I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPAN

RESEARCH ACTIVITIES

(1) Chromosome partitioning mechanism of Escherichia coli; Involvement of a cisacting site on the chromosome

Yoshiharu Yamaichi and Hironori Niki

--During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization.

(2) Partitioning mechanism of F plasmid; elucidation of motive force for bipolar migration of the plasmid DNA

Toshiyuki Hatano and Hironori Niki

--In a bacterial cell, replicated chromosomal segments including the replication origin and the F plasmid actively move from midcell toward cell poles and are located at cell quarter positions. The Sop ABC partitioning system of F plasmid has been characterized genetically and biochemically to elucidate molecular mechanism of DNA partitioning. The SopA protein is a hypothetical motor protein because of its walker-type ATPase activity. Moreover, some proteins homologous to SopA change subcellular location periodically. We have constructed an active SopA-YFP and observed oscillation of YFP fluorescence from one cell pole to the other with an interval of a few minutes. Simultaneously migration of the plasmid DNA was detected using fluorescent labeling technique with lacO array and LacI-CFP. The analyses of movement of both the fluorescence suggest that SopA should not be the motor protein to move the plasmid DNA. Another new possibility is that the SopA protein could govern the direction of movement of the plasmid DNA.

(3) Subcellular localization of Escherichia coli proteins in living cells

Yasuyuki Ogata, Toshiyuki Hatano and Hironori Niki

--Bacterial cells are too small to see details of their structure using a light microscope because of the ultimate limit to the resolution of the microscope set by the wavelength of visible light. In recent years, the development of methods for the specific labeling and imaging of individual cell components and the reconstruction of their three-demensional architecture allows us to understand the structural organization of bacterial cells. Cytoskeletal filaments that bacteria were long thought to lack were found by means of these methods and visualization of DNA polymerase of the living Bacillus subtilis cells by tagging the catalytic subunit with fluorescent protein supported the model in which the polymerase is stationary like a factory and DNA is pulled through.
--Escherichia coli genome has been fully sequenced and the genes of unknown function revealed. To estimate their function, we used a plasmid library, archive clone library, that expresses fusion genes containing open reading flame (ORF) of E. coli W3110 strain and green fluorescent protein (GFP) gene and we observed their subcellular localizations in the living E. coli cells. These proteins were classified into five categories: (i) at cell periphery, (ii) in whole cell, (iii) spot, (iv) filament, and (v) on nucleoid.
--We subsequently investigated the localizations of the proteins of known function and found the correlations between subcellular localization and protein function. Most of the cell peripheral proteins have either trans membrane elements or signal peptides. Proteins with spots at cell quarter positions like a replication factory include replication machineries and recombination enzymes and proteins with several spots participate in transcription. Some proteins with a ring at mid cell are involved in cell division. Nucleoid proteins contain nucleoid constituents such as histone-like proteins and various enzymes involved in DNA-related events including DNA supercoiling, recombination, and repair.
--We further replaced stop codon of these genes with gfp gene by the method of Warner and confirmed subcellular localization of these proteins.

(4) Stationary phase-induced illegitimate recombination in Escherichia coli

Yasuyuki Ogata and Hideo Ikeda

--In nature, bacterial cells are usually in stationary stages, in which the cells alter their genetic inheritance. Adaptive mutations are induced when Escherichia coli cells are exposed to conditions of stationary-phase starvation, resulting in a variety of genetic changes, some of which allow the cells to survive under such conditions. Gene amplification of the lac operon of E. coli also occurs during stationary-phase starvation. Whether or not stationary-phase starvation induces chromosomal rearrangements has remained obscure.
--We found that illegitimate recombination is induced when growth phase is changed from log to stationary phase. Illegitimate recombination occurred more frequently in early-stationary phase than in mid-log phase during formation of λbio-transducing phage. Moreover, we found that illegitimate recombination increased in the tag alkA1 double mutant in early-stationary phase, thereby implying that the alkylated lesion may be responsible for the stationary phase-induced illegitimate recombination.
--When we did a quantitative analysis of deletion formation using a miniF-based plasmid, we found that the stationary phase-induced illegitimate recombination can be also detected in this assay system. In addition, we indicated that the frequency of deletion of the tag alkA1 double mutant was higher than that of its isogenic wild-type strain in early-stationary phase.
--Since illegitimate recombination results in chromosomal rearrangements including deletion, duplication, insertion, or translocation, it seems likely that stationary phase may induce chromosomal rearrangements. Thus, when bacterial cells confront unfavorable conditions for their growth, the cells seem to increase diverse mutations as if they struggle for adaptation to the environment. If the situation takes a favorable turn, a suitable mutant clone is likely to grow preferentially, which may lead to evolution.

PUBLICATIONS

Papers
1. Yamaichi, Y. and Niki, H. (2004). migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome. EMBO J. 23, 221-233.
2. Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M., Kubota, T., Ogata, Y., Sekimizu, K. and Katayama, T. (2004). DiaA, a novel DnaA-binding protein, ensures the timely initiation of E. coli chromosome replication. J. Biol. Chem., 279, 45546-45555.
3. Ikeda, H., Shiraishi, K. and Ogata, Y. (2004). Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli. Adv. Biophys., 38, 3-20.

Reviews
4. 仁木宏典（2004）「原核生物の染色体の分配起点」蛋白質,核酸,酵素49, 2017-2023.

Books
5. 仁木宏典,ゲノミクス・プロテオミクスの新展開 生物情報の解析と応用,エヌ・ティ・エス,47-56.