Dynamic organization of chromosomal DNA in Escherichia coli.
Journal: 2000/February - Genes and Development
ISSN: 0890-9369
PUBMED: 10652275
Abstract:
We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.
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Genes Dev 14(2): 212-223

Dynamic organization of chromosomal DNA in <em>Escherichia coli</em>

“Unit Process and Combined Circuit,” PRESTO, Japan Science and Technology Corporation (JST); Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, 862-0976, Japan
Corresponding author.
Received 1999 Oct 18; Accepted 1999 Dec 8.

Abstract

We have revealed the subcellular localization of different DNA segments that are located at ∼230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.

Keywords: Prokaryote, cell division, FISH, nucleoid, partitioning, segregation
Abstract

The bacterial chromosome, 1000 times the length of the bacterium, is folded by an unknown mechanism and organized in a compact form called the nucleoid. Carefully isolated nucleoids from Escherichia coli have been investigated in vitro (for review, see Pettijohn 1996). The nucleoid is a closed duplex structure with a series of loops. The DNA loops are negatively supercoiled, resulting in further compaction. Moreover, several DNA-binding proteins, including HU, IHF, and H-NS, may promote a compact state of DNA in nucleoids. Interestingly, mutants defective in HU and/or H-NS accumulate anucleate cells (Wada et al. 1988; Kaidow et al. 1995). The compaction of nucleoids may be related to the mechanism of chromosome partitioning. This possibility is strongly supported by investigations of bacterial homologs of the eukaryotic SMC (structural maintenance of chromosome) protein. The SMC protein is involved in chromosome condensation, pairing, and partitioning in eukaryotic cells from yeast to human (for review, see Hirano 1999). In Bacillus subtilis, smc null mutations cause a temperature-sensitive lethal phenotype and failure to partition the chromosome (Britton et al. 1998; Moriya et al. 1998), similar to mukB null mutants in E. coli (Niki et al. 1991). The MukB protein does not share the common amino acid motif found in the SMC family. Nevertheless, the morphological structure of the MukB protein molecules is remarkably similar to that of SMC proteins (Niki et al. 1992; Melby et al. 1998). Thus, elucidation of the structure of compacted nucleoids and chromosome organization in vivo is important for understanding the mechanism of chromosome partitioning.

The bacterial nucleoid is amorphous in vivo because the folded chromosome is not highly condensed throughout the cell division cycle. Replication and transcription take place simultaneously, even during chromosome partitioning. The DNA packing density in the nucleoid has been estimated in exponentially grown E. coli cells (Kellenberger 1990). It is similar to that of interphase nuclei of eukaryotes. Therefore, whole chromosomes seem to migrate to daughter cells with coupling to cell elongation but not by an active mechanism such as a mitotic apparatus. Recently, it has become apparent that specific DNA segments on bacterial chromosomes migrate rapidly during chromosome partitioning (Glaser et al. 1997; Gordon et al. 1997; Lin et al. 1997; Webb et al. 1997, 1998; Niki and Hiraga 1998). The results revealed that the oriC DNA segment in newborn cells is localized at a nucleoid border and the replication terminus DNA segment is localized at the opposite nucleoid border. One copy of the replicated oriC segment remains at its nucleoid border and the other copy migrates to the opposite nucleoid border. On the other hand, the terminal DNA segment migrates from the nucleoid border to mid-cell in the early stage of the cell division cycle. Moreover, chromosomal DNA segments midway between oriC and the replication terminus tend to be localized at subcellular positions between oriC and the terminus (Teleman et al. 1998). Cytological methods, based on subcellular localization of a few chromosome segments, have thus revealed a dynamic organization of bacterial chromosomes. Further examination of various segments on the chromosome clearly reveals the organization of the whole nucleoid.

The cell division cycle of bacteria can be defined in terms of two constants, the C and D periods. During the E. coli cell division cycle, the C period is the time during which a round of chromosome replication takes place, lasting 40 min at 37°C. The D period is the period between completion of chromosome replication and the subsequent cell division—20 min at 37°C. When cells duplicate every 60 min at 37°C (doubling time of 60 min), chromosome replication initiates soon after the cells divide. Cells growing at doubling times of >60 min may have an additional stage, the B period, between birth of the newborn cell and the initiation of replication, which resembles the G1 phase of eukaryotic cell cycle.

In the present work, we examine the subcellular localization of 22 DNA segments of the E. coli chromosome by the FISH method. The series of chromosome segments was localized within the cell in the same order as the chromosome map. Our results demonstrate that the circular chromosomal DNA is organized to form a compact ring structure in the cell. The dynamic organization of the nucleoid plays a critical role in chromosome replication, partitioning, and cell division in the bacterial cell cycle.

Acknowledgments

We are grateful to K. Hayashi, T, Horiuchi, and H. Mori for providing the Kohara phage DNAs, T. Onogi for photomicrographs of DAPI-stained cells, and J.-M. Louarn for providing the inversion mutants. We thank Richard D'Ari for critical reading of the manuscript and comments. This work was supported by a grant from the Grants-in-Aid for Scientific Research (C), Grants-in-Aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports, and Culture of Japan, and a grant from the Human Frontiers Science Program (RG-386/95M). H. N. is supported by Ciba-Geigy Foundation (Japan) for the Promotion of Science.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL pj.ca.u-otomamuk.opg@ikin; FAX 81-96-371-2408.

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