Journal of Structural Biology 156 (2006) 273–283 www.elsevier.com/locate/yjsbi 1047-8477/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2006.04.013 Minireview Structural and physical aspects of bacterial chromosome segregation Conrad L. Woldringh ¤ , Nanne Nanninga Molecular Cytology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands Received 31 March 2006; accepted 27 April 2006 Abstract Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase sepa- rate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid- binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. SpeciWc DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diVusion coeYcient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be eVectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. DiVerences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diVusion coeYcient could explain the linear positioning of genes in the nucleoid and that diVerential transcriptional activity could induce diVerent mobilities between either replich- ores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move speciWc chromosomal regions and the nucleoid as a whole without invoking a dedicated mech- anism.  2006 Elsevier Inc. All rights reserved. Keywords: Escherichia coli; Caulobacter crescentus; DNA segregation; Excluded-volume interaction; ConWned Brownian diVusion; DiVerential transcrip- tion; Replichores; Leading–lagging strand; MreB; Transertion 1. Introduction In spite of our detailed knowledge of the enzymology of DNA replication (Kornberg and Baker, 1992) and of the topology of gene expression (Willenbrock and Ussery, 2004; Peter et al., 2004), we do not understand how, on a larger scale, bacterial DNA is organized within cell or nucleoid. Also, in the process of segregation, we hardly know what force(s) move the newly replicated DNA strands faithfully to the prospective daughter cells. Many groups in the Weld of bacterial chromosome segrega- tion assume the involvement of a dedicated, “mitotic-like” mechanism as “the process is far too important to leave to chance” (Gitai et al., 2005a). This view is based on the obser- vation of a rapid movement of oriC-GFP spots (Gordon et al., 1997), and on the possible involvement of actin-related proteins (MreB; Kruse et al., 2006) such as those that segre- gate R1 plasmids (ParM; Garner et al., 2004). We will discuss the various observations on the dynamics of DNA within the nucleoids of Escherichia coli , Bacillus sub- tilis, and Caulobacter crescentus. Operationally, we distinguish three stages: (i) initial separation of origins; (ii) separation and positioning of replicated DNA regions; (iii) Wnal separation of the nucleoid. We will Wrst review DNA organization from global microscopic observations and then discuss the physical consequences of macromolecular crowding for chromosome compaction because we consider these issues as the basis for our understanding of DNA segregation. 2. Microscopic observations An important feature of the structural organization of the bacterial chromosome, we believe, is phase separation of DNA and cytoplasm. An indication for such a phase separation was already present in the time-lapse images of * Corresponding author. Fax: +31 20 525 6271. E-mail address: woldringh@science.uva.nl (C.L. Woldringh).