H, Shimasaki T, and Aiba H
- Subjects
- Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins metabolism, Transcription Factors metabolism, Gene Expression Regulation, Fungal, Longevity, Protein Interaction Maps, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics, Signal Transduction, Transcription Factors genetics
- Abstract
So far, more than 70 genes involved in the chronological lifespan (CLS) of Schizosaccharomyces pombe (fission yeast) have been reported. In this mini-review, we arrange and summarize these genes based on the reported genetic interactions between them and the physical interactions between their products. We describe the signal transduction pathways that affect CLS in S. pombe: target of rapamycin complex 1, cAMP-dependent protein kinase, Sty1, and Pmk1 pathways have important functions in the regulation of CLS extension. Furthermore, the Php transcription complex, Ecl1 family proteins, cyclin Clg1, and the cyclin-dependent kinase Pef1 are important for the regulation of CLS extension in S. pombe. Most of the known genes involved in CLS extension are related to these pathways and genes. In this review, we focus on the individual genes regulating CLS extension in S. pombe and discuss the interactions among them., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)
- Published
- 2021
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5. Sulfur restriction extends fission yeast chronological lifespan through Ecl1 family genes by downregulation of ribosome.
- Author
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Ohtsuka H, Takinami M, Shimasaki T, Hibi T, Murakami H, and Aiba H
- Subjects
- Down-Regulation genetics, Gene Expression Regulation, Fungal genetics, Ribosomes metabolism, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Sulfur metabolism, Transcription Factors metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Schizosaccharomyces pombe Proteins genetics, Schizosaccharomyces pombe Proteins metabolism
- Abstract
Nutritional restrictions such as calorie restrictions are known to increase the lifespan of various organisms. Here, we found that a restriction of sulfur extended the chronological lifespan (CLS) of the fission yeast Schizosaccharomyces pombe. The restriction decreased cellular size, RNA content, and ribosomal proteins and increased sporulation rate. These responses depended on Ecl1 family genes, the overexpression of which results in the extension of CLS. We also showed that the Zip1 transcription factor results in the sulfur restriction-dependent expression of the ecl1
+ gene. We demonstrated that a decrease in ribosomal activity results in the extension of CLS. Based on these observations, we propose that sulfur restriction extends CLS through Ecl1 family genes in a ribosomal activity-dependent manner., (© 2017 John Wiley & Sons Ltd.)- Published
- 2017
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6. Hfq binding at RhlB-recognition region of RNase E is crucial for the rapid degradation of target mRNAs mediated by sRNAs in Escherichia coli.
- Author
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Ikeda Y, Yagi M, Morita T, and Aiba H
- Subjects
- Endoribonucleases genetics, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Protein Binding, Protein Interaction Mapping, Sequence Deletion, DEAD-box RNA Helicases metabolism, Endoribonucleases metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Host Factor 1 Protein metabolism, RNA Stability, RNA, Small Interfering metabolism
- Abstract
An RNA chaperon Hfq along with Hfq-binding sRNAs stably binds to RNase E in Escherichia coli. The role of the Hfq-RNase E interaction is to recruit RNase E to target mRNAs of sRNAs resulting in the rapid degradation of the mRNA-sRNA hybrid. The C-terminal scaffold region of RNase E is responsible for the interaction with Hfq. Here, we demonstrate that the scaffold region can be deleted up to residue 750 without losing the ability to cause the rapid degradation of target mRNAs mediated by Hfq/sRNAs. The truncated RNase E750 can still bind to Hfq although the truncation significantly reduces the Hfq-binding ability. We conclude that the subregion between 711 and 750 is sufficient for the functional interaction with Hfq to support the rapid degradation of ptsG mRNA although additional subregions within the scaffold are also involved in Hfq binding. Deletion of the 702-750 region greatly impairs the ability of RNase E to cause the degradation of ptsG mRNA. In addition, a polypeptide corresponding to the scaffold region binds to Hfq without the help of RNA. Finally, we demonstrate that overexpression of RhlB partially inhibits the Hfq binding to RNase E and the rapid degradation of ptsG mRNA., (© 2010 Blackwell Publishing Ltd.)
- Published
- 2011
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7. A minimal base-pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA.
- Author
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Maki K, Morita T, Otaka H, and Aiba H
- Subjects
- Base Sequence, Down-Regulation, Escherichia coli chemistry, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Phosphoenolpyruvate Sugar Phosphotransferase System chemistry, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, RNA, Bacterial metabolism, Base Pairing, Escherichia coli genetics, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Protein Biosynthesis, RNA, Bacterial genetics
- Abstract
Escherichia coli SgrS is an Hfq-binding small RNA that is induced under glucose-phosphate stress to cause translational repression and RNase E-dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31-nt-long stretch in the 3' region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre-annealed with ptsG mRNA by a high-temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base-pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base-pairing region is sufficient to inhibit ptsG translation in the context of full-length SgrS in vivo. We conclude that SgrS 168-181 is a minimal base-pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high-temperature pre-treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.
- Published
- 2010
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8. Cleavage of mRNAs and role of tmRNA system under amino acid starvation in Escherichia coli.
- Author
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Li X, Yagi M, Morita T, and Aiba H
- Subjects
- Bacterial Toxins metabolism, Cyclic AMP Receptor Protein metabolism, Escherichia coli genetics, Escherichia coli Proteins metabolism, Gene Deletion, RNA, Bacterial genetics, Transcription Factors metabolism, Amino Acids metabolism, Escherichia coli metabolism, RNA, Bacterial metabolism, RNA, Messenger metabolism
- Abstract
We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin-antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions.
- Published
- 2008
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9. Reduced action of polypeptide release factors induces mRNA cleavage and tmRNA tagging at stop codons in Escherichia coli.
- Author
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Li X, Yokota T, Ito K, Nakamura Y, and Aiba H
- Subjects
- Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Peptides metabolism, RNA, Bacterial metabolism, Codon, Terminator, Escherichia coli metabolism, Peptide Termination Factors metabolism, RNA, Bacterial genetics, RNA, Messenger metabolism, Transcription Factors
- Abstract
Certain C-terminal sequences of nascent peptide cause an efficient protein tagging by tmRNA system at stop codons in Escherichia coli. Here, we demonstrate that both mRNA cleavage and tmRNA tagging occur at UAG stop codon recognized specifically by polypeptide release factor 1 (RF-1) when the activity of RF-1 is reduced by a mutation in the prfA gene without requirement of particular C-terminal sequences of nascent peptide. The tmRNA tagging and mRNA cleavage in the prfA mutant were eliminated when the wild-type RF-1 but not RF-2 was supplied from plasmid. In addition, depletion of either RF-1 or RF-2 induces endonucleolytic cleavage and tmRNA tagging at UAG or UGA stop codons respectively. We conclude that ribosome stalling at the cognate stop codon caused by reduced activity or expression of RF-1 or RF-2 is responsible for mRNA cleavage. The present data along with our previous studies strongly suggest that ribosome stalling leads to endonucleolytic cleavage of mRNA in general resulting in non-stop mRNA and that the 3' end of non-stop mRNA is probably only target for the tmRNA system.
- Published
- 2007
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10. Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq.
- Author
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Kawamoto H, Koide Y, Morita T, and Aiba H
- Subjects
- Base Pairing, Blotting, Northern, Blotting, Western, Electrophoretic Mobility Shift Assay, Escherichia coli chemistry, Escherichia coli physiology, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Point Mutation, RNA Interference, RNA, Antisense genetics, RNA, Bacterial genetics, RNA, Double-Stranded metabolism, RNA, Messenger genetics, Escherichia coli genetics, Escherichia coli Proteins physiology, Host Factor 1 Protein physiology, RNA, Antisense metabolism, RNA, Bacterial metabolism, RNA, Messenger metabolism
- Abstract
SgrS is an Hfq-binding small antisense RNA that is induced upon phosphosugar stress. It forms a ribonucleoprotein complex with RNase E through Hfq to mediate silencing of the target ptsG mRNA encoding the membrane component of the glucose-specific phosphoenolpyruvate phosphotransferase system. Although SgrS is believed to act on ptsG mRNA through base pairing between complementary regions, this was not previously tested experimentally. We addressed the question of whether SgrS indeed forms an RNA-RNA duplex with ptsG mRNA to exert its regulatory function. Specific single nucleotide substitutions around the Shine-Dalgarno (SD) sequence of ptsG completely eliminated SgrS action while compensatory mutations in SgrS restored it. A systematic mutational analysis of both ptsG and SgrS RNAs revealed that six base pairs around SD sequence of ptsG are particularly important for SgrS action. We also showed in vitro that SgrS forms a stable duplex with the ptsG mRNA, and that Hfq markedly facilitates the rate of duplex formation.
- Published
- 2006
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11. Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli.
- Author
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Morita T, Kawamoto H, Mizota T, Inada T, and Aiba H
- Subjects
- Escherichia coli Proteins genetics, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Glucose metabolism, Multienzyme Complexes, Polyribonucleotide Nucleotidyltransferase metabolism, Promoter Regions, Genetic, RNA Helicases metabolism, Sugar Phosphates metabolism, Escherichia coli Proteins metabolism, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, Phosphopyruvate Hydratase metabolism, RNA, Messenger metabolism
- Abstract
The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.
- Published
- 2004
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12. Membrane localization itself but not binding to IICB is directly responsible for the inactivation of the global repressor Mlc in Escherichia coli.
- Author
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Tanaka Y, Itoh F, Kimata K, and Aiba H
- Subjects
- Base Sequence, Cell Membrane metabolism, DNA Primers, Escherichia coli genetics, Genotype, Molecular Sequence Data, Monosaccharide Transport Proteins genetics, Polymerase Chain Reaction, RNA, Bacterial genetics, Repressor Proteins metabolism, Transcription, Genetic, Escherichia coli metabolism, Escherichia coli Proteins antagonists & inhibitors, Monosaccharide Transport Proteins metabolism, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, Repressor Proteins antagonists & inhibitors
- Abstract
Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc-Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc-IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc-Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc., (Copyright 2004 Blackwell Publishing Ltd)
- Published
- 2004
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13. Metabolic block at early stages of the glycolytic pathway activates the Rcs phosphorelay system via increased synthesis of dTDP-glucose in Escherichia coli.
- Author
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El-Kazzaz W, Morita T, Tagami H, Inada T, and Aiba H
- Subjects
- Escherichia coli genetics, Escherichia coli growth & development, Gene Expression Regulation, Bacterial, Gene Silencing, Genes, Bacterial, Glucose metabolism, Glucose-6-Phosphate metabolism, Glucose-6-Phosphate Isomerase genetics, Glucose-6-Phosphate Isomerase physiology, Glucosephosphate Dehydrogenase genetics, Glucosephosphate Dehydrogenase metabolism, Mutation, Polysaccharides biosynthesis, RNA, Messenger analysis, Repressor Proteins genetics, Repressor Proteins physiology, Signal Transduction, Transcription, Genetic genetics, Transcription, Genetic physiology, Bacterial Proteins physiology, Escherichia coli metabolism, Escherichia coli Proteins physiology, Glucose analogs & derivatives, Glucose biosynthesis, Glycolysis genetics, Multienzyme Complexes physiology, Phosphoprotein Phosphatases physiology, Phosphotransferases physiology, Protein Kinases physiology, Thymine Nucleotides biosynthesis, Transcription Factors
- Abstract
A mutational block in the early stages of the glycolytic pathway facilitates the degradation of the ptsG mRNA encoding the major glucose transporter IICBGlc in Escherichia coli. The degradation is RNase E dependent and is correlated with the accumulation of either glucose-6-P or fructose-6-P (Kimata et al., 2001, EMBO J 20: 3587-3595; Morita et al., 2003, J Biol Chem 278: 15608-15614). In this paper, we investigate additional physiological effects resulting from the accumulation of glucose-6-P caused by a mutation in pgi encoding phosphoglucose isomerase, focusing on changes in gene expression. The addition of glucose to the pgi strain caused significant growth inhibition, in particular in the mlc background. Cell growth then gradually resumed as the level of IICBGlc decreased. We found that the transcription of the cps operon, encoding a series of proteins responsible for the synthesis of colanic acid, was markedly but transiently induced under this metabolic stress. Both genetic and biochemical studies revealed that the metabolic stress induces cps transcription by activating the RcsC/YojN/RcsB signal transduction system. Overexpression of glucose-6-P dehydrogenase eliminated both growth inhibition and cps induction by reducing the glucose-6-P level. Mutations in genes responsible for the synthesis of glucose-1-P and/or dTDP-glucose eliminated the activation of the Rcs system by the metabolic stress. Taken together, we conclude that an increased synthesis of dTDP-glucose activates the Rcs phosphorelay system, presumably by affecting the synthesis of oligosaccharides for enterobacterial common antigen and O-antigen.
- Published
- 2004
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14. Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.
- Author
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Oshima T, Aiba H, Masuda Y, Kanaya S, Sugiura M, Wanner BL, Mori H, and Mizuno T
- Subjects
- Bacterial Proteins genetics, Escherichia coli growth & development, Escherichia coli physiology, Gene Expression Profiling, Oligonucleotide Array Sequence Analysis, Proteome, Transcription, Genetic, Bacterial Proteins metabolism, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Mutation, Signal Transduction
- Abstract
We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components).
- Published
- 2002
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15. A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC --> YojN --> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour.
- Author
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Takeda S, Fujisawa Y, Matsubara M, Aiba H, and Mizuno T
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Culture Media, Escherichia coli genetics, Molecular Sequence Data, Phosphorylation, Plasmids, Signal Transduction, Transcription Factors genetics, Bacterial Capsules biosynthesis, Bacterial Proteins genetics, Bacterial Proteins metabolism, Escherichia coli physiology, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Multienzyme Complexes, Phosphoprotein Phosphatases, Phosphotransferases, Protein Kinases, Transcription Factors metabolism
- Abstract
In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --> RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His --> Asp phosphorelay, this RcsC --> RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His --> Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC --> YojN --> RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.
- Published
- 2001
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16. Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc.
- Author
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Hogema BM, Arents JC, Bader R, Eijkemans K, Yoshida H, Takahashi H, Aiba H, and Postma PW
- Subjects
- Bacterial Proteins metabolism, Biological Transport physiology, Carbohydrate Metabolism, Carbohydrates pharmacology, Escherichia coli enzymology, Glucose-6-Phosphate pharmacology, Methylgalactosides metabolism, Mutation genetics, Phosphoenolpyruvate metabolism, Phosphoproteins metabolism, Phosphorylation, Pyruvic Acid metabolism, Thiogalactosides metabolism, Enzyme Induction physiology, Escherichia coli metabolism, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism
- Abstract
The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.
- Published
- 1998
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17. A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli.
- Author
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Kimata K, Inada T, Tagami H, and Aiba H
- Subjects
- Base Sequence, Binding Sites genetics, Carrier Proteins, Cyclic AMP Receptor Protein genetics, Cyclic AMP Receptor Protein metabolism, DNA, Bacterial genetics, DNA, Bacterial metabolism, Escherichia coli drug effects, Gene Expression drug effects, Glucose pharmacology, Molecular Sequence Data, Mutagenesis, Insertional, Promoter Regions, Genetic, Protein Binding, RNA, Bacterial biosynthesis, RNA, Bacterial genetics, RNA, Messenger biosynthesis, RNA, Messenger genetics, Repressor Proteins genetics, Bacterial Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins, Genes, Bacterial drug effects, Monosaccharide Transport Proteins genetics, Repressor Proteins metabolism
- Abstract
Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.
- Published
- 1998
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18. Inducer exclusion by glucose 6-phosphate in Escherichia coli.
- Author
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Hogema BM, Arents JC, Bader R, Eijkemans K, Inada T, Aiba H, and Postma PW
- Subjects
- Biological Transport, Escherichia coli Proteins, Gluconates metabolism, Methylgalactosides metabolism, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, Phosphorylation, Thiogalactosides metabolism, beta-Galactosidase metabolism, Escherichia coli metabolism, Glucose-6-Phosphate metabolism, Lactose metabolism
- Abstract
The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc). In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources.
- Published
- 1998
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19. Catabolite repression by glucose 6-phosphate, gluconate and lactose in Escherichia coli.
- Author
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Hogema BM, Arents JC, Inada T, Aiba H, van Dam K, and Postma PW
- Subjects
- Adenylyl Cyclases metabolism, Carbon metabolism, Cyclic AMP metabolism, Glucose-6-Phosphate genetics, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, Receptors, Cyclic AMP metabolism, Escherichia coli metabolism, Gene Expression Regulation, Bacterial, Gluconates metabolism, Glucose-6-Phosphate metabolism, Lactose metabolism
- Abstract
While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA(Glc) of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of beta-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.
- Published
- 1997
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20. Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth.
- Author
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Ohmiya R, Yamada H, Nakashima K, Aiba H, and Mizuno T
- Subjects
- Adaptation, Physiological, Amino Acid Sequence, Base Sequence, Cloning, Molecular, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Genetic Complementation Test, Glycerol metabolism, Molecular Sequence Data, Osmolar Concentration, Phenotype, Schizosaccharomyces enzymology, Schizosaccharomyces growth & development, Sequence Homology, Amino Acid, Glycerolphosphate Dehydrogenase genetics, Schizosaccharomyces genetics
- Abstract
Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osms) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1+ and gpd2+, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1+ was regulated at the mRNA level in response to osmotic upshift. (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) delta gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osms phenotype. On the other hand, the gpd2+ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.
- Published
- 1995
- Full Text
- View/download PDF
21. Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP.
- Author
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Tagami H, Inada T, Kunimura T, and Aiba H
- Subjects
- Adenylyl Cyclases physiology, Carrier Proteins, Cyclic AMP metabolism, Cyclic AMP Receptor Protein biosynthesis, Enzyme Repression, Escherichia coli enzymology, Escherichia coli genetics, Gene Amplification, Genes, Bacterial, Plasmids genetics, Point Mutation, Promoter Regions, Genetic genetics, RNA, Bacterial biosynthesis, RNA, Messenger biosynthesis, beta-Galactosidase biosynthesis, beta-Galactosidase metabolism, Cyclic AMP Receptor Protein genetics, Gene Expression Regulation, Bacterial drug effects, Glucose pharmacology, Lac Operon genetics
- Abstract
CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.
- Published
- 1995
- Full Text
- View/download PDF
22. A novel gene whose expression is regulated by the response-regulator, SphR, in response to phosphate limitation in Synechococcus species PCC7942.
- Author
-
Aiba H and Mizuno T
- Subjects
- Amino Acid Sequence, Bacterial Proteins biosynthesis, Base Sequence, Cyanobacteria drug effects, Cyanobacteria physiology, Membrane Proteins biosynthesis, Molecular Sequence Data, Molecular Weight, Phosphates metabolism, Phosphates pharmacology, Promoter Regions, Genetic, Sequence Alignment, Signal Transduction drug effects, Transcription, Genetic drug effects, Bacterial Proteins genetics, Bacterial Proteins physiology, Cyanobacteria genetics, DNA-Binding Proteins physiology, Gene Expression Regulation, Bacterial drug effects, Genes, Bacterial, Genes, Plant, Membrane Proteins genetics, Membrane Transport Proteins, Phosphotransferases, Transcription Factors physiology
- Abstract
In Synechococcus species PCC7942, the production of a subset of proteins is induced when it is grown in a phosphate-limited medium. We previously suggested that a pair of cyanobacterial two-component regulatory proteins, SphS (sensory-kinase) and SphR (response-regulator), may be involved in this particular response to phosphate limitation. Here it was found that a protein with an apparent molecular mass of 33 kDa became particularly abundant when the total cellular proteins from cells grown in a phosphate-limited medium were analysed by SDS-PAGE. A deletion mutant lacking both the sphS and the sphR genes failed to produce this 33 kDa protein in response to phosphate limitation. Thus it was reasonable to assume that this protein is a member of the group of proteins involved in the Synechococcus phosphate regulatory circuit (hence, it was named SphX). The SphX protein was purified to near homogeneity, and the corresponding structural gene was cloned. The determined nucleotide sequence revealed that the sphX gene encodes a novel protein with a calculated molecular mass of 36,374 Da, which was demonstrated to be located in the cytoplasmic membrane. Structural features of the sphX promoter were then clarified by determining its transcription start site, from which transcription was triggered in response to phosphate limitation. Furthermore, the putative response-regulator, SphR, was demonstrated to bind to the upstream region of the sphX promoter by means of in vitro DNase I footprinting. From these results, we conclude that the sphX gene is a member of the Synechococcus phosphate regulatory circuit, in which the two signal-transduction components, SphS and SphR, are crucially involved as transcriptional regulators. The SphX protein may play a role in phosphate assimilation in Synechococcus.
- Published
- 1994
- Full Text
- View/download PDF
23. A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli.
- Author
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Ishizuka H, Hanamura A, Kunimura T, and Aiba H
- Subjects
- Bacterial Proteins biosynthesis, Carrier Proteins, Cyclic AMP Receptor Protein genetics, DNA-Binding Proteins metabolism, Enzyme Repression, Escherichia coli drug effects, Escherichia coli enzymology, Recombinant Proteins biosynthesis, Cyclic AMP metabolism, Cyclic AMP Receptor Protein biosynthesis, Escherichia coli physiology, Glucose pharmacology, beta-Galactosidase biosynthesis
- Abstract
A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.
- Published
- 1993
- Full Text
- View/download PDF
24. Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation.
- Author
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Aiba H, Nagaya M, and Mizuno T
- Subjects
- Adaptation, Physiological, Alkaline Phosphatase biosynthesis, Alkaline Phosphatase genetics, Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Bacterial Proteins genetics, Base Sequence, Cyanobacteria enzymology, Cyanobacteria genetics, Escherichia coli genetics, Escherichia coli metabolism, Genes, Bacterial, Molecular Sequence Data, Molecular Weight, Multigene Family, Phenotype, Plant Proteins genetics, Protein Kinases genetics, Sequence Alignment, Sequence Homology, Amino Acid, Transcription Factors genetics, Bacterial Proteins metabolism, Cyanobacteria physiology, Gene Expression Regulation, Bacterial, Phosphates metabolism, Phosphotransferases, Plant Proteins metabolism, Protein Kinases metabolism, Signal Transduction, Transcription Factors metabolism
- Abstract
A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypically to complement a phoR creC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5 kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46,389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29,012 Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate-limitation in the medium. These results are probably involved, either directly or indirectly, in the signal-transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (Synechococcus phosphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.
- Published
- 1993
- Full Text
- View/download PDF
25. A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation.
- Author
-
Hanamura A and Aiba H
- Subjects
- Base Sequence, Binding Sites physiology, Cyclic AMP Receptor Protein genetics, Homeostasis physiology, Molecular Sequence Data, Promoter Regions, Genetic physiology, Cyclic AMP physiology, Cyclic AMP Receptor Protein physiology, Escherichia coli genetics, Gene Expression Regulation, Bacterial physiology, Genes, Bacterial physiology, Transcription, Genetic physiology
- Abstract
Transcription of the Escherichia coli crp gene is negatively regulated by CRP-cAMP that binds to a specific site located downstream of the crp promoter. A second binding site for CRP-cAMP (CRP site II) exists upstream of the crp promoter. Using an in vitro transcription assay, we have demonstrated that CRP-cAMP activates transcription of crp in certain conditions. A promoter which carries an altered CRP-binding site II is no longer activated by CRP-cAMP, indicating that CRP site II mediates the activation of crp transcription. The concentrations of cAMP that are required for positive autoregulation are higher than those for negative autoregulation. Evidence for positive and negative autoregulation in vivo is presented by a quantitative S1 nuclease analysis.
- Published
- 1992
- Full Text
- View/download PDF
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