Biomedical subjects

Hirotada Mori

Publications and source records attributed to Hirotada Mori.

At least 19 recordsLinked to original sources

PubMed2006

Experimental and computational assessment of conditionally essential genes in Escherichia coli.

Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of 3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regulatory model of E. coli, and it was found that the model made the correct prediction in approximately 91% of the cases. The discrepancies between model predictions and experimental results were analyzed in detail to indicate where model improvements could be made or where the current literature lacks an explanation for the observed phenotypes. The identified set of essential genes and their model-based analysis indicates that our current understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately lead to a better understanding of the organism.

Bacterial Proteins↗

PubMed2006

Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family.

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.

Catalysis↗

PubMed2006

Comparative analysis of argK-tox clusters and their flanking regions in phaseolotoxin-producing Pseudomonas syringae pathovars.

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181-10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064-11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named "tox islands." The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437-457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands.

3' Flanking Region↗

PubMed2006

The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase.

BACKGROUND: Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT. RESULTS: As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases. CONCLUSION: Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).

Escherichia coli Proteins↗

PubMed2006

Large-scale identification of protein-protein interaction of Escherichia coli K-12.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.

Escherichia coli K12↗

PubMed2006

Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG.

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.

Amino Acid Sequence↗

PubMed2006

Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110.

With the goal of solving the whole-cell problem with Escherichia coli K-12 as a model cell, highly accurate genomes were determined for two closely related K-12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single-nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino-acid residue deletion, five amino-acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13,000 bases) were mostly within portions sequenced with out-dated technology based on radioactive chemistry.

Base Sequence↗

PubMed2006

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Escherichia coli↗

PubMed2006

Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research.

Based on the genomic sequence data of Escherichia coli K-12 strain, we have constructed a complete set of cloned individual genes encoding Histidine-tagged proteins with or without GFP fused for functional genomic analysis. Each clone encodes a protein of predicted ORF attached by Histidines and seven spacer amino acids at the N-terminal end, and five spacer amino acids and GFP at the C-terminal end. SfiI restriction sites are generated at both the N- and C-terminal boundaries of ORF upon cloning, which enables easy transfer of ORF to other vector systems by cutting with SfiI. Expression of cloned ORF is under the control of an IPTG-inducible promoter, which is strictly repressed by lacI(q) repressor gene product. The set of cloned ORFs described here should provide unique resources for systematic functional genomic approaches including (i) construction of DNA microarray, (ii) production and purification of proteins, (iii) analysis of protein localization by monitoring GFP fluorescence and (iv) analysis of protein-protein interaction.

Escherichia coli K12↗

PubMed2006

Escherichia coli K-12: a cooperatively developed annotation snapshot--2005.

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Congresses as Topic↗

PubMed2006

Metabolomics approach for enzyme discovery.

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.

Electrophoresis, Capillary↗

PubMed2005

Identification of human calphoglin-induced phosphoglucomutase phosphorylation in Escherichia coli.

Orthologous proteomes, universal protein networks conserved from bacteria to mammals, dictate the core functions of cells. To isolate mammalian protein sequences that interact with bacterial signaling proteins, a BLASTP genome search was performed using catalytic domains of bacterial phosphoryl-transfer enzymes as probes. A [32P]phosphoryl-transfer assay of these mammalian cDNA-expressing Escherichia coli cells was used to screen proteins retrieved from the database. Here we report that the expression of a human protein, named calphoglin, resulted in a significant increase in the phosphorylation of a 55-kDa protein in E. coli. The phosphorylation of the 55-kDa protein was acid-stable and its isoelectric point was determined to be 5.4. The 55-kDa protein was sequentially purified from an E. coli extract using three chromatography and two-dimensional polyacrylamide gel electrophoresis. Finally, the 55-kDa protein was purified 830-fold to homogeneity and the N-terminal amino acid sequence was analyzed. The sequence obtained, AIHNRAGQPAQQ, was identical to the N-terminal amino acids of E. coli phosphoglucomutase (PGM). This method may be applicable to the detection and analysis of other orthologous proteomes.

Amino Acid Sequence↗

PubMed2005

Use of GFP tags to monitor localization of different luciferases in E. coli.

The utility of the green fluorescent protein (GFP) as a probe to monitor protein localization in living cells is gaining a great deal of attention. In this study, to understand the localization of luciferases in E. coli, we have attached GFP tags at both the N- and the C-terminus of firefly luciferase (FF-Luc)(from Pyrocoelia miyako) and of red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), respectively. There was no significant change in the bioluminescence emission spectrum for any of the three luciferases following the tagging with GFP at either the N- or C-terminus, confirming the absence of energy transfer between one another. Using confocal imaging microscopy, we observed that all three luciferases expressed in the E.coli cultured at 37 degrees C tend to aggregate and are seen to localize in the poles, thus confirming their poor folding properties. In contrast, in the E.coli cultured at 18 degrees C FF-Luc was found to be highly expressed in the soluble form when compared to RE-Luc and GR-Luc. These results support our previous finding that the folding properties of FF-Luc and RE/GR-Luc are totally different.

Cytosol↗

PubMed2005

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.

Base Sequence↗

PubMed2005

Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays.

Escherichia coli is one of the best elucidated organisms. However, about 40% of E. coli genes have not been assigned to their function yet. We analyzed 1440 single gene knock-out mutants using the GN2-MicroPlate, which permits assay of 95 carbon-source utilizations simultaneously. In the knock-out library there are 1044 of so called y-genes with no apparent function. The raw dataset was analyzed and genes were interrelated by the clustering method of the GeneSpring software. In the resulted dendrogram of genes, a group of genes with known and related function tended to be assembled into a cluster. Our clustering method would be useful for functional assignment of so called y-genes with no apparent function, since the resulted dendrogram could connect y-genes to phenotype and function of well-studied genes.

Escherichia coli↗

PubMed2005

Cell lysis directed by sigmaE in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli.

It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding sigma(E) exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a sigma(E)-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified sigma(E) regulon genes, suggesting that a large number of genes are under the control of sigma(E) in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.

Escherichia coli↗

PubMed2005

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli.

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.

Amino Acid Sequence↗

PubMed2005

Identification and functional analysis of Escherichia coli cysteine desulfhydrases.

In Escherichia coli, three additional proteins having L-cysteine desulfhydrase activity were identified as O-acetylserine sulfhydrylase-A, O-acetylserine sulfhydrylase-B, and MalY protein, in addition to tryptophanase and cystathionine beta-lyase, which have been reported previously. The gene disruption for each protein was significantly effective for overproduction of L-cysteine and L-cystine. Growth phenotype and transcriptional analyses suggest that tryptophanase contributes primarily to L-cysteine degradation.

Cystathionine gamma-Lyase↗