Tomoya Baba

Publications

• Environmental dependency of gene knockouts on phenotype microarray analysis in Escherichia coli.

  Yukako Tohsato, Tomoya Baba, Yusaku Mazaki, Masahiro Ito, Barry L Wanner, Hirotada Mori

  Journal of bioinformatics and computational biology. 12/2010; 8 Suppl 1:83-99.

  Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene delet... [more] Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray™ (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.
• 4.02

  Impact points

  Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments.

  Eiji Aono, Tomoya Baba, Takeshi Ara, Tatsunari Nishi, Tomoko Nakamichi, Eiji Inamoto, Hiromi Toyonaga, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Masaru Tomita, Chiaki Kato, Taku Oshima, Kaoru Nakasone, Hirotada Mori

  Molecular bioSystems. 07/2010; 6(7):1216-26.

  Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic gamma-proteobacterium that was isolated from the surface layer of deep sea sediment at ... [more] Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic gamma-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor.
• 2.20

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  Glutathione production by efficient ATP-regenerating Escherichia coli mutants.

  Kiyotaka Y Hara, Natsuka Shimodate, Yasutaka Hirokawa, Mikito Ito, Tomoya Baba, Hirotada Mori, Hideo Mori

  FEMS microbiology letters. 07/2009;

  There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identif... [more] There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene deletion strains that had over 150% higher total cellular ATP synthetic activity relative to the parental strain. Most of them also showed higher ATP-driven glutathione synthesis. The deleted genes of the identified strains that showed increased efficiency of ATP regeneration for glutathione production could be divided into the following four groups: (1) glycolytic pathway-related genes, (2) genes related to degradation of ATP or adenosine, (3) global regulatory genes, and (4) genes whose contribution to the ATP regeneration is unknown. Furthermore, the high glutathione productivity of DeltanlpD, the highest glutathione-producing mutant strain, was due to its reduced sensitivity to the externally added ATP for ATP regeneration. This study showed that the Permeable Cell Assay was useful for improving the ATP-regenerating activity of E. coli for practical applications in various ATP-driven bioprocesses, much as that of glutathione production.
• 4.73

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  Systematic genome-wide scanning for genes involved in ATP generation in Escherichia coli.

  Kiyotaka Y Hara, Natsuka Shimodate, Mikito Ito, Tomoya Baba, Hirotada Mori, Hideo Mori

  Metabolic engineering. 08/2008;

  Adenosine 5'-triphosphate (ATP) generation is an essential biological reaction for all living cells. Recently, we developed a Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity, mainly resulting from glycolysis [Hara, K.Y., Mori, H., 2006. An efficient method... [more] Adenosine 5'-triphosphate (ATP) generation is an essential biological reaction for all living cells. Recently, we developed a Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity, mainly resulting from glycolysis [Hara, K.Y., Mori, H., 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. J. Biomol. Screen. 11, 310-317]. By using this method, we determined the cellular ATP synthetic activity in the stationary phase of a complete set of single-gene deletion strains of Escherichia coli. Their activities ranged from a minimum of 2% to a maximum of 445%, relative to parental strains. Deletions of metabolism-related genes frequently caused an increase in the rate of ATP synthetic activity, while activity was reduced by deletions of a variety of functional genes, including many poorly characterized genes. We also demonstrated that the deletion of the ptsG gene doubled ATP-driven glutathione synthesis and increased cellular ATP synthetic activity. Our study also indicated that it should be easy to obtain active strains for ATP synthesis from deletion strains. Overall, the data set of this study may be useful to improve E. coli strains for ATP-dependent industrial processes and, therefore, may be important for the design of so-called cell factories.
• 4.92

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  Genome Structure of the Legume, Lotus japonicus.

  Shusei Sato, Yasukazu Nakamura, Takakazu Kaneko, Erika Asamizu, Tomohiko Kato, Mitsuteru Nakao, Shigemi Sasamoto, Akiko Watanabe, Akiko Ono, Kumiko Kawashima, [......], Chika Takahashi, Tsuyuko Wada, Manabu Yamada, Nobuko Ohmido, Makoto Hayashi, Kiichi Fukui, Tomoya Baba, Tomoko Nakamichi, Hirotada Mori, Satoshi Tabata

  DNA research : an international journal for rapid publication of reports on genes and genomes. 06/2008;

  The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies cor... [more] The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.

• The construction of systematic in-frame, single-gene knockout mutant collection in Escherichia coli K-12.

  Tomoya Baba, Hirotada Mori

  Methods in molecular biology (Clifton, N.J.). 02/2008; 416:171-81.

  Here we describe the systematic construction of well-defined, in-frame, single-gene deletions of all nonessential genes in Escherichia coli K-12. The principal strategy is based on the method for one-step inactivation of chromosomal genes in E. coli K-12 established by Datsenko and Wanner (1), namel... [more] Here we describe the systematic construction of well-defined, in-frame, single-gene deletions of all nonessential genes in Escherichia coli K-12. The principal strategy is based on the method for one-step inactivation of chromosomal genes in E. coli K-12 established by Datsenko and Wanner (1), namely, the replacement of a target gene with a selectable antibiotic-resistant marker generated by polymerase chain reaction (PCR) using oligonucleotide DNA primers homologous to the gene flanking regions. The advantages of this method include complete deletion of an entire open reading frame and precise design eliminating polar effects for the downstream genes on E. coli chromosome.

• The applications of systematic in-frame, single-gene knockout mutant collection of Escherichia coli K-12.

  Tomoya Baba, Hsuan-Cheng Huan, Kirill Datsenko, Barry L Wanner, Hirotada Mori

  Methods in molecular biology (Clifton, N.J.). 02/2008; 416:183-94.

  The increasing genome sequence data of microorganisms has provided the basis for comprehensive understanding of organisms at the molecular level. Besides sequence data, a large number of experimental and computational resources are required for genome-scale analyses. Escherichia coli K-12 has been o... [more] The increasing genome sequence data of microorganisms has provided the basis for comprehensive understanding of organisms at the molecular level. Besides sequence data, a large number of experimental and computational resources are required for genome-scale analyses. Escherichia coli K-12 has been one of the best characterized organisms in molecular biology. Recently, the whole-genome sequences of two closely related E. coli K-12 strains, MG1655 (1) and W3110 (2), were compared and confirmed by resequencing selected regions from both strains (2). The availability of highly accurate E. coli K-12 genomes provided an impetus for the cooperative reannotation of both MG1655 and W3110 (3). A set of precisely defined, single-gene knockout mutants of all nonessential genes in E. coli K-12 was constructed based on the recent accurate genome sequence data ([4] and Chapter 11). These mutants were designed to create in-frame (nonpolar) deletions upon elimination of the resistance cassette. These mutants have provided new key information on E. coli biology. First, the vast majority of the 3985 genes that were independently disrupted at least twice are probably nonessential, at least under the conditions of selection. Second, the 303 genes that we repeatedly failed to disrupt are candidates for E. coli essential genes. Lastly, phenotypic effects of all these mutations in the uniform genetic background of E. coli BW25113 were assessed by profiling mutants' growth yields on rich and minimal media (4). These mutants should provide not only a basic resource for systematic functional genomics but also an experimental data source for systems biology applications. The mutants can serve as fundamental tools for a number of reverse genetics approaches, permitting analysis of the consequences of the complete loss of gene function, in contrast with forward genetics approaches in which mutant phenotypes are associated with a corresponding gene or genes.
• 29.75

  Impact points

  Multiple high-throughput analyses monitor the response of E. coli to perturbations.

  Nobuyoshi Ishii, Kenji Nakahigashi, Tomoya Baba, Martin Robert, Tomoyoshi Soga, Akio Kanai, Takashi Hirasawa, Miki Naba, Kenta Hirai, Aminul Hoque, [......], Yuki Takai, Katsuyuki Yugi, Kazuharu Arakawa, Nayuta Iwata, Yoshihiro Toya, Yoichi Nakayama, Takaaki Nishioka, Kazuyuki Shimizu, Hirotada Mori, Masaru Tomita

  Science (New York, N.Y.). 05/2007; 316(5824):593-7.

  Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disr... [more] Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.
• 1.95

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  High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system.

  Yuko Kawahashi, Nobuhide Doi, Yo Oishi, Chizuru Tsuda, Hideaki Takashima, Tomoya Baba, Hirotada Mori, Takashi Ito, Hiroshi Yanagawa

  Journal of biochemistry. 02/2007; 141(1):19-24.

  Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein... [more] Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.
• 4.43

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  Escherichia coli proteome chips for detecting protein-protein interactions.

  Yo Oishi, Shuichi Yunomura, Yuko Kawahashi, Nobuhide Doi, Hideaki Takashima, Tomoya Baba, Hirotada Mori, Hiroshi Yanagawa

  Proteomics. 01/2007; 6(24):6433-6.
• 3.94

  Impact points

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

  Andrew R Joyce, Jennifer L Reed, Aprilfawn White, Robert Edwards, Andrei Osterman, Tomoya Baba, Hirotada Mori, Scott A Lesely, Bernhard Ø Palsson, Sanjay Agarwalla

  Journal of bacteriology. 01/2007; 188(23):8259-71.

  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-supp... [more] 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.
• 2.32

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

  Hiroyuki Genka, Tomoya Baba, Masataka Tsuda, Shigehiko Kanaya, Hirotada Mori, Takanobu Yoshida, Masako Tsujimoto Noguchi, Kenichi Tsuchiya, Hiroyuki Sawada

  Journal of molecular evolution. 10/2006; 63(3):401-14.

  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 the... [more] 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.
• 11.34

  Impact points

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

  Mohammad Arifuzzaman, Maki Maeda, Aya Itoh, Kensaku Nishikata, Chiharu Takita, Rintaro Saito, Takeshi Ara, Kenji Nakahigashi, Hsuan-Cheng Huang, Aki Hirai, [......], Taku Oshima, Tomoya Baba, Natsuko Yamamoto, Tomoyo Kawamura, Tomoko Ioka-Nakamichi, Masanari Kitagawa, Masaru Tomita, Shigehiko Kanaya, Chieko Wada, Hirotada Mori

  Genome research. 06/2006; 16(5):686-91.

  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 ... [more] 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.
• 12.13

  Impact points

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

  Tomoya Baba, Takeshi Ara, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Kirill A Datsenko, Masaru Tomita, Barry L Wanner, Hirotada Mori

  Molecular systems biology. 02/2006; 2:2006.0008.

  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 chromosom... [more] 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/).
• 12.13

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

  Koji Hayashi, Naoki Morooka, Yoshihiro Yamamoto, Katsutoshi Fujita, Katsumi Isono, Sunju Choi, Eiichi Ohtsubo, Tomoya Baba, Barry L Wanner, Hirotada Mori, Takashi Horiuchi

  Molecular systems biology. 02/2006; 2:2006.0007.

  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, includin... [more] 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.
• 2.95

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

  Masami Ueta, Hideji Yoshida, Chieko Wada, Tomoya Baba, Hirotada Mori, Akira Wada

  Genes to cells : devoted to molecular & cellular mechanisms. 01/2006; 10(12):1103-12.

  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 ... [more] 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.
• 4.73

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

  Mikito Ito, Tomoya Baba, Hirotada Mori, Hideo Mori

  Metabolic engineering. 08/2005; 7(4):318-27.

  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 l... [more] 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.
• 3.94

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  Identification of the two missing bacterial genes involved in thiamine salvage: thiamine pyrophosphokinase and thiamine kinase.

  Jonathan Melnick, Ewa Lis, Joo-Heon Park, Cynthia Kinsland, Hirotada Mori, Tomoya Baba, John Perkins, Ghislain Schyns, Olga Vassieva, Andrei Osterman, Tadhg P Begley

  Journal of bacteriology. 07/2004; 186(11):3660-2.

  The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.... [more] The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.
• 4.73

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  Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate.

  Jiao Zhao, Tomoya Baba, Hirotada Mori, Kazuyuki Shimizu

  Metabolic engineering. 05/2004; 6(2):164-74.

  The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers. The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or... [more] The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers. The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or acetate. The decreased rates of substrate uptake and CO(2) production were found for the mutant grown on acetate, whereas these two rates were increased during the growth on glucose. The metabolic flux analysis based on (13)C-labeling experiments indicates that the metabolism of the mutant grown on glucose is related to the higher flux via tricorboxylic acid (TCA) cycle to generate anabolic reducing equivalents normally provided by the oxidative pentose phosphate pathway. However, the metabolism of the mutant grown on acetate shows a lower flux towards the TCA cycle as compared with the parent strain. The decreased flux through TCA cycle is associated with an increased flux via the glyoxylate shunt, by which the carbon source can bypass the two decarboxylative steps of TCA cycle in which CO(2) is released, thus conserving more carbon for biosynthesis in response to the decreased uptake rate of the carbon source.
• 3.94

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

  Qiang Hua, Chen Yang, Tomoya Baba, Hirotada Mori, Kazuyuki Shimizu

  Journal of bacteriology. 01/2004; 185(24):7053-67.

  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 ... [more] 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.