A recent development in the field of E. coli DNA replication is the identification of weak (incognito) binding sites for the initiator DnaA in the origin of replication, called I-sites. These sites play a key role in timing of replication initiation in the cell cycle. Because of the difficulty of recognizing I-sites, the generality of their occurrence has remained obscure. Richard Fekete and Tatiana Venkova-Canova have succeeded in localizing sequences with characteristics of an I-site in a second system (in the replication origin of plasmid P1). The sequence information should help to define an I-site better and thus help identification of such sites in other systems and study their roles in replication control. Bacteria capable of rapid proliferation such as E. coli and B. subtilis usually amplify the origin proximal region of their chromosome. In V. cholerae, Preeti Srivastava found this to be true for chromosome I but not II. The growth-rate insensitive behavior of the latter turned out to be due to homeostatic controls on its initiator RctB. Relaxing the control increased the copy number of chromosome II but decreased the cell growth rate, suggesting that this chromosome might serve as a repository for potentially deleterious genes. These genes thus could play a role in maintaining relative copy number of the two chromosomes. Ryosuke Kadoya is standardizing conditions to selectively inhibit replication initiation of one of the two Vibrio chromosomes and study the consequences to replication and segregation of the other chromosome, as well as to cell growth and division. These studies are expected to provide initial evidence for communication between the two chromosomes and whether mechanisms exist in bacteria analogous to check-point controls prevalent in eukaryotes. To study the regulation of replication of chromosome II, Tatiana Venkova-Canova has defined the bounds of the replication origin and adjoining sequences that serve to control replication initiation negatively. The origin resembles those of some plasmids but the negative control locus is more extended and complex compared to the plasmids. A novel mechanism was found that controls the level of chromosome II specific initiator, RctB, homeostatically, and thereby the replication initiation frequency. Unlike in E. coli, where DNA adenine methylase (Dam) plays a facilitatory role in DNA replication, the enzyme appears essential for replication of both the chromosomes of V. cholerae. The exact role of Dam in replication is not known but most likely it alters the structure of origin DNA that eases its melting, a crucial step in replication initiation. Dam can also modulate gene expression by methylating promoter DNA, and could play an indirect role by supplying a gene product crucial for replication initiation. Gaelle Demarre is testing these alternate possibilities on how Dam could be playing its obligatory role in V. cholerae. The equivalent of microtubules of the eukaryotic mitotic apparatus has not been found in bacteria, and how the bacterial chromosomes segregate has remained largely enigmatic. The recent discovery in bacteria of an actin homolog, MreB, is of special interest because of its suggested role in chromosome segregation. Preeti Srivastava found that the properties of V. cholerae MreB to be similar to its homolog in other bacteria, and that alteration of the protein led to gross distortion in nucleoid morphology and localization of centromeric regions for both the chromosomes. In V. cholerae, Ranajit Ghosh has identified the centromere for chromosome I. A couple of centromere-associated proteins (ParA and ParB) have been purified. To understand how the centromere is utilized to segregate and retain the sister chromosomes in opposite cell halves, Ranajit Ghosh is trying to identify proteins that may serve as mitotic motors or chromosomal anchors. He has been able to co-immuno-precipitate several proteins from crude cell extracts using antibodies against ParAB proteins. The proteins are being identified using LCMS/MS.