Controls of DNA replication and segregation of replicated DNA to daughter cells are essential for chromosome maintenance in all organisms. Subversion of these controls leads to cancer. Dr Chattoraj is studying these controls in E. coli and in V. cholerae, the latter having two chromosomes provides an opportunity to study coordination of replication and segregation in a genetically tractable bacterium. Unlike low copy number plasmids and many other bacteria, E. coli contains no homology to any known segregation system. Richard Fekete is trying to identify cis-acting sequences in the E. coli chromosome that would behave in a centromere-like manner. He has labeled different loci on the same E. coli chromosome using LacI-YFP and Lambda cI-CFP fusion proteins bound to arrays of their respective binding sites. Analysis of migration of a few such pairs of loci using fluorescence microscopy suggests a potential centromere site since it migrates towards the cell pole ahead of the other loci. He is attempting time-lapse photography to track the movement of the centromere to its final destination. He is also trying to identify centromere binding proteins. Preeti Srivastava is doing similar studies to follow the migration of the two cholera chromosomes.To study chromosome maintenance in V. cholerae, Ranajit Ghosh made a genomic library from which the origin of replication and potential """"""""centromere"""""""" sequences for chromosome I were mined. He purified a chromosomal homolog of ParB, a centromere binding protein for plasmids, and developed antibody against the protein. This is being used to verify ParB binding to the candidate centromeric sequences in vivo using the ChIP assay. Role of ParB in chromosome stability is also in progress.We are close to understanding replication control of plasmid P1. Nilangshu Das with some help from a mathematician from Cambridge (UK), Johan Paulsson, has characterized control-defective initiator mutants that confer higher copy number to the plasmid. The properties of the mutants are best explained assuming initiators to be limiting for replication. Tatiana Venkova-Kanova is in the process of testing the applicability of the plasmid control models to the V. cholerae chromosome II whose origin has features similar to those of plasmid P1. The control of the cholera origin has to be also novel as, unlike P1, it involves a small RNA. The role of the small RNA is being studied by Debasish Pal.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
United States
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Chattoraj, Dhruba K (2007) Tryptophanase in sRNA control of the Escherichia coli cell cycle. Mol Microbiol 63:1-3
Srivastava, Preeti; Demarre, Gaelle; Karpova, Tatiana S et al. (2007) Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae. J Bacteriol 189:7450-63
Srivastava, Preeti; Chattoraj, Dhruba K (2007) Selective chromosome amplification in Vibrio cholerae. Mol Microbiol 66:1016-28
Srivastava, Preeti; Fekete, Richard A; Chattoraj, Dhruba K (2006) Segregation of the replication terminus of the two Vibrio cholerae chromosomes. J Bacteriol 188:1060-70
Das, Nilangshu; Valjavec-Gratian, Majda; Basuray, Ashish N et al. (2005) Multiple homeostatic mechanisms in the control of P1 plasmid replication. Proc Natl Acad Sci U S A 102:2856-61
Pal, Debasish; Venkova-Canova, Tatiana; Srivastava, Preeti et al. (2005) Multipartite regulation of rctB, the replication initiator gene of Vibrio cholerae chromosome II. J Bacteriol 187:7167-75
Fekete, Richard A; Chattoraj, Dhruba K (2005) A cis-acting sequence involved in chromosome segregation in Escherichia coli. Mol Microbiol 55:175-83