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Hirotada Mori

Publications and source records attributed to Hirotada Mori.

At least 19 recordsLinked to original sources
PubMed2004

From the sequence to cell modeling: comprehensive functional genomics in Escherichia coli.

As a result of the enormous amount of information that has been collected with E. coli over the past half century (e.g. genome sequence, mutant phenotypes, metabolic and regulatory networks, etc.), we now have detailed knowledge about gene regulation, protein activity, several hundred enzyme reactions, metabolic pathways, macromolecular machines, and regulatory interactions for this model organism. However, understanding how all these processes interact to form a living cell will require further characterization, quantification, data integration, and mathematical modeling, systems biology. No organism can rival E. coli with respect to the amount of available basic information and experimental tractability for the technologies needed for this undertaking. A focused, systematic effort to understand the E. coli cell will accelerate the development of new post-genomic technologies, including both experimental and computational tools. It will also lead to new technologies that will be applicable to other organisms, from microbes to plants, animals, and humans. E. coli is not only the best studied free-living model organism, but is also an extensively used microbe for industrial applications, especially for the production of small molecules of interest. It is an excellent representative of Gram-negative commensal bacteria. E. coli may represent a perfect model organism for systems biology that is aimed at elucidating both its free-living and commensal life-styles, which should open the door to whole-cell modeling and simulation.

Computational Biology↗
PubMed2004

The ATPase domain of HscC (DnaK homolog) is essential for interfering sigma70 activity in E. coli.

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains. Overexpression of HscC markedly inhibits growth of host cell and reduces the sigma(70)-dependent promoter activity presumably by forming a complex with sigma(70). To identify the region(s) of HscC responsible for growth inhibition and complex formation with sigma(70), domain swapping experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the sigma(70)-dependent promoter activity and form a complex with sigma(70). These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is important for determining its functional specificity.

Adenosine Triphosphatases↗
PubMed2003

Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout.

The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C(6)]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed.

Acetyl Coenzyme A↗
PubMed2003

Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation.

To understand the nature and function of bacterial biofilm and the process of its formation, we have performed systematic screening of a complete set of Escherichia coli genes/open reading frames (ORFs) to identify those that affect biofilm development upon over-expression. In contrast to the biofilm of strain AG1 used as a control, some of the genes/ORFs when over-expressed led to the formation of an abnormal biofilm such as thin, mat-like, filamentous or one easily detaching from various surfaces. Disruptants of selected genes were constructed in order to clarify their roles in the different stages of biofilm formation. Our results suggest that diverse metabolic pathways contribute to the development of biofilm.

Bacterial Outer Membrane Proteins↗
PubMed2003

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement.

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.

Carbon Isotopes↗
PubMed2003

Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus.

DNA microarray covering the whole genome of Staphylococcus aureus strain N315 was prepared to investigate transcription profiles. The microarray analyses revealed that vancomycin induces transcription of 139 genes. Forty-six genes among them failed to be induced in the vraSR null mutant KVR. Part of the genes regulated by VraSR system is associated with cell-wall biosynthesis, such as PBP2, SgtB and MurZ. Other cell-wall synthesis inhibitors also induced VraSR, suggesting that the sensor kinase VraS responds to the damage of cell-wall structure or inhibition of cell-wall biosynthesis. Additionally, the vraSR null mutants derived from hetero- and homo-methicillin-resistant S. aureus showed significant decrease of resistance against teicoplanin, beta-lactam, bacitracin and fosfomycin but not of D-cycloserine and levofloxacin. The observation strongly indicates that VraSR constitutes a positive regulator of cell-wall peptidoglycan synthesis, and that is deeply involved in the expression of beta-lactam and glycopeptide resistance in S. aureus.

Anti-Bacterial Agents↗
PubMed2003

Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli.

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 (Mg(2+)-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.

Bacterial Outer Membrane Proteins↗
PubMed2003

Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). 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, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) 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 Mg(2+) stimulon in E. coli.

Bacterial Proteins↗
PubMed2003

Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli.

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

Bacterial Capsules↗
PubMed2003

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts.

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

Citric Acid Cycle↗
PubMed2003

Escherichia coli response to exogenous pyrophosphate and analogs.

The addition of exogenous pyrophosphate increases the growth yield and cAMP synthesis in stationary phase when Escherichia coli is grown in minimal medium. Pyrophosphate increases the yield by altering the enterobactin uptake system. We studied the physiological effects and examined how the E. coli transcriptome was modified when two structural analogs of pyrophosphate were added to the growth medium. Methylenediphosphonic acid or a high concentration of iron had the same positive effects as pyrophosphate on growth yield, cAMP synthesis and the repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect these cellular processes significantly. The transcriptome modifications generated by pyrophosphate or methylenediphosphonic acid were more similar than those generated by imidodiphosphate or excess iron. The transcriptome data also indicated that processes other than iron uptake might be involved in the cellular response to exogenous pyrophosphate or methylenediphosphonic acid.

Culture Media↗
PubMed2002

Periodicity in prokaryotic and eukaryotic genomes identified by power spectrum analysis.

We used a power spectrum method to identify periodic patterns in nucleotide sequence, and characterized nucleotide sequences that confer periodicities to prokaryotic and eukaryotic genomes and genomes. A 10-bp periodicity was prevalent in hyperthermophilic bacteria and archaebacteria, and an 11-bp periodicity was prevalent in eubacteria. The 10-bp periodicity was also prevalent in the eukaryotes such as the worm Caenorhabditis elegans. Additionally, in the worm genome, a 68-bp periodicity in chromosome I, a 59-bp periodicity in chromosome II, and a 94-bp periodicity in chromosome III were found. In human chromosomes 21 and 22, approximately 167- or 84-bp periodicity was detected along the entire length of these chromosomes. Because the 167-bp is identical to the length of DNA that forms two complete helical turns in nucleosome organization, we speculated that the respective sequences may correspond to arrays of a special compact form of nucleosomes clustered in specific regions of the human chromosomes. This periodic element contained a high frequency of TGG. TGG-rich sequences are known to form a specific subset of folded DNA structures, and therefore, the sequences might have potential to form specific higher order structures related to the clustered occurrence of a specific form of the speculated nucleosomes.

Animals↗
PubMed2002

Distribution of repetitive sequences on the leading and lagging strands of the Escherichia coli genome: comparative study of Long Direct Repeat (LDR) sequences.

In the present study, we developed a method for detecting sequences whose similarity to a target sequence is statistically significant and we examined the distribution of these sequences in the E. coli K-12 genome. Target sequences examined are as follows: (i) short repeat: Crossover hot-spot instigator (Chi) sequence, replication termination (Ter) sequence, and DnaA binding sequence (DnaA box); (ii) potential stem-loop structure repeats: palindromic unit (PU), boxC sequences, and intergenic repeat unit (IRU); (iii) potential RNA coding repeats: rRNAs, PAIR, TRIP, and QUAD; and (iv) potential protein coding repeats: insertion elements (ISs) and Long Direct Repeats (LDRs). We also examined the distribution of these sequences on leading and lagging strands. We obtained another four statistically significant LDR sequences with more than 187 bp matched to LDR-A near the LDR loci, suggesting that these regions might be used as high recombination hot spots for LDR. Adaptation of individual LDRs to E. coli genome is also discussed on the basis of codon usage.

Bacterial Proteins↗
PubMed2002

Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon.

Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides. Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes. Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively.

Base Sequence↗
PubMed2002

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.

Amino Acid Sequence↗
PubMed2002

Characterization of HscC (Hsc62), homologue of Hsp70 in Escherichia coli: over-expression of HscC modulates the activity of house keeping sigma factor sigma70.

BACKGROUND: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with sigma32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown. RESULTS: We observed that the over-expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a beta-galactosidase assay and found that HscC reduced the sigma70-dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited sigma70-dependent transcription. In addition, the co-purification analysis showed that sigma70 co-eluted with HscC. CONCLUSION: These results indicate that HscC forms a complex with sigma70 and may function as its negative modulator.

DNA-Directed RNA Polymerases↗
PubMed2002

Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli.

Deoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5'-GATC-3' sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a high-density DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam-16::kam mutant strain grown under four different conditions. We also used two-dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expression of a large number of genes was affected by the dam deficiency. Genes involved in aerobic respiration, stress and SOS responses, amino acid metabolism and nucleotide metabolism were expressed at higher levels in the mutant cells, especially in aerobic conditions. In contrast, transcription of genes participating in anaerobic respiration, flagella biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and low aerobic conditions. Thus, Dam-controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Dam-controlled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam-mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites.

Amino Acids↗