F.GENETIC STRAINS RESEARCH CENTER
F-f. Microbial Genetics Laboratory - Akiko Nishimura Group

RESEARCH ACTIVITIES

(1) Dynamics of bacterial tubulin: coupling model between DNA replication and cell division

Ippei Inoue, Kenji Yasuda and Akiko Nishimura

--The most approaches understanding cell division have asked whether cell division occurs coupling with DNA replication. To solve this problem, we examined the dynamics of FtsZ, an essential key protein of Escherichia coli cell division, in relation to the stage of DNA replication by creating a fusion protein containing FtsZ and a green fluorescent protein (GFP). FtsZ started to assemble at potential division site coincident with initiation of DNA replication, all FtsZ assembled in the ring structure coincident with termination of replication, and the ring constricted after nucleoids separation. These results suggest that cell division occurs in association with DNA replication.

(2) A complete set of Escherichia coli open reading frames in mobile plasmids and their successful application to the systematic identification of cell division mutant (fts) genes

Kimiko Saka, Maki Tadenuma, Shinsuke Nakade, Noriko Tanaka, Hideaki Sugawara, Ken Nishikawa, Nobuyuki Ichiyoshi, Masanari Kitagawa, Hirotada Mori, Naotake Ogasawara and Akiko Nishimura

--To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). The vector for mobile plasmid clones carries the following genes and sequences; (i) bom, rom, and mob of ColE1, which enable the plasmid to transfer from F⁺ to F^- by F-mediated conjugation; (ii)P_tac/ lacI^q, which enables control of the expression of the cloned ORF by IPTG; (iii) the ColE1 replication origins, ori and pri, to maintain a low plasmid copy number in the cell under normal growth condition; (iv) the ampicillin resistant gene, bla, for selection; (v) the unique multi-cloning site of pJF118HE; (vi) universal primer recognition sites -21M13 and SP6 for confirmation of the cloned ORF by sequencing; and (vii) rrnB with a transcriptional terminator which prevents read-through. Since replication of the plasmid in vivo does not require protein synthesis, the plasmid can be enriched during inhibition of protein synthesis that leads to inhibition of chromosomal replication without blocking the plasmid replication. To clone an ORF with its native SD into pNT3, we amplified the predicted ORF along with 20 bp of the upstream region by PCR using primer sets designed to contain the appropriate restriction sites. With this procedure, native products should be produced even if fragments longer than necessary are cloned. 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. In order to simplify the screening method, we investigated the possibility of searching for positive clones from a mixed pool of unrelated clones. We first made a test stock composed of subpools of 48, 24, 12, or 6 clones within single wells of a 96-well microtiter plate. A complementation test was carried out using ftsA, ftsI, ftsW, and ftsZ mutants. All four mutants were complemented by the subpools of 48, 24, 12, or 6 clones, or by a single clone. There were fewer and smaller colonies in the ftsZ-positive patches on the 1 mM IPTG plate than on the 0.1 mM IPTG or no IPTG plates. The results are supported by the facts that low-level expression of ftsZ (less than two times) can complement the ftsZ mutant, but high level expression of ftsZ (more than four times) causes inhibition of growth of the ftsZ mutant. The size of the ftsA-positive patches was the largest on the 1 mM IPTG-supplemented plate and very small on a plate without IPTG. The ftsA mutants might need high-level expression of FtsA for normal growth when it is provided from plasmid-born ftsA. The results for ftsI were similar to ftsA. Changes in IPTG concentration did not affect the size of the ftsW-positive patches. These results indicate that the 48-clone pool is effective for complementation studies if we use the three selection plates containing 0, 0.1, and 1 mM of IPTG. Having demonstrated that we can identify the positive ORF within a mixture of 48 clones, we created two types of stock: a single microtiter plate containing pools of 48 clones in each well, and a second stock composed of 45 microtiter plates containing the individual clones. In summary, to find the clone that complements the mutant, we first identify the positive mixed pool and then determine which clone in the mixture complements the mutant. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations.

(3) Global regulation of cell division: Isolation of a whole set of cell division mutants

Kimiko Saka, Noriko Matsumoto and Akiko Nishimura

--The entire nucleotide sequence of E. coli has been analyzed, and 4311 ORFs have been demonstrated, but the functions of more than half of these ORFs remain unknown. The greater part of these ORFs are considered to be involved in coordinating cell proliferation. To thoroughly analyze the hierarchy and network responses in expression of cell division genes, as one of the model cases for post-genome analysis, we have identified a whole set of cell division genes using above mobile plasmid clone sets by their ability to complement a filament-temperature-sensitive (fts) cell division mutants. A total of 339 fts strains from the Hirota thermosensitive (Ts) mutant bank, which form multi-cellular filaments at 41℃ without immediate arrest of DNA synthesis or an increase in cell mass were tested. We found that 278 fts mutants were complemented by 403 of ORF clones. Of these, 69% of the fts mutants were complemented by one ORF each. Sequence analysis of genomic DNA of 10 of these fts mutants showed that all had missense mutation in the corresponding ORF. However, 15% of the fts mutants were complemented by two ORFs, and 16% by three ORFs. These may contain the allele of the fts mutant gene and a high-dosage suppressor gene(s). Sequence analysis of five of these fts mutants showed that all five had missense mutation corresponding to one ORF and no mutation in the remaining ORF(s). The cog database (http://www.ncbi.nlm.nih.gov/COG/new/) was consulted for functional annotation of individual genes. From the functional annotation analysis, known cell division genes were found in only 6% of the fts mutants, while 30% were unknown. Of all genes identified, 24% were involved in a basic process, such as DNA replication or protein synthesis, despite the fact that the mutants involved in this category had presumably been excluded by the process of selection of the fts mutants. 19% were involved in cellular processes, such as ion transport and signal transduction; and 21% were involved in metabolism. These results suggest that various intracellular reactions are coordinated with cell division and that the E. coli cell cycle is the result of the coordination of multiple independent processes, so that each daughter cell receives a complete copy of cell components. We are currently planning to purify these mutations in cells with wild-type background, and thoroughly analyze the hierarchy and network responses in expression of cell division genes using DNA chip technologies along with these mutants, as one of the models for post-genome analysis for global cellular regulation in E. coli.

PUBLICATIONS

Papers
1. Saka, K., Tadenuma, N., Nakade, S., Tanaka, N., Sugawara, H., Nishikawa, K., Ichiyoshi, N., Kitagawa, M., Mori, H., Ogasawara, N. and Nishimura, A. (2004). A complete set of Escherichia coli open reading frames in mobile plasmids and their successful application to the systematic identification of cell division mutant (fts) genes. (DNA res., imprinting).

Database
2. http://shigen.lab.nig.ac.jp/ecoli/strain/top/top.jsp

ORAL PRESENTATIONS

池内与志穂、野間章子、相馬亜希子、加藤潤一、西村昭子、三木健良、小林和夫、小笠原直毅、関根靖彦、鈴木努「リボヌクレオソーム解析を用いたRNA修飾遺伝子の網羅的探索」第27回日本分子生物学会・神戸・2004年12月
西村昭子・「NBRP中核機関整備:大腸菌」第27回日本分子生物学会・神戸・2004年12月

POSTER PRESENTATIONS

井之上一平、安田賢二、西村昭子「バクテリアのDNA複製と細胞分裂の共役」第27回日本分子生物学会・神戸・2004年12月
岡本元子、米澤寿美子、西村昭子「大腸菌ftsX遺伝子の機能解析」第27回日本分子生物学会・神戸・2004年12月
伊藤敬子、麻生文子、末永由美子、西村昭子「NBRP中核拠点整備:大腸菌」第27回日本分子生物学会・神戸・2004年12月

SOCIAL CONTRIBUTIONS AND OTHERS

特許
1. 出願番号未定,マルチウェルプレート,西村昭子・梶谷隆文,大学共同利用機関法人情報システム研究機構・有限会社カジックストレーディング
2. 特許2004-195247,超薄型マルチウェルプレートの製造法,西村昭子・富川宗博・相吉三宏,大学共同利用機関法人情報システム研究機構・静宏産業株式会社

学会活動
Dr. A. Nishimura was appointed for a member of editorial board of “Microbiology and Culture Collections".