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Molecular Genetics
Plant Development and Physiology
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Photosynthetic Microbial Consortia
Photo Molecular Genetics Laboratory
"How many genes are essential for the simplest cellular organism to live?" "What are the functions of the genes included in the minimum gene set?" We are trying to answer these questions using the bacteria, Escherichia coli, which is well understood and can be investigated in detail at the molecular level.
Faculty
Prof Jun-ichi Kato e-mail
Asc Prof Shigeki Ehira e-mail
Ast Prof Nobuhisa Furuya e-mail
Identification of the E. coli minimum gene set
To identify the minimum set of genetic information which is essential for cell proliferation, several types of long chromosomal deletions were systematically constructed. Using these long chromosomal deletions, we identified all of the essential genetic information including small essential genes and essential chromosome regions encoding no proteins or no RNA. The deletion mutations we have constructed cover more than 90% of the whole genome, and during construction of these deletions, we identified novel essential genes. We also succeeded in constructing an E. coli strain that lacks about 30% of the parental chromosome; there are no organisms which have such significantly reduced genome. Our final goal is to construct "minimum E. coli" which has the minimum gene set.
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Systematic Construction of E. coli deletion mutants
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Large-scale genome reduction
Functional analysis of non-characterized essential genes
We are trying not only to identify all of the essential genes but also to clarify their cellular functions. Our first approach to understanding the functions of the non-characterized essential genes is genetic analysis. Isolation of their mutants (temperature-sensitive mutants) and analyses of their phenotypes at the non-permissive temperature sometimes provide clues, and further isolation of suppressor mutants may enable identification of functionally relevant genes and yield hints. On the basis of these genetic analyses, biochemical studies at the molecular level are necessary to understand in detail the activity of the gene products. We are especially interested in the mechanism of chromosome replication, partition and cell division. We have identified many important essential factors, topoisomerase IV, Hda protein and so on. Recently, we have also investigated the regulatory mechanism of gene expression and RNA degradation.
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Identification of essential DNA topoisomerase, topo IV
Regulatory network of cellular differentiation in cyanobacteria
Cyanobacteria are a large and morphologically diverse group of phototrophic prokaryotes with wide ecological tolerance that they occur in almost every habitat on earth. Some cyanobacteria can use not only CO2 as a carbon source by photosynthesis, but also N2 as a nitrogen source by nitrogen fixation. Filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, form differentiated cells called heterocysts, which are specialized cells for nitrogen fixation. Upon limitation of combined nitrogen in the medium, particular vegetative cells within linear multicellular filaments differentiate into heterocysts with a regular spacing of 10-15 cells. Heterocyst differentiation takes about 24 h to complete, when approximately 10% of chromosomal genes are upregulated with spatiotemporal regulation. We are analysing the molecular mechanisms of cellular differentiation and pattern formation using cyanobacteria.
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Analysis of the mechanism of plasmid conjugation
It is known that certain plasmids mediate the transfer of genetic information of one cell into another cell. This process is called conjugation. We are analyzing the mechanism of DNA replication during conjugation using the R64 plasmid.
Recent Publications
  1. Kurata, T., Nakanishi, S., Hashimoto, M., Taoka, M., Yamazaki, Y., Isobe, T., and Kato, J. (2015) Novel essential gene involved in 16S rRNA processing in Escherichia coli. J Mol. Biol. 427: 955-965.
  2. Ehira, S., Miyazaki, S. (2015) Regulation of genes involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 by a group 2 sigma factor SigC. Life 5:587-603.
  3. Nishimura, T., Hayashi, K., Suzuki, H., Gyohda, A., Takaoka, C., Sakaguchi, Y., Matsumoto, S., Kasahara, H., Sakai, T., Kato, J., Kamiya, Y., and Koshiba, T. (2014) Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis. The Plant Journal. 77: 352-366.
  4. Ehira, S., Kimura, S., Miyazaki, S., Ohmori, M. (2014) Sucrose synthesis in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 is controlled by the two-component response regulator OrrA. Appl. Environ. Microbiol. 80:5672-5679.
  5. Halimatul, H.S.M., Ehira, S., Awai, K. (2014) Fatty alcohols can complement functions of heterocyst specific glycolipids in Anabaena sp. PCC 7120. Biochem. Biophys. Res. Commun. 450:178-183.
  6. Ehira, S. and Ohmori, M. (2014) NrrA directly regulates expression of the fraF gene and antisense RNAs for fraE in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Microbiology 160:844-850.
  7. Watanabe, M., Semchonok, D.A., Webber-Birungi, M.T., Ehira, S., Kondo, K., Narikawa, R., Ohmori, M., Boekema, E.J. and Ikeuchi, M. (2014) Attachment of phycobilisomes in an antenna-photosystem I supercomplex of cyanobacteria. Proc. Natl. Acad. Sci. USA 111:2512-2517.
  8. Hashimoto, C., Hashimoto, M., Honda, H., and Kato, J. (2013) Effects on IS1 transposition frequency of a mutation in the ygjD gene involved in an essential tRNA Modification in Escherichia coli. FEMS Microbiology Letter. 347: 140-148.
  9. Ehira, S. (2013) Transcriptional regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120. Russ. J. Plant Physiol. 60:443-452.
  10. Kushige, H., H. Kugenuma, M. Matsuoka, S. Ehira, M. Ohmori, and H. Iwasaki. (2013) Genome-wide and heterocyst-specific circadian gene expression in the filamentous cyanobacterium, Anabaena sp. PCC 7120. J. Bacteriol. 195:1276-1284.
  11. Iwamoto, A., Osawa, A., Kawai, M., Honda, H., Yoshida, S., Furuya, N., and Kato, J. (2012) Mutations in the essential Escherichia coli gene, yqgF, and their effects on transcription. J. Mol. Microbiol. Biotechnol. 22: 17-23.
  12. Ehira, S., and M. Ohmori. (2012) The redox-sensing transcriptional regulator RexT controls expression of thioredoxin A2 in the cyanobacterium Anabaena sp. strain PCC 7120. J. Biol. Chem. 287:40433-40440.
  13. Ehira, S., and M. Ohmori. (2012) The pknH gene restrictively expressed in heterocysts is required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120. Microbiology 158:1437-1443.
  14. Tanaka, Y., S. Ehira, H. Teramoto, M. Inui, and H. Yukawa. (2012) Coordinated regulation of gnd encoding 6-phosphogluconate dehydrogenase by two transcriptional regulators GntR1 and RamA in Corynebacterium glutamicum. J. Bacteriol. 194: 6527-6536.
  15. Hashimoto, C., Sakaguchi, K., Taniguchi, Y., Honda, H., Oshima, T., Ogasawara, N., and Kato, J. (2011) Effects on transcription of mutations in ygjD, yeaZ, and yjeE genes involved in a universal tRNA modification in Escherichia coli. J. Bacteriol. 193: 6075-6079.
  16. Iwadate, Y., Honda, H., Sato, H., Hashimoto, M. and Kato, J. (2011) Oxidative stress sensitivity of engineered Escherichia coli cells with a reduced genome. FEMS Microbiology Letter. 322: 25-33.
  17. Tachikawa, T., and Kato, J. (2011) Suppression of the temperature-sensitive mutation of the bamD gene required for the assembly of outer membrane proteins by multicopy of the yiaD gene in Escherichia coli. Biosci. Biotechnol. Biochem. 75: 162-164.
  18. Ehira, S., and M. Ohmori. (2011) NrrA, a nitrogen-regulated response regulator protein, controls glycogen catabolism in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. J. Biol. Chem. 286:38109-38114.
  19. Ehira, S., H. Teramoto, M. Inui, and H. Yukawa. (2010) A novel redox-sensing transcriptional regulator CyeR controls expression of an old yellow enzyme family protein in Corynebacterium glutamicum. Microbiology 156:1335-1341.
  20. Yoshimura, H., Y. Kaneko, S. Ehira, S. Yoshihara, M. Ikeuchi, and M. Ohmori. (2010) CccS and CccP are involved in construction of cell surface components in the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Cell Physiol. 51:1163-1172.
  21. Fujisawa, T., R. Narikawa, S. Okamoto, S. Ehira, H. Yoshimura, I. Suzuki, T. Masuda, M. Mochimaru, S. Takaichi, K. Awai, M. Sekine, H. Horikawa, I. Yashiro, S. Omata, H. Takarada, Y. Katano, H. Kosugi, S. Tanikawa, K. Ohmori, N. Sato, M. Ikeuchi, N. Fujita, and M. Ohmori. (2010) Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39. DNA Res. 17:85-103.
  22. Toyoshima, M., N. Sasaki, M. Fujiwara, S. Ehira, M. Ohmori, and N. Sato. (2010) Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120. Arch. Microbiol. 192:23-31.
  23. Ehira, S., H. Ogino, H. Teramoto, M. Inui, and H. Yukawa. (2009) Regulation of quinone oxidoreductase by a redox-sensing transcriptional regulator QorR in Corynebacterium glutamicum. J. Biol. Chem. 284:16736-16742.
  24. Ehira, S., H. Teramoto, M. Inui, and H. Yukawa. (2009) Regulation of Corynebacterium glutamicum heat shock response by the extracytoplasmic-function sigma factor SigH and transcriptional regulators HspR and HrcA. J. Bacteriol. 191:2964-2972.
  25. Asai, H., S. Iwamori, K. Kawai, S. Ehira, J. Ishihara, K. Aihara, S. Shoji, and H. Iwasaki. (2009) Cyanobacterial cell lineage analysis of the spatiotemporal hetR expression profile during heterocyst pattern formation in Anabaena sp. PCC 7120. PLoS ONE 4:e7371.
  26. Ohmori, K., S. Ehira, S. Kimura, and M. Ohmori. (2009) Changes in the amount of cellular trehalose, the activity of maltooligosyl trehalose hydrolase, and the expression of its gene in response to salt stress in the cyanobacterium Spirulina platensis. Microbes Environ. 24:52-56.
  27. Inoue, A., Murata, Y., Takahashi, H., Tsuji, N., Fujisaki, S., and Kato, J. (2008) Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli. J. Bacteriol. 190: 7298-7301.
  28. Mizoguchi, H., Sawano, Y., Kato, J., and Mori, H. (2008) Superpositioning of deletions promotes growth of Escherichia coli with a reduced genome. DNA Research 15: 277-284.
  29. Yoshida, H., Furuya, N., Lin, Y.-J., Güntert, P., Komano, T., Kainosho, M. (2008) Structural basis of the role of the NikA ribbon-helix-helix domain in initiating bacterial conjugation. J. Mol. Biol. 384: 690-701.
  30. Kato, J. and Hashimoto, M. (2008) Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome. Methods in Molecular Biology (Microbial Gene Essentiality - Protocols and Bioinformatics) 416: 279-293. (Osterman, A. L. and Gerdes, S. Y. (ed.), Humana Press, Totowa, New Jersey)
  31. Ehira, S., T. Shirai, H. Teramoto, M. Inui, and H. Yukawa. (2008) Group 2 sigma factor SigB of Corynebacterium glutamicum positively regulates glucose metabolism under conditions of oxygen deprivation. Appl. Environ. Microbiol. 74:5146-5152.
  32. Kato, J. and Hashimoto, M. (2007) Construction of consecutive deletions of the Escherichia coli chromosome, Mol. Syst. Biol. 3: Article number 132
  33. Shimoda, E., Muto, T., Horiuchi, T., Furuya, N., and Komano T. (2007) Novel class of mutations of pilS mutants, encoding plasmid R64 type IV prepilin: interface of PilS-PilV interactions. J. Bacteriol. 190: 1202-1208.
  34. Gyohda, A., Zhu, S., Furuya, N. and Komano, K. (2006) Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences. J. Biol. Chem. 281:20772-20779.
  35. Ikeuchi, Y., Shigi, N., Kato, J., Nishimura, A., and Suzuki, T. (2006) Mechanistic insights into sulfur-relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol Cell 21: 97-108.
  36. Ote, T., Hashimoto, M., Ikeuchi, Y., Su'etsugu, M., Suzuki, T., Katayama, T., and Kato, J. (2006) Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation . Mol. Microbiol. 59: 265-275.
  37. Ehira, S., and M. Ohmori. (2006) NrrA directly regulates expression of hetR during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 188:8520-8525.
  38. Ehira, S., and M. Ohmori. (2006) NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Mol. Microbiol. 59:1692-1703.
  39. Kato, J. (2005) Regulatory network of the initiation of chromosomal replication in Escherichia coli. Crit. Rev. Biochem. Mol. Biol. (Critical Reviews in Biochemistry and Molecular Biology) 40: 331-342.
  40. Ikeuchi, Y., Soma, A., Ote, T., Kato, J., Sekine, Y., and Suzuki, T. (2005) Molecular mechanism of lysidine synthesis that determines tRNA identity and codon recognition. Mol. Cell 19: 235-246.
  41. Hashimoto, M., Ichimura, T., Mizoguchi, H., Tanaka, K., Fujimitsu, K., Keyamura, K., Ote, T., Yamakawa, T., Yamazaki, Y., Mori, H., Katayama, T. and Kato, J. (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol. Microbiol. 55: 137-149.
  42. Akahane, K., D. Sakai, N. Furuya, and T. Komano (2005) Analysis of the pilU gene encoding prepilin peptidase for type IV pilus biogenesis in plasmid R64. Mol. Gen. Genomics 273:350-359.
  43. Gyohda, A., Furuya, N., Ishiwa, A., Zhu, S. and Komano, T. (2004) Structure and function of the shufflon in plasmid R64. Adv. Biophys. 38:183-213.
  44. Soma, A., Ikeuchi, Y., Kanemasa, S., Kobayashi, K., Ogasawara, N., Ote, T., Kato, J., Watanabe, K., Sekine, Y., and Suzuki, T. (2003) An RNA-modifying enzyme that governs both the codon and amino acid specificities of isoleucine tRNA. Mol. Cell 12:689-698.
  45. Hashimoto M, Kato J. (2003) Indispensabilityof the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure. Biosci Biotech Biochem. 67: 919-922.
  46. Furuya, N. and T. Komano (2003) NikAB- or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors. J. Bacteriol. 185:3871-3877.
  47. Ehira, S., M. Ohmori, and N. Sato. (2003) Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 10:97-113.
  48. Kato, J. and Katayama, T. (2001) Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli. EMBO Journal 20: 4253-4262.
  49. Kato, J., Fujisaki, S., Nakajima, K., Nishimura, Y., Sato, M., and Nakano, A. (1999) The E. coli homologue of yeast Rer2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis. J. Bacteriol. 181: 2733-2738.
  50. Tsukamoto, Y., Kato, J., and Ikeda, H. (1997) Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 388: 900-903.
  51. Shimizu, H., Yamaguchi, H., Ashizawa, Y., Kohno, Y., Asami, M., Kato, J., and Ikeda, H. (1997) Short-homology-independent illegitimate recombination in Escherichia coli: distinct mechanism from short-homology-dependent illegitimate recombination. J. Mol. Biol. 266: 297-305.
  52. Yokochi, T., Kato, J., and Ikeda, H. (1996) DNA nicking by Escherichia coli topoisomerase IV with a substitution mutation from tyrosine to histidine at the active site. Genes to Cells 1: 1069-1075.
  53. Kato, J., Suzuki, H., and Ikeda, H. (1992). Purification and characterization of DNA topoisomerase IV in Escherichia coli. J. Biol. Chem. 267: 25676-25684.
  54. Kato, J., Nishimura, Y., Imamura, R., Niki, H., Hiraga, S., and Suzuki, H. (1990). New topoisomerase essential for chromosome segregation in E. coli. Cell 63: 393-404.
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