Asc Prof

Junichi Kato

e-mail

jkato@comp.metro-u.ac.jp

Ast Prof

Nobuhisa Furuya

e-mail

furuya-nobuhisa@c.metro-u.ac.jp

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.

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.

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.

Related Links

Recent Publications

1. Kato, J. and Hashimoto, M. (2006) Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome. Methods in Molecular Biology (Gene Essentiality at Genome Scale: Protocols and Bioinformatics), in press.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Ishiwa, A. and Komano, T. (2004) PilV adhesins of the plasmid R64 thin pili specifically bind to the lipopolysaccharides of recipient cells. J. Mol. Biol. 343:615-625.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. Kato, J. and Katayama, T. (2001) Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli. EMBO Journal 20: 4253-4262.
15. 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.
16. Tsukamoto, Y., Kato, J., and Ikeda, H. (1997) Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 388: 900-903.
17. 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.
18. 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.
19. Kato, J., Suzuki, H., and Ikeda, H. (1992). Purification and characterization of DNA topoisomerase IV in Escherichia coli. J. Biol. Chem. 267: 25676-25684.
20. 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.