Genes Genet. Syst. (2009) 84 477 Symposia (S1-1 – S2-10) 3E EMOTO, Kazunori1, WADA, Takeo1, KUTSUKAKE, S1 IKEDA, Masato1 (1Dept. BioSci. and BioTech., Faclt. -11 Kazuhiro1 (1Grad. Sch. Natural Sci. Tech., Okayama -1 Agr., Shinshu Univ.) Univ.) Genome-based strain reconstruction for amino acid produc- Target protein of the RpoS-induced toxin in Salmonella tion enterica serovar Typhimurium The classical approach to strain improvement involves random Although toxin-antitoxin (TA) systems are ubiquitously present mutagenesis and screening, but this technology cannot avoid on bacterial chromosomes, their biological significance has accumulating detrimental or unnecessary mutations. Genome- remained enigmatic. We previously reported that Salmonella based strain reconstruction is a technology that identifies the enterica serovar Typhimurium possesses a toxin gene rsaB, useful mutations in such strains through genome sequencing and which is induced by a stress-specific sigma factor RpoS and then systematically and precisely engineers those mutations into causes growth arrest. Together with its cognate antitoxin gene the wild-type genome, creating a strain with only the useful rsaA, this gene seems to constitute a TA system on the mutations. Strains derived by this reverse engineering method Salmonella chromosome. In this study, as the first step to are expected to be more robust, give higher fermentation yields in elucidate the physiological role of this TA system, we attempted a shorter time and resist stressful conditions. Here we describe to identify a target protein of the toxin. From the cell lysate of the technology by using L-lysine fermentation as a model. Salmonella, an RNA-binding protein CsrA was found to be co- purified with the GST-tagged RsaB protein. Because CsrA is known to be essential for Salmonella growth, this protein is a plausible candidate of the target protein of RsaB. CsrA is known also to inhibit glycogen synthesis through its binding to mRNA of the glgC gene. We showed that RsaB inhibits CsrA from binding to glgC mRNA in vitro. Consistent with this, expression of RsaB within the cell enhanced glycogen synthesis in Salmonella.On the basis of the obtained results, we discuss the physiological role of this TA system. 3E FUKUSHIMA, Tatsuya1, FURIHATA, Isako1, S1 SEKIGUCHI, Junichi1,2, FUKUSHIMA, Tatsuya2, -12 TOKORO, Hideyuki1, SZURMANT, Hendrik2, -2 HASHIMOTO, Masayuki3, YAMAMOTO, Hiroki4 HOCH, James A.2 (1Div. Gene Res., Dept. Life Sci., (1Interdisciplinary Grad. Sch. Sci. and Tech., Shinshu Res. Cent. for Human and Env. Sci.s, Shinshu Univ., Univ., 2Div. Gene Res., Res. Cent. for Human and Env. 2The Scripps Res. Inst.) Sci.s, Shinshu Univ., 3Internatl. Young Researchers Empowerment Center, Shinshu Univ., 4Experimental Activation mechanism of cell division sensor, YycG Farm, Faclt. Textile Sci. and Tech., Shinshu Univ.) The recognition of cell division is very important for the Functions of cell wall binding proteins investigated on the microorganisms. However, the question is how the microorgan- genome analysis isms recognize the timing of cell division. As the answer for this question, recently we demonstrated the essential two-component Among cell wall binding proteins, the largest group consists of system (TCS), YycFG in Bacillus subtilis, senses the cell division. cell wall degrading enzymes. In Bacillus subtilis more than 35 Moreover, the inhibitors, YycH and YycI for the TCS, were gene products are predicted to be cell wall degrading enzymes, identified. Now we are studying the activation mechanism of cell and more than 20 products have been analyzed. Some cell wall division sensor, YycG. degrading enzyme genes are located in pro-phage regions and Immnofluorescent microscopic observation showed the sensor integrative conjugative element. Although the enzymes are kinase, YycG, localizes at cell division site and the localization is associated with cell lysis, the other physiological functions, such independent of the inhibitors, YycHI. Immunoprecipitation as cell differentiation (spore formation and germination), cell wall experiment demonstrated YycH interacts to YycI without YycG turnover and cell separation during the vegetative cell growth, and the YycH-I complex is associated with YycG in non-dividing have been reported. Various substrate specificities for peptido- cells. Bacterial two-hybrid system for YycG and cell division glycan (PG) are found in cell wall hydrolases: 3 are associated proteins indicates YycG interacts to the cell division proteins. with the glycan portion and 5 are associated with the peptide Since the signal transduction of the TCS is occurred by cell portion. Recently it was reported the essential two-component division, the switch of inactivation and activation of YycG may be system, YycFG in B. subtilis, positively regulates two PG dependent on the interaction partners, YycHI and division hydrolases (CwlO and LytE) and negatively regulates PG hydro- proteins. lase-inhibitory protein (IseA). In this presentation, we will report the characterization of CwlO and LytE-double mutant and domain exchange of these proteins, and we will discuss with the lateral PG biosynthesis. 478 Genes Genet. Syst. (2009) 84 S1 SASAKI, Hiroyuki1 (1Dept. Integrated Genetics, Natl. S1 SHIRAHIGE, Katsuhiko1, BANDO, Masashige1, -3 Inst. Genet., Research Org. of Inform. and Systems) -5 SAKAI, Akiko1, KATOU, Yuki1, ITOH, Takehiko1 (1Dept. Biol. Sci.s, Tokyo Inst. Tech.) Identification and characterization of functional small RNAs by next generation sequencers Genomic Analyses of Cohesinopathy Functional small RNAs are highly conserved through evolution Cohesin regulates sister chromatid cohesion during the mitotic and have important roles in various biological processes, such as cell cycle. In addition to this role, cohesin has also been cell differentiation and development, defense against viruses and demonstrated to play a critical role in regulation of gene other parasitic agents, and heterochromatin formation. The small expression in nondividing cells as a transcriptional insulator. RNAs are classified into three major categories, i.e. miRNA, Heterozygous mutations in the cohesin loader NIPBL or cohesin siRNA and piRNA, depending on the biogenesis pathway and structural components result in the multisystem developmental interacting factors. The emergence of the next generation disorder Cornelia de Lange Syndrome (CdLS). Genome-wide sequencers now enables us to know the composition of small assessment of transcription in 16 mutant cell lines from severely RNA repertoire in a given cell, mode of their biogenesis, target affected CdLS probands has identified a unique profile of genes, and presence/absence of RNA editing. In this symposium, I dysregulated gene expression. In B cell lines established from will present some of our recent results and discuss the power of CdLS patients, 60% of cohesin binding sites were lost compared the next generation sequencers in small RNA studies. to healthy control B cell lines, suggesting that 40% of cohesin sites are sufficient for mitotic function of cohesin. Cohesin localization analysis also demonstrates a preference for inter- genic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregu- lated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor. S1 SUZUKI, Yutaka1 (1Dept. of Med. Genome Sci., Grad. S1 GOJOBORI, Takashi1 (1Natl. Inst. Genet.) -4 Sch. of Sci, The Univ. Tokyo) -6 Integrated Analysis of Human Alternative Promtoers using Database in the time of Next Generation Sequencers Next Generation Sequencer We carried out genome-wide analysis of the putative alternative promoters in human genes by multifaceted use of massive paralleled sequencer. We characterized 130 million transcrip- tional start site (TSS) sequence tags which were identified by TSS Seq analysis in 12 human cell types. Although the number of the TSS tag clusters (TSCs) is generally large throughout the cell types, TSCs showing significant expression levels were relatively rare and had distinctive several sequence features from other lowly expressed TSS tag clusters. ChIP-Seq and Nucleosome-Seq analyses revealed highly-ordered nucleosome structure and strong signals of the binding of polymerase II predominantly around the TSCs of the former group, possibly allowing their deterministic transcriptions. Evaluation of 16080 previously sequenced and 846 newly shotgun-sequenced complete cDNA sequences also showed that transcripts associated with those TSCs are more likely to be translated. By RNA Seq analysis using polysome fraction, we directly observed at least one-third of those transcript products are incorporated in translating ribosome. Genes Genet. Syst. (2009) 84 479 S1 UCHIYAMA, Ikuo1 (1Natl. Inst. Basic Biol., Natl. S1 HATTORI, Masahira1, OSHIMA, Kenshiro1, KIM, -7 Institutes of Natural Sci.) -9 Seokwon1, KUROKAWA, Ken2, TOH, Hidehiro3, TAYLOR, Todd3 (1Center for Omics and Bioinform., Comparative genome informatics in the era of high-through- Grad. Sch. Front. Sci.s, The Univ. Tokyo, 2Dept. Biol. put sequencing Inform., Tokyo Inst. Tech., 3RIKEN Adv. Sci. Inst.) Recent development of massively parallel sequencing technolo- Metagenomics and genomics decoding human gut micro- gies leads us to a new era of high-throughput sequencing, and is biomes revolutionizing biological research based